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over the past several decades , combination chemotherapy has been serving as a primary treatment option for many types of cancers . compared to the use of a single cytotoxic agent , the use of two or more properly selected agents in combination can lead to a decrease in systemic toxicity and an increase in efficacy due to synergistic or cooperative effects of the drugs on the tumor eradication . furthermore , a sequential treatment with chemotherapeutic agents bearing different mechanisms of action has been shown to avert drug resistance and lead to synergistic enhancement of efficacy . despite the profound advantages of combination chemotherapy , the lack of tumor - specificity continues to be a serious issue for cancer treatment . in the past decade , significant advancement has been made on the development of tumor - targeted drug delivery systems ( ttddss ) , especially for monoclonal antibody drug conjugates ( adcs ) and small molecule drug conjugates ( smdcs ) . however , only one such tumor - targeting dual - drug conjugate has been reported for the combination of mitomycin c and desacetylvinblastine using folate as the tumor - targeting module . thus , there is an unmet need for the development of efficacious ttddss amenable to anticancer drug combinations . because a ttdds delivers anticancer drugs through receptor - mediated endocytosis ( rme ) , it should be able to circumvent multidrug resistance caused by abc transporter efflux pumps such as p - glycoprotein in the cancer cell membrane , which is one of the beneficial features of ttddss . we set out to design a versatile ttdds platform for two - drug combinations and construct dual - warhead guided molecular missiles for tumor - targeted combination chemotherapy . such a ttdds would have only single pharmacokinetics for the delivery of two different drugs to tumor because this conjugate is a single molecule , which can get around potentially complicated treatment regimen to deal with two different pharmacokinetic parameters for two drugs . this is a salient feature of the dual - warhead ttdds . for the selection of two drugs ( warheads ) for the novel dual - warhead molecular missiles , we focused on the combination of a microtubule - stabilizing agent ( taxanes ) and a topoisomerase i inhibitor ( camptothecin and its analogues ) in this study . one of the primary reasons for the selection of this combination is the fact that these two classes of anticancer drugs possess clearly different and well - defined molecular targets to investigate their synergistic or additive effects . combinations of paclitaxel or docetaxel ( taxane ) with topotecan or irinotecan ( camptothecin analogue ) have been extensively studied for preclinical and clinical drug development , exhibiting synergistic or additive effects depending on the sequence of drug administration . for taxane anticancer agents , we have been developing highly potent next - generation taxoids which exhibit 23 orders of magnitude greater potency than paclitaxel and docetaxel against multidrug - resistant and paclitaxel - resistant cancer cell lines and tumors . thus , we have used one of these next - generation taxoids for combination with camptothecin in this study . we report here the design , synthesis , and biological evaluations of novel tumor - targeting dual - warhead conjugates , bearing a next - generation taxoid , 1 ( sb - t-1214 ) , and camptothecin as warheads , and biotin as the tumor - targeting module ( figure 1 ) . we designed a versatile platform , consisting of 1,3,5-triazine as the key tripod splitter module , self - immolative linkers with tetraethylene glycol diamine spacers to improve water solubility , and a propargylamine arm for the attachment of second warhead module . then , novel dual - warhead conjugate , 2 ( dw-1 ) , was synthesized based on this platform using click chemistry to attach the camptothecin module with a self - immolative linker and a tetraethylene glycol diamine spacer ( figure 2 ) . another dual - warhead conjugate , 3 ( dw-2 ) , bearing the camptothecin module with a simple ester linkage , was also synthesized to secure stepwise drug release ( figure 2 ) . in addition to these dual - warhead conjugates , we also designed two more drug conjugates using the same platform , wherein one of the warheads was replaced with a surrogate , i.e. , phenol . thus , a conjugate with taxoid 1 and phenol , 4 ( sw - tax ) , and another with camptothecin and phenol , 5 ( sw - cpt ) , were synthesized in order to properly compare the efficacy of dual - warhead vs single - warhead in the same conjugate scaffold ( figure 3 ) . all living cells depend on vitamins for survival , but cancer cells require certain vitamins substantially more than most normal cells do in order to sustain their rapid cell growth and enhanced proliferation . thus , receptors for vitamins are overexpressed on the cell surfaces of cancer cells to maintain sufficient vitamin uptake . therefore , these cell surface vitamin receptors serve as useful biomarkers for tumor - targeting drug delivery . the folate receptor ( fr ) has been studied extensively for its overexpression in cancer cells and solid tumors , as well as the relevant target for tumor - directed drug delivery , as exemplified by a highly promising smdc , vintafolide . in addition to the fr , recent studies have disclosed that the biotin receptor ( br ) is overexpressed even more than the fr in a good number of cancer cell lines . thus , the br has emerged as a promising biomarker for tumor - targeted drug delivery , and we have successfully employed biotin as a tumor - targeting module for small - molecule - based as well as single - walled carbon nanotube - based drug conjugates . in these previous studies , we designed , synthesized , and used several fluorescent and fluorogenic probes for the br by employing fluorescein isothiocyanate ( fitc ) , fluorescein , and coumarin . among those fluorescent probes , biotin - nhnh - fitc was successfully used to verify efficient receptor - mediated endocytosis ( rme ) , targeting the br . in the present study , we designed fluorescent probe 7 , which is more water - soluble than biotin - nhnh - fitc , for the assessment of the br overexpression level in several cancer cell lines to select appropriate cancer cell lines for evaluation of biotin drug conjugates , targeting the br . probe 7 was synthesized through coupling of fitc with n - biotinyl - nh - peg3-(ch2)2nh2 , which was readily prepared from biotin and commercially available 11-azido-3,6,9-trioxaundecan-1-amine through amide coupling , followed by staudinger reduction of the resulting azide ( scheme 1 ) . although russell - jones et al . reported a dozen cell lines overexpressing the br ( br+ ) , most of those cell lines are from murine cancers . accordingly , we used probe 7 to assess the br expression levels in several human cancer cell lines by means of flow cytometry and confocal fluorescence microscopy ( cfm ) . internalization of probe 7 into mx-1 ( breast ) , mcf-7 ( breast ) , id8 ( ovary ) , and wi38 ( lung fibroblast , normal ) cell lines via rme was monitored at 0 , 1 , and 3 h periods . previously studied murine leukemia cell lines l1210fr ( br+ ) and l1210 ( br ) were also used for comparison . as figure 4 clearly indicates , the br is highly expressed in mcf-7 and id8 at the same level as known br cell line , l1210fr , while the expression level is a little lower in mx-1 ( br ) . as anticipated , the br expression level is negligible in human normal cell line , wi38 , and known br- cell line , l1210 . the flow cytometry histograms as well as the corresponding cfm images of probe 7 internalization into id8 and mcf-7 are shown in figure 5 as typical examples . internalization of probe 7 into various cell lines at 0 , 1 , and 3 h at 37 c . fluorescence intensity is a geometric mean of values obtained from a flow cytometry histogram for each cell line , which is provided in the supporting information . internalization of probe 7 monitered by flow cytometry and cfm : ( a ) flow cytometry analysis of 7 in id8 at 0 h ( red , control ) , 1 h ( green ) , and 3 h ( blue ) ; ( b , c ) cfm images of 7 in id8 at 1 and 3 h , respectively ; ( d ) flow cytometry analysis of 7 in mcf-7 at 0 h ( red , control ) , 1 h ( green ) , 3 h ( blue ) ; ( e , f ) cfm images of 7 in mcf-7 at 1 and 3 h , respectively . we also performed the assessment of the br expression level in additional human breast cancer cell lines and found that these cell lines also overexpress the receptor : bt-20 ( br ) , lcc6-wt ( br ) , lcc6-mdr ( br ) , mda - mb 231 ( br ) , and skbr3 ( br ) ( see supporting information ) . it was also found that the br and fr are equally overexpressed in bt-20 , lcc6-wt , mda - mb 231 , and skbr3 cell lines , while the fr is more expressed than the br in lcc6-mdr and mcf-7 cell lines ( see supporting information ) . disulfide linkers , which can be conjugated to a cytotoxic warhead on one end and a tumor - targeting module on the other end . smart linkers were designed to be stable during circulation in bloodstream but readily cleavable inside the tumor microenvironment . linkers consist of a 2-sulfhydrylphenylacetic acid component and a sulfanylalkanoic acid component , and the former is connected to a cytotoxic agent and the latter to a tumor - targeting module or a splitter unit linked to a tumor - targeting module ( see figure 2 ) . in this study , 4-sulfanylpentanoic acid was used as the sulfanylalkanoic acid component , since it has been shown that linkers bearing 4-disulfanylpentanoic acid moiety are more stable than disulfanylpropanoic acid moiety , which was used in our previous studies . disulfide linker was designed to be self - immolative for releasing a cytotoxic warhead through disulfide thiol exchange reaction with endogenous thiols , represented by glutathione ( gsh ) , followed by facile benzothiolactonization . the phenyl moiety was placed to electronically direct the cleavage of the disulfide linkage by intracellular thiol and entropically drive thiolactionization . thus , when the tumor - targeting module navigates the drug - conjugate to the target biotin receptors on the tumor surface , the whole conjugate is internalized via receptor - mediated endocytosis ( rme ) . then , an intracellular thiol - triggered cascade drug - release takes place through thiolactonization . because it has been shown that the glutathione level in tumor tissues ( 28 mm ) is more than 1000 times higher than that in bloodstream ( 12 m ) , glutathione and other endogenous intracellular thiols would act as excellent tumor - specific agents to trigger the drug release . the preparation of bifunctional disulfide linker unit 11 and its use for the synthesis of coupling - ready ( 14 t , 14p ) or click-ready constructs ( 15c , 15p ) of drug - linker conjugates with taxoid 1 , camptothecin ( cpt ) , and phenol are illustrated in scheme 2 . first , 4-sulfanylpentanoic acid ( 9 ) was prepared by ring - opening substitution of -valerolactone ( 8) with hydrobromic acid and thiourea , followed by basic hydrolysis . the thiol disulfide exchange reaction of 9 with 4,4-dipyridinedisulfide , followed by tips protection of the carboxylic acid moiety , gave tips pyridinyldisulfanylpentanoate 10 in 92% yield for two steps . disulfide exchange reaction of 10 with 2-sulfanylphenylacetic acid afforded 11 in 68% yield . the coupling reactions of 11 with 1 , cpt , and phenol in the presence of dic and dmap gave the corresponding drug disulfide linker constructs 12 t , 12c , and 12p , respectively , in 5779% yields . tips groups of 12 t , 12c , and 12p were removed with hf - pyridine to afford 13 t , 13c , and 13p , respectively . then , the esterification of 13 t and 13p with n - hydroxysuccinimide ( hosu ) gave the corresponding coupling - ready taxoid - linker construct 14 t and phenol - linker construct 14p , respectively , as activated esters in high yields . finally , the dic coupling of 13c and 13p with 1-amino-11-azido-3,6,9-trioxaundecane afforded the corresponding click-ready drug - linker constructs , 15c and 15p , respectively , in good yields . to investigate the effects of sequential drug release on the potency of dual warhead drug conjugates , cpt - linker construct 18 , bearing a simple ester linker , was designed to secure that the taxoid is released prior to cpt inside the cancer cell . as scheme 3 illustrates , succinic anhydride ( 16 ) was reacted with h2n - peg3-(ch2)2n3 to give 17 , which was coupled to cpt in the presence of dic and dmap to give 18 in good yield . among possible tripod splitter modules , we chose 1,3,5-triazine for novel dual - warhead conjugates with three arms . also , we decided to implement water - soluble linker components by incorporating poly(ethylene glycol ) oligomers , i.e. , peg3 , as well as the use of click chemistry to introduce the third arm at the end of the synthesis . thus , dual - warhead conjugates , 2 and 3 , as well as their single - warhead surrogates , i.e. , 4 and 5 , were designed for construction by incorporating those components and strategies ( see figure 2 ) . the syntheses of conjugates 2 , 3 , and 4 are illustrated in scheme 4 . first , cyanuric acid ( 19 ) was reacted with n - boc - ethylenediamine ( 1.0 equiv ) in the presence of n , n - diisopropylethylamine ( dipea ) at 0 c , followed by the subsequent substitution reaction with biotinyl - nh - peg3-nh2 ( 6 ) ( 1.0 equiv ) in the presence of dipea at 40 c to give 20 in moderate yield for 2 steps . the reaction of 20 with excess propargylamine in the presence of dipea at 50 c afforded fully functionalized triazine 21 in 69% yield . the deprotection of 21 with tfa gave 22 , which was coupled with taxoid - linker - osu 14 t in the presence of pyridine at 25 c to afford 23 in 73% yield . reactions with azides 15c , 18 , and 15p in the presence of cupric sulfate and ascorbic acid at 25 c to give the corresponding conjugates , 2 , 3 , and 4 , respectively , in 7392% yields . for the synthesis of the single - warhead surrogate 5 bearing camptothecin , triazine 22 was reacted with phenol - linker - osu 14p in the presence of pyridine at 25 c to afford 24 in 52% yield . reaction of 24 with azide 15c under the standard conditions gave 5 in 54% yield ( scheme 5 ) . the effect of the combinations of taxoid 1 and camptothecin ( 1:1 ) on cytotoxicity was examined using three different timings for drug mixing against human breast carcinoma ( mx-1 and mcf-7 ) , murine ovarian ( id8 ) , and murine leukemia ( l1210fr ) cell lines . as table 1 shows , the cytotoxicity of taxoid 1 was 148846 times stronger than camptothecin against four cancer cell lines examined ( entries 1 and 2 ) . the most significant and interesting observation was the fact that the order of exposure to these two drugs , i.e. , ( a ) simultaneous exposure ( entry 3 ) , ( b ) taxoid first for 24 h , followed by camptothecin ( entry 4 ) , and ( c ) camptothecin first for 24 h , followed by taxoid ( entry 5 ) , exhibited a marked difference in the potency . when cancer cells were exposed to camptothecin first for 24 h , followed by equimolar concentrations of taxoid 1 ( entry 5 ) , drastic increases in the ic50 values as compared to those values for simultaneous exposure ( entry 3 ) were observed . these ic50 values ( entry 5 ) were even higher than those for camptothecin alone except for that against the id8 cell line ( entry 2 ) . the results clearly indicate that these two drugs act antagonistically in this case , and camptothecin appears to block the action of taxoid 1 . this is probably ascribed to the cell cycle control , e.g. , synchronization , by camptothecin , which causes detrimental effects on the action of taxoid 1 . in sharp contrast , when cancer cells were exposed to equimolar mixture of the two drugs ( entry 3 ) or to taxoid 1 first for 24 h , followed by equimolar concentrations of camptothecin ( entry 4 ) , ic50 values were essentially the same as those for taxoid 1 alone within errors ( entry 1 ) . the results indicate that in these two cases , these two drugs act at least cooperatively and taxoid 1 might be potentiating camptothecin . cells were incubated with an equimolar mixture of two drugs for 72 h at 37 c . cells were incubated with 1 at a given concentration for 24 h at 37 c . then , an equimolar amount of cpt was added and the cells were incubated for additional 48 h at 37 c . cells were incubated with cpt at a given concentration for 24 h at 37 c . then , an equimolar amount of 1 was added and the cells were incubated for additional 48 h at 37 c . we have shown the efficient internalization of fluorescence - labeled biotin probe 7 into several cancer cell lines via rme ( see figures 4 and 5 ) . we have also previously reported the proofs for highly efficient rme of a biotin - linker-(taxoid - fluorescein ) conjugate and a ( biotin)n swnt(taxoid - fluorescein)m conjugate . in this study , we examined the receptor - mediated internalization of dual - warhead conjugate 2 by taking advantage of the intrinsic fluorescence of camptothecin using cfm . id8 and mcf-7 cancers were incubated with conjugate 2 ( 10 m ) at 37 c for 10 h. after ample washing of the drug media , the cells were analyzed by cfm . as figure 6 shows , the intense signature blue fluorescence of camptothecin was observed in both cancer cells , confirming the anticipated efficient internalization of conjugate 2 . confocal fluorescence microscopy images showing internalization of conjugate 2 in id8 ( left ) and mcf-7 ( right ) after incubation at 37 c for 10 h. cytotoxicities of novel dual - warhead conjugates , 2 and 3 , as well as their single - warhead surrogates , 4 and 5 , were evaluated in two ways against four br(+ ) cancer cell lines , mx-1 , mcf-7 , id8 and l1210fr , as well as normal human lung fibroblast cell line , wi38 , using the standard mtt assay . taxoid 1 and camptothecin were also examined for comparison ( table 2 , entries 1 and 2 , respectively ) . cells were incubated with a drug or a conjugate at 37 c for 72 h. cells were initially incubated with conjugates 25 for 24 h , followed by washing of the drug media with pbs , then addition of gsh - oet ( 6 equiv . to a conjugate ) for drug release and additional incubation at 37 c for 48 h. human breast carcinoma cell line ( br+ ) . human breast carcinoma cell line ( br+ ) . human lung fibroblast cell line ( br ) . in the first experiment , mx-1 ( br ) , mcf7 ( br ) , id8 ( br ) , and l1210fr ( br ) cancer cells were incubated with conjugate 25 for 72 h , and the corresponding ic50 values were determined . as table 2 shows , the cytotoxicities of conjugates 2 and 3 based on their ic50 values were found to be in a range of 1965 nm ( entries 3 and 4 ) , which makes a sharp contrast with that against normal cell line wi38 ( ic50 742868 nm ) ( entries 3 and 4 ) . the results indicate that these conjugates were selectively internalized into br(+ ) cancer cells via rme , and the drug was partially released inside cancer cells . single - warhead surrogate 4 showed a similar potency as that of conjugate 3 ( entry 5 ) , while the other surrogate 5 did not exhibit appreciable cytotoxicity ( mx-1 and id8 ) or very weak potency ( mcf-7 , wi38 ) , with l1210fr as an exception ( entry 6 ) . for conjugates 24 bearing taxoid 1 as a warhead , the cytotoxicity level appeared to be markedly reduced as compared to that for free taxoid 1 . because we have confirmed that these four cancer cell lines overexpress br ( see figures 3 and 4 ) and also observed marked selectivity for br+ cancer cell lines as compared to normal wi38 cell line ( br ) , the internalization of conjugate 25 via rme should not be the major issue for these cancer cell lines . thus , it is very likely that the level of gsh ( and/or other endogenous thiols ) is not sufficient to release the warhead(s ) via disulfide linker cleavage under in vitro conditions . this situation makes a sharp contrast to that in vivo where there are sufficient endogenous thiols ( e.g. , gsh ) in tumors , which are under hypoxic conditions . to clarify these points , glutathione ethyl ester ( gsh - oet ) ( 6 mol equiv to a conjugate ) was added to the resuspended cancer cells after the cells had been incubated with a conjugate for 24 h , followed by thorough washing with phosphate - buffered saline ( pbs ) , and further incubated for 48 h in the second experiment ( table 3 ) . it should be noted that the resuspended cancer cells only included a conjugate internalized in the first 24 h period . as table 3 shows , this protocol , including washing of cells and addition of gsh - oet , did not make any appreciable difference in the cytotoxicity of these conjugates against wi38 normal fibroblast cells , based on the comparison of the results in table 2 with those in table 3 . conjugate 2 showed ic50 values of 3.29.8 nm against four br+ cancer cell lines , but that against wi38 was 742 nm ( entry 1 ) . thus , there is two order of magnitude difference in cytotoxicity between cancer cells and normal cells , which is unambiguously attributed to the highly efficient receptor - targeting by conjugate 2 . there was also considerable difference in potency between conjugate 2 and conjugate 3 ( entries 1 and 2 ) . this difference is attributable to full release of both taxoid 1 and camptothecin in conjugate 2 via cleavage of a disulfide linkage versus insufficient release of camptothecin in conjugate 3 , due to a more stable ester linkage between the linker and the drug , under the conditions examined . the results clearly indicate that there were synergistic or cooperative effects between taxoid 1 and camptothecin when these two drugs were delivered to cancer cells simultaneously and released inside cancer cells . because surrogate 4 , bearing taxoid 1 and phenol , exhibited a similar but systematically slightly weaker potency that that of conjugate 3 ( entry 3 ) , it is reasonable to assume that the release of camptothecin occurred to some extent under the experimental conditions examined . it appears that all four br+ cancer cell lines are highly drug resistant to camptothecin and the potency was further decreased substantially when camptothecin and phenol were delivered to mx-1 , mcf-7 , and id8 cells via rme and released inside cancer cells ( table 3 , entry 4 ) as compared to that observed in the standard mtt assay of camptothecin ( table 2 , entry 2 ) . the results may indicate that phenol interferes with the action of camptothecin in those cancer cells . interestingly , surrogate 5 exhibited higher potency than camptothecin against l1210fr leukemia cell line ( ic50 319 nm in table 2 and 177 nm in table 3 for 5 vs 510 nm for cpt in table 2 ) . the results may suggest that phenol has cytotoxic effect on this leukemia cell line . we have designed and successfully synthesized novel tumor - targeting dual - warhead conjugates , 2 and 3 , which consist of taxoid 1 and camptothecin as two warheads , self - immolative disulfide linkers for drug release , biotin as the tumor - targeting moiety , and 1,3,5-triazine as the tripod splitter module . conjugates 2 and 3 incorporated tetraethylene glycol diamine moieties to increase the water solubility of the conjugates . the functionalized 1,3,5-triazine - based ttdds platform is versatile and applicable to a variety of two - drug combinations as well as the use of various tumor - targeting modules . for the introduction of the second warhead - linker module to the 1,3,5-triazine splitter module then , its acetylene moiety was used for click reaction , with the second drug - linker construct bearing an -azido group to install the second warhead module as the third arm . since biotin was used as the tumor - targeting moiety , we screened the expression levels of the biotin receptor ( br ) in various human breast cancer cell lines , mx-1 , mcf-7 , lcc6-wt , lcc6-mdr , mda - mb 231 , and skbr3 , which had not been reported previously in addition to the known br - overexpressing cancer cell lines , id8 ( human ovary ) and l1210fr ( murine leukemia ) , by means of flow cytometry and confocal fluorescence microscopy ( cfm ) . then , we confirmed that these human breast cancer cell lines overexpress br at variable levels . also , we confirmed that there was no appreciable expression of br in l1210 ( murine leukemia ) and wi38 ( normal , human lung fibroblast ) cell lines . the potencies of conjugates , 2 and 3 , were evaluated against mx-1 , mcf-7 , id8 , l1210fr , and wi38 . it was found that the amount of endogenous thiols such as glutathione ( gsh ) was insufficient to trigger the drug release via cleavage of the self - immolative disulfide linkers under in vitro assay conditions ( this is not an issue in vivo ) . accordingly , glutathione ethyl ester ( gsh - oet ) ( 6 equiv to a conjugate ) was added after the cells were incubated with 2 or 3 for 24 h , thoroughly washed with saline ( pbs ) , and resuspended . then , the cells were further incubated for 48 h. thus , only the conjugate molecules internalized into the cancer cells in the first 24 h were treated with gsh - oet in this protocol . the addition of gsh - oet showed remarkable effect , as anticipated , and conjugate 2 exhibited ic50 values of 3.229.80 nm against all br+ cancer cell lines , while that against normal cell line , wi38 , was 705 nm . thus , there were two orders of magnitude difference in selectivity to cancer cells , which was impressive . also , the potency of 1 was 23 times as high as those of 3 and surrogate conjugate 4 , which clearly indicates the synergistic or cooperative effect of the taxoid it is noteworthy that this substantial enhancement in potency in this combination was observed only when these two drugs were internalized via rme . the ic50 values observed for conjugate 2 were 3.57.3 times larger than that of taxoid 1 . the observed reduced potencies can be ascribed to insufficient internalization of 1 to those cells in the first 24 h , which can be attributed to ( a ) a saturation phenomenon for br - binding and internalization , depending on the expression level of br on the cell surface and ( b ) slow recycling of the receptors in the first 24 h. this is , however , not an issue in vivo . further studies along this line , as well as the evaluation of the efficacy of 2 and 3 in vivo are actively underway in our laboratory . thus , these drugs and all structurally related compounds and derivatives must be considered as mutagens and potential reproductive hazards for both males and females . appropriate precautions , such as the use of gloves , goggles , labware , and fume hood , must be taken while handling the compounds at all times . h and c nmr spectra were measured on a varian 300 , 400 , or 500 mhz spectrometer or a bruker 500 mhz nmr spectrometer . melting points were measured on a thomas hoover capillary melting point apparatus and are uncorrected . tlc was performed on sorbent technologies aluminum - backed silica g tlc plates ( sorbent technologies , 200 m , 20 cm 20 cm ) , and column chromatography was carried out on silica gel 60 ( merck , 230400 mesh astm ) . purity was determined with a shimadzu l-2010a hplc ht series hplc assembly , using a kinetex pfp column ( 4.6 mm 100 mm , 2.6 m ) with acetonitrile water gradient solvent system . two analytical conditions were used and noted as a part of the characterization data and purity for literature unknown compounds , i.e. , hplc ( 1 ) , flow rate 0.4 ml / min , a gradient of 15 95% acetonitrile for the 012 min period ; hplc ( 2 ) , flow rate 0.5 ml / min , a gradient of 595% acetonitrile for the 012 min period and 95% acetonitrile for the 1115 min period . high resolution mass spectrometry analysis was carried out on an agilent lc - uv - tof mass spectrometer at the institute of chemical biology and drug discovery , stony brook , ny or at the mass spectrometry laboratory , university of illinois at urbana champaign , urbana , il . the chemicals were purchased from sigma - aldrich , fisher scientific , and vwr international and used as received or purified before use by standard methods . 2,2-dipyridyldisulfide , ( 2-sulfanylphenyl)acetic acid , taxoid 1 , 1-amino-11-azido-3,6,9-trioxaundecane , n - boc - ethylenediamine , n - biotinyl - peg3-diamine ( 6 ) , and 4-sulfanylpentanoic acid ( 9 ) were prepared by literature methods . 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) was obtained from sigma chemical co. biological materials including rpmi-1640 and dmem cell culture media , fetal bovine serum , nuserum , penstrep , and tryple were obtained from gibco and vwr international , and used as received for cell - based assays . to a solution of fluorescein isothiocyanate ( fitc ) ( 20 mg , 0.0502 mmol ) and n - biotinyl-3,6,9-trioxaundecamethylene-1,11-diamine ( 30 mg , 0.0717 mmol ) in dmf ( 0.6 ml ) the mixture was allowed to react for 48 h at room temperature in the dark with stirring , and the reaction mixture was directly loaded onto a silica gel column . purification of the reaction mixture by column chromatography on silica gel using 15% ch3oh in ch2cl2 as eluent gave probe 7 ( 27 mg , 68% yield ) as an orange solid ; mp 191192 c . h nmr ( 500 mhz , cd3od ) 1.40 ( m , 2h ) , 1.62 ( m , 2h ) , 1.69 ( m , 2h ) , 2.19 ( t , j = 7.4 hz , 2h ) , 2.67 ( d , j = 12.7 hz , 1h ) , 2.88 ( dd , j = 5.0 , 12.7 hz , 1h ) , 3.15 ( m , 1h ) , 3.32 ( m , 2h ) , 3.51 ( t , j = 5.2 hz , 2h ) , 3.59 ( m , 2h ) , 3.63 ( m , 2h ) , 3.68 ( s , 4h ) , 3.71 ( t , j = 5.2 hz , 2h ) , 3.82 ( bs , 2h ) , 4.26 ( dd , j = 4.5 , 7.9 hz , 1h ) , 4.45 ( dd , j = 4.5 , 7.9 hz , 1h ) , 6.55 ( dd , j = 2.4 , 8.8 hz , 2h ) , 6.67 ( d , j = 2.4 hz , 2h ) , 6.69 ( d , j = 8.8 hz , 2h ) , 7.16 ( d , j = 8.3 hz , 1h ) , 7.77 ( d , j = 7.8 hz , 1h ) , 7.95 ( bs , 1h ) , 8.15 ( d , 1.7 hz , 1h ) . c nmr ( 125 mhz , cd3od ) 7.21 , 12.43 , 15.43 , 21.71 , 24.82 , 27.47 , 27.73 , 30.77 , 34.74 , 38.32 , 39.05 , 43.49 , 45.89 , 54.97 , 59.61 , 61.35 , 68.58 , 69.24 , 69.32 , 69.56 , 69.61 , 101.54 , 109.74 , 112.02 , 123.99 , 128.46 , 140.43 , 152.40 , 164.11 , 169.25 , 174.15 , 180.88 . hrms ( tof ) for c39h46n5o16s2 calcd , 808.2681 ; found , 808.2691 ( = 1.2 ppm ) . hplc ( 1 ) : t = 9.1 min , purity > 97% . to a solution of 9 ( 709 mg , 5.29 mmol ) in ethanol ( 20 ml ) was added 2,2-dipyridinedisulfide ( 7.0 g , 31.7 mmol ) in ethanol ( 100 ml ) , and the mixture was refluxed at 80 c for 2 h. the reaction mixture was cooled to 25 c and concentrated in vacuo to afford a yellow precipitate . the crude product was purified by column chromatography on silica gel to afford a mixture of 4-(pyridin-2-yldisulfanyl)pentanoic acid and pyridine-2-thiol . to a cooled solution of this mixture in dichloromethane ( 40 ml ) at 0 c was added triethylamine ( 2.1 ml , 15.1 mmol ) and chlorotriisopropylsilane ( 1.93 ml , 9.04 mmol ) . the mixture was allowed to react for 24 h with stirring and concentrated in vacuo to afford a crude product as an oil . purification of the crude product by column chromatography on silica gel with hexanes / ethyl acetate ( 2:1 ) as eluent gave 10 ( 2.71 g , 92% yield for 2 steps ) , as a colorless oil . h nmr ( 600 mhz , cdcl3 ) 1.06 ( m , 21 h ) , 1.34 ( d , j = 6.8 hz , 3h ) , 1.89 ( m , 1h ) , 2.00 ( m , 1h ) , 2.51 ( m , 2h ) , 3.03 ( m , 1h ) , 7.06 ( ddd , j = 1.0 , 4.8 , 7.4 hz , 1h ) , 7.62 ( dt , j = 1.5 , 8.2 hz , 1h ) , 7.72 ( d , j = 8.2 hz , 1h ) , 8.44 ( d , j = 4.8 hz , 1h ) . h nmr spectrum was consistent with the reported data . to a cooled solution of 10 ( 300 mg , 0.752 mmol ) in tetrahydrofuran ( 1.5 ml ) at 10 c was added ( 2-sulfanylphenyl)acetic acid ( 130 mg , 0.752 mmol ) in tetrahydrofuran ( 1.5 ml ) , and the mixture was allowed to react for 2 h at room temperature with stirring . purification the crude produce by column chromatography on silica gel with hexanes / ethyl acetate ( 3:1 ) as eluent gave 11 ( 166 mg , 68% yield ) as an orange oil . h nmr ( 300 mhz , cdcl3 ) 1.06 ( m , 21h ) , 1.25 ( d , j = 6.8 , 3h ) , 1.81 ( m , 1h ) , 1.92 ( m , 1h ) , 2.44 ( m , 2h ) , 2.90 ( m , 1h ) , 3.90 ( s , 2h ) , 7.24 ( m , 2h ) , 7.32 ( m , 1h ) , 7.82 ( d , j = 7.7 hz , 1h ) . hrms ( tof ) for c22h37o4s2si calcd , 457.1897 ; found , 457.1892 ( = 1.1 ppm ) . to a solution of taxoid 1 ( 600 mg , 0.703 mmol ) , 11 ( 350 mg , 0.768 mmol ) , and 4-n , n - dimethylaminopyridine ( dmap ) ( 38 mg , 0.308 mmol ) in ch2cl2 ( 13 ml ) was added n , n-diisopropylcarbodiimide ( dic ) ( 135 l , 0.877 mmol ) , and the mixture was allowed to react for 24 h at room temperature with stirring . the reaction was quenched with saturated nh4cl ( 10 ml ) , and the reaction mixture was diluted with water ( 20 ml 3 ) , washed with brine ( 20 ml 3 ) , dried over mgso4 , and concentrated in vacuo to afford a yellow oil . the crude product was dissolved in ether ( 20 ml ) , and the insoluble urea byproduct was removed by gravity filtration on a filter paper . purification of the crude product by column chromatography on silica gel with hexanes / ethyl acetate ( 3:1 ) as eluent gave 12 t ( 697 mg , 77% yield ) , as a white solid . h nmr ( 400 mhz , cdcl3 ) 0.97 ( m , 2h ) , 1.06 ( d , j = 7.2 hz , 18h ) , 1.14 ( m , 5h ) , 1.25 ( s , 6h ) , 1.30 ( d , j = 6.8 hz , 3h ) , 1.33 ( s , 9h ) , 1.66 ( s , 3h ) , 1.69 ( s , 3h ) , 1.72 ( s , 3h ) , 1.76 ( m , 2h ) , 1.85 ( m , 1h ) , 1.90 ( s , 3h ) , 1.93 ( m , 2h ) , 2.30 ( m , 2h ) , 2.35 ( s , 3h ) , 2.42 ( m , 2h ) , 2.53 ( m , 2h ) , 2.59 ( m , 1h ) , 2.94 ( m , 1h ) , 3.81 ( d , j = 7.2 hz , 1h ) , 3.94 ( dd , j = 1.7 , 16.8 hz , 1h ) , 4.10 ( dd , j = 1.7 , 16.8 hz , 1h ) , 4.18 ( d , j = 8.4 hz , 1h ) , 4.30 ( d , j = 8.4 hz , 1h ) , 4.42 ( m , 1h ) , 4.79 ( d , j = 8.4 hz , 1h ) , 4.92 ( s , 1h ) , 4.96 ( d , j = 7.2 hz , 1h ) , 5.07 ( d , j = 8.4 hz , 1h ) , 5.67 ( d , j = 7.2 hz , 1h ) , 6.19 ( t , j = 8.7 hz , 1h ) , 6.28 ( s , 1h ) , 7.26 ( m , 3h ) , 7.47 ( t , j = 7.9 hz , 2h ) , 7.60 ( t , j = 7.4 hz , 1h ) , 7.80 ( d , j = 7.4 hz , 1h ) , 8.11 ( d , j = 7.1 hz , 2h ) . hrms ( tof ) for c67h94no18s2si calcd , 1292.5676 ; found , 1292.5676 ( = 0 ppm ) . to a solution of camptothecin ( 158 mg , 0.431 mmol ) , 10 ( 489 mg , 1.072 mmol ) , and dmap ( 53 mg , 0.431 mmol ) in ch2cl2 ( 20 ml ) was added dic ( 73 l , 0.474 mmol ) , and the mixture was allowed to react for 16 h at room temperature with stirring . the reaction mixture was cooled to 0 c , and the urea byproduct was removed via filtration . purification of the crude product by column chromatography on silica gel with 1.5% ch3oh in ch2cl2 as eluent gave 12c ( 192 mg , 57% yield ) as an off - white solid . h nmr ( 500 mhz , cdcl3 ) 0.98 ( t , j = 7.5 hz , 3h ) , 1.05 ( m , 21h ) , 1.45 ( d , j = 6.8 hz , 2h ) , 1.85 ( m , 1h ) , 2.18 ( m , 1h ) , 2.33 ( m , 2h ) , 2.87 ( m , 1h ) , 4.00 ( dd , j = 4.5 , 16.5 hz , 1h ) , 4.02 ( d , j = 22.0 hz , 1h ) , 4.17 ( dd , j = 9.2 , 16.5 hz , 1h ) , 5.21 ( m , 2h ) , 5.38 ( d , j = 17.5 hz , 1h ) , 5.64 ( d , j = 17.2 hz , 1h ) , 7.21 ( d , j = 2.5 hz , 1h ) , 7.26 ( m , 4h ) , 7.66 ( t , j = 7.5 hz , 1h ) , 7.85 ( t , j = 8.0 hz , 1h ) , 7.92 ( d , j = 8.0 hz , 1h ) , 8.27 ( d , j = 8.5 hz , 1h ) , 8.35 ( s , 1h ) . c nmr ( 125 mhz , cdcl3 ) 7.60 , 11.85 , 17.78 , 20.39 , 20.98 , 22.55 , 23.53 , 30.94 , 31.78 , 32.96 , 38.59 , 45.82 , 49.92 , 53.51 , 67.13 , 96.26 , 120.18 , 127.62 , 127.67 , 127.77 , 127.92 , 128.05 , 128.13 , 128.23 , 128.27 , 128.42 , 129.69 , 129.72 , 129.94 , 130.04 , 130.40 , 130.54 , 130.59 , 130.86 , 130.89 , 131.06 , 132.56 , 132.63 , 134.65 , 137.09 , 137.44 , 145.60 , 146.20 , 148.83 , 152.30 , 153.82 , 157.27 , 167.33 , 169.91 , 172.91 . hrms ( tof ) for c42h51n2o7s2si calcd , 787.2901 ; found , 787.2897 ( = 0.5 ppm ) . to a solution of phenol ( 135 mg , 1.434 mmol ) , 11 ( 545 mg , 1.195 mmol ) , and dmap ( 14 mg , 0.119 mmol ) in ch2cl2 ( 15 ml ) was added dic ( 210 l , 1.314 mmol ) , and the mixture was allowed to react for 10 h at room temperature with stirring . the reaction was quenched with saturated nh4cl ( 10 ml ) , and the reaction mixture was diluted with water ( 5 ml ) and extracted with ch2cl2 ( 20 ml 3 ) . the combined organic layers were dried over mgso4 and concentrated in vacuo to afford a yellow oil . purification of the crude product by column chromatography on silica gel with 3% ethyl acetate in hexanes as eluent gave 12p ( 505 mg , 79% yield ) as a colorless oil . h nmr ( 400 mhz , cdcl3 ) 1.06 ( m , 21h , tips ) , 1.30 ( d , j = 6.8 hz , 3h ) , 1.85 ( m , 1h ) , 1.93 ( m , 1h ) , 2.42 ( m , 2h ) , 2.96 ( m , 1h ) , 4.11 ( s , 2h ) , 7.10 ( d , j = 7.4 hz , 2h ) , 7.21 ( t , j = 7.4 hz , 1h ) , 7.26 ( t , j = 7.3 hz , 1h ) , 7.31 ( t , j = 7.3 hz , 2h ) , 7.36 ( t , j = 7.4 hz , 2h ) , 7.82 ( d , j = 7.9 , 1h ) . c nmr ( 100 mhz , cdcl3 ) 11.89 , 17.79 , 20.57 , 30.99 , 33.03 , 39.41 , 46.11 , 121.48 , 125.84 , 127.65 , 128.27 , 129.36 , 130.23 , 130.83 , 133.38 , 137.67 , 150.79 , 169.40 , 173.01 . hrms ( tof ) for c28h41o4s2si calcd , 533.2210 ; found , 533.2222 ( = 2.3 ppm ) . to a solution of 12 t ( 649 mg , 0.502 mmol ) in a 1:1 mixture of ch3cn pyridine ( 1:1 ) ( 50 ml ) and cooled to 0 c under nitrogen was added hf / pyridine ( 6.5 ml ) , slowly , and the mixture was stirred for 24 h at room temperature . the reaction was quenched with 10% citric acid ( 10 ml ) , and the reaction mixture was diluted with ethyl acetate ( 20 ml ) . the reaction mixture was extracted with ethyl acetate ( 20 ml 2 ) and washed with saturated cuso4 ( 3 20 ml ) and brine ( 3 20 ml ) . the organic layer was dried over mgso4 and concentrated in vacuo to afford a clear oil . purification of the crude product by column chromatography on silica gel with hexanes / ethyl acetate ( 1:4 ) as eluent gave 13 t ( 516 mg , 91% yield ) as a white solid . h nmr ( 500 mhz , cdcl3 ) 1.02 ( m , 2h ) , 1.16 ( m , 2h ) , 1.18 ( m , 3h ) , 1.29 ( s , 6h ) , 1.39 ( s , 9h ) , 1.70 ( s , 3h ) , 1.75 ( s , 3h ) , 1.76 ( s , 3h ) , 1.80 ( m , 2h ) , 1.89 ( bt , 1h ) , 1.93 ( s , 3h ) 1.94 ( m , 2h ) , 2.39 ( m , 2h ) , 2.42 ( s , 3h ) , 2.56 ( m , 1h ) , 3.03 ( m , 1h ) , 3.83 ( m , 1h ) , 3.96 ( dd , j = 1.7 , 16.8 hz , 1h ) , 4.15 ( dd , j = 1.7 , 16.8 hz , 1h ) , 4.20 ( d , j = 8.4 hz , 1h ) , 4.34 ( d , j = 8.5 hz , 1h ) , 4.45 ( m , 1h ) , 5.00 ( m , 3h ) , 5.18 ( d , j = 7.1 hz , 1h ) , 5.70 ( d , j = 7.2 hz , 1h ) , 6.25 ( m , 1h ) , 6.32 ( m , 1h ) , 7.33 ( m , 3h ) , 7.51 ( t , j = 7.8 hz , 2h ) , 7.63 ( t , j = 7.5 hz , 1h ) , 7.84 ( d , j = 7.8 hz , 1h ) , 8.14 ( d , j = 7.1 hz , 2h ) , 8.66 ( bs , 1h ) . hrms for ( tof ) c58h74no18s2 calcd , 1136.4342 ; found , 1136.4365 ( = 2.0 ppm ) . to a cooled solution of 12c ( 192 mg , 0.244 mmol ) in ch3cn pyridine ( 1:1 ) ( 30 ml ) at 0 c was added 70% hf / pyridine ( 3 ml ) dropwise , and the mixture was allowed to gradually warm to room temperature over 5 h with stirring . the reaction was quenched with 10% citric acid ( 10 ml ) , and the reaction mixture was diluted with water ( 10 ml ) . the reaction mixture was extracted with ch2cl2 ( 3 20 ml ) , and the combined organic layers were washed with saturated cuso4 ( 60 ml ) , water ( 60 ml ) , and brine ( 60 ml ) . the organic layers were dried over mgso4 and concentrated in vacuo to give a green solid . purification of the crude product by column chromatography on silica gel with 1% ch3oh in ch2cl2 as eluent gave 13c ( 131 mg , 85% yield ) as a pale - green solid . h nmr ( 500 mhz , dmso - d6 ) 0.91 ( t , j = 7.5 hz , 3h ) , 1.05 ( dd , j = 6.8 , 8.4 hz , 3 h ) , 1.53 ( m , 1h ) , 1.70 ( m , 1h ) , 2.18 ( t , j = 7.5 hz , 2h ) , 2.20 ( m , 2h ) , 2.88 ( m , 1h ) , 4.05 ( d , j = 16.5 hz , 1h ) , 4.20 ( dd , j = 4.0 , 16.5 hz , 1h ) , 5.30 ( m , 2h ) , 5.46 ( m , 2h ) , 7.10 ( s , 1h ) , 7.27 ( t , j = 7.5 hz , 1h ) , 7.35 ( m , 2h ) , 7.74 ( m , 2h ) , 7.91 ( t , j = 7.3 hz , 1h ) , 8.15 ( d , j = 8.0 hz , 1h ) , 8.23 ( d , j = 8.0 hz , 1h ) , 8.70 ( s , 1h ) , 12.10 ( s , 1h ) . c nmr ( 125 mhz , dmso - d6 ) 8.00 , 20.13 , 20.24 , 30.69 , 30.79 , 30.86 , 31.29 , 31.35 , 38.57 , 45.75 , 45.83 , 50.70 , 55.40 , 66.78 , 76.61 , 95.61 , 119.37 , 127.95 , 127.97 , 128.17 , 128.46 , 128.89 , 129.00 , 129.57 , 129.78 , 129.86 , 130.16 , 130.84 , 131.70 , 131.96 , 133.11 , 133.16 , 137.59 , 137.63 , 145.48 , 146.41 , 148.43 , 152.71 , 156.98 , 167.51 , 169.84 , 174.22 . hrms ( tof ) for c33h31n2o7s2 calcd , 631.1567 ; found , 631.1561 ( = 0.9 ppm ) . to a cooled solution of 12p ( 505 mg , 0.947 mmol ) in ch3cn pyridine ( 1:1 ) ( 80 ml ) at 0 c , was added 70% hf / pyridine ( 5 ml ) , and the mixture was stirred for 6 h at room temperature . the reaction was quenched with 10% citric acid and extracted with ethyl acetate ( 50 ml 3 ) . the combined organic layers were washed with saturated cuso4 ( 50 ml 3 ) and brine ( 50 ml 3 ) , dried over mgso4 , and concentrated in vacuo to afford a pale - yellow oil . purification of the crude product by column chromatography on silica gel with 50% ethyl acetate in hexanes as eluent gave 14p ( 314 mg , 88% yield ) as a colorless oil . h nmr ( 500 mhz , cdcl3 ) 1.29 ( d , j = 6.8 hz , 3h , ch3 ) , 1.86 ( m , 1h ) , 1.95 ( m , 1h ) , 2.42 ( m , 2h ) , 2.94 ( m , 2h ) , 4.12 ( s , 2h ) , 7.11 ( d , j = 7.5 hz , 2h ) , 7.21 ( t , j = 7.5 hz , 1h ) , 7.26 ( m , 1h ) , 7.31 ( t , j = 7.5 hz , 2h ) , 7.35 ( t , j = 7.5 hz , 2h ) , 7.81 ( d , j = 7.5 hz , 1h ) . c nmr ( 400 mhz , cdcl3 ) 20.48 , 30.35 , 31.03 , 39.42 , 45.90 , 121.47 , 125.88 , 127.80 , 128.31 , 129.39 , 130.45 , 130.94 , 133.53 , 137.51 , 150.77 , 169.50 , 178.29 . hrms ( tof ) for c19h21o4s2 calcd , 377.0876 ; found , 377.0888 ( = 3.2 ppm ) . to a solution of 13 t ( 500 mg , 0.441 mmol ) and n - hydroxysuccinimide ( hosu ) ( 0.160 g , 1.32 mmol ) in thf pyridine ( 1:1 ) ( 9 ml ) was added 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride ( edchcl ) ( 0.100 g , 0.485 mmol ) , and the mixture was allowed to react for 36 h at room temperature with stirring . the reaction was quenched with saturated nh4cl ( 10 ml ) , washed with brine ( 3 20 ml ) , dried over mgso4 , and concentrated in vacuo to afford a yellow oil . purification of the crude product by column chromatography on silica gel with hexanes / ethyl acetate ( 1:1 ) as eluent gave 14 t ( 376 mg , 70% yield ) as a sticky white solid . h nmr ( 500 mhz , cdcl3 ) 0.98 ( m , 2h ) , 1.12 ( m , 2h ) , 1.14 ( s , 3h ) , 1.25 ( m , 3h ) , 1.31 ( dd , j = 4.8 , 6.8 hz , 3h ) , 1.33 ( s , 2h ) , 1.34 ( s , 9h ) , 1.66 ( s , 3h ) , 1.71 ( s , 3h ) , 1.73 ( s , 3h ) , 1.86 ( t , j = 13.8 hz , 1h ) , 1.90 ( s , 3h ) , 1.97 ( m , 2h ) , 2.32 ( m , 2h ) , 2.35 ( s , 3h ) , 2.53 ( m , 1h ) , 2.64 ( t , j = 7.0 hz , 2h ) , 2.83 ( s , 4h ) , 2.99 ( m , 1h ) , 3.80 ( d , j = 7.2 hz , 1h ) , 3.97 ( dd , j = 1.7 , 16.8 hz , 1h ) , 4.08 ( dd , j = 1.7 , 16.8 hz , 1h ) , 4.13 ( m , 1h ) , 4.18 ( d , j = 8.4 hz , 1h ) , 4.30 ( d , j = 8.4 hz , 1h ) , 4.42 ( m , 1h ) , 4.81 ( m , 1h ) , 4.92 ( s , 1h ) , 4.96 ( d , j = 8.5 hz , 1h ) , 5.09 ( d , j = 8.5 hz , 1h ) , 5.67 ( d , j = 7.2 hz , 1h ) , 6.18 ( t , j = 8.5 hz , 1h ) , 6.28 ( s , 1h ) , 7.30 ( m , 3h ) , 7.47 ( t , j = 7.8 hz , 2h ) , 7.60 ( t , j = 7.4 hz , 1h ) , 7.79 ( d , j = 8.2 hz , 1h ) , 8.11 ( d , j = 7.5 hz , 2h ) . c nmr ( 500 mhz , cdcl3 ) 9.19 , 9.39 , 9.58 , 13.04 , 13.75 , 14.23 , 14.87 , 18.54 , 19.15 , 20.40 , 22.25 , 22.45 , 23.51 , 25.61 , 25.75 , 26.70 , 28.24 , 30.26 , 30.66 , 35.45 , 35.51 , 38.80 , 42.29 , 43.20 , 45.60 , 58.48 , 64.40 , 71.83 , 72.16 , 75.01 , 75.22 , 75.49 , 76.42 , 79.31 , 80.97 , 84.52 , 119.98 , 128.03 , 128.48 , 128.66 , 129.29 , 130.21 , 130.64 , 130.69 , 131.00 , 132.40 , 133.45 , 133.63 , 154.94 , 156.89 , 167.01 , 168.16 , 169.10 , 169.64 , 170.19 , 170.21 , 175.12 , 204.18 . hrms ( tof ) for c62h77n2o20s2 calcd , 1233.4506 ; found , 1233.4496 ( = 0.8 ppm ) . hplc ( 2 ) : t = 14.1 min , purity > 98% . to a solution of 13p ( 154 mg , 0.410 mmol ) and hosu ( 0.141 g , 1.23 mmol ) in ch2cl2 ( 1.6 ml ) was added edchcl ( 94 mg , 0.493 mmol ) in ch2cl2 ( 1 ml ) , and the mixture was allowed to react for 16 h at room temperature with stirring . the reaction was quenched with saturated nh4cl ( 10 ml ) , and the reaction mixture was diluted with water ( 10 ml ) , extracted with ch2cl2 ( 20 ml 3 ) , and washed with brine ( 20 ml 3 ) . the combined organic layers were dried over mgso4 and concentrated in vacuo to afford a yellow oil . purification the crude product by column chromatography on silica gel with 50% ethyl acetate in hexanes as eluent gave 14p ( 162 mg , 84% yield ) as a colorless oil . h nmr ( 500 mhz , cdcl3 ) 1.30 ( d , j = 6.8 hz , 3h ) , 1.98 ( m , 1h ) , 2.06 ( m , 1h ) , 2.66 ( t , j = 7.8 hz , 2h ) , 2.82 ( bs , 4h ) , 3.00 ( m , 1h ) , 4.13 ( s , 2h ) , 7.10 ( d , j = 7.5 hz , 2h ) , 7.21 ( m , 1h ) , 7.26 ( m , 1h ) , 7.34 ( m , 4h ) , 7.82 ( d , j = 7.5 hz , 1h ) . c nmr ( 500 mhz , cdcl3 ) 20.41 , 25.60 , 28.31 , 30.28 , 39.44 , 45.60 , 121.50 , 125.88 , 127.92 , 128.42 , 129.41 , 130.56 , 130.94 , 133.57 , 137.43 , 150.76 , 168.16 , 169.01 , 169.46 . hrms ( tof ) for c23h24no6s2 calcd , 474.1040 ; found , 474.1044 ( = 0.8 ppm ) . to a solution of 13c ( 110 mg , 0.175 mmol ) and 1-amino-11-azido-3,6,9-trioxaundecane ( 76 mg , 0.349 mmol ) in ch2cl2 ( 60 ml ) was added dic ( 35 l , 0.229 mmol ) , and the mixture was allowed to react for 4 h at room temperature with stirring . the reaction mixture was concentrated in vacuo and directly loaded on to a silica gel column . purification of the reaction mixture by column chromatography on silica gel with 2% ch3oh in ch2cl2 as eluent gave 15c ( 103 mg , 71% yield ) as a pale - white solid ; mp 104105 c . h nmr ( 500 mhz , cdcl3 ) 1.00 ( m , 3h ) , 1.18 ( d , j = 6.5 hz , 3h ) , 1.74 ( m , 4h ) , 2.04 ( m , 1h ) , 2.21 ( m , 1h ) , 2.33 ( m , 1h ) , 2.82 ( m , 1h ) , 3.34 ( m , 1h ) , 3.40 ( m , 2h ) , 3.49 ( m , 2h ) , 3.61 ( m , 2h ) , 3.69 ( m , 10h ) , 4.04 ( d , j = 16.5 hz , 1h ) , 4.22 ( dd , j = 4.0 , 16.5 hz , 1h ) , 5.31 ( s , 2h ) , 5.42 ( d , j = 17.3 hz , 1h ) , 5.66 ( dd , j = 1.4 , 17.3 hz , 1h ) , 5.98 ( m , 1h ) , 7.25 ( m , 5h ) , 7.72 ( m , 2h ) , 7.89 ( t , j = 7.4 hz , 1h ) , 7.98 ( d , j = 8.1 hz , 1h ) , 8.30 ( m , 1h ) , 8.42 ( s , 1h ) . c nmr ( 100 mhz , cdcl3 ) 7.57 , 14.20 , 20.60 , 20.73 , 21.05 , 23.50 , 31.21 , 31.42 , 31.82 , 33.50 , 38.56 , 39.10 , 42.15 , 46.36 , 49.95 , 50.67 , 53.44 , 60.39 , 67.15 , 69.74 , 70.03 , 70.23 , 70.56 , 70.61 , 70.69 , 96.30 , 120.20 , 120.29 , 127.89 , 128.01 , 128.17 , 128.19 , 128.24 , 128.46 , 129.71 , 130.62 , 130.69 , 131.12 , 131.15 , 131.21 , 133.07 , 137.55 , 145.59 , 146.22 , 148.86 , 152.32 , 157.29 , 167.32 , 170.20 , 171.14 , 172.05 , 172.12 . hrms ( tof ) for c41h47n6o9s2 calcd , 831.2840 ; found , 831.2838 ( = 0.2 ppm ) . hplc ( 2 ) : t = 12.4 min , purity > 95% . to a solution of 13p ( 149 mg , 0.396 mmol ) and 1-amino-11-azido-3,6,9-trioxaundecane ( 259 mg , 1.19 mmol ) in ch2cl2 ( 1 ml ) was added dic ( 67 l , 0.436 mmol ) , and the mixture was stirred for 10 h at room temperature . purification of the crude product by column chromatography on silica gel with 1.5% ch3oh in ch2cl2 as eluent gave 15p ( 0.127 g , 56% ) , as a colorless oil . h nmr ( 500 mhz , cdcl3 ) 1.34 ( d , j = 6.8 hz , 3h ) , 1.95 ( m , 2h ) , 2.17 ( t , j = 7.6 hz , 1h ) , 2.57 ( m , 1h ) , 2.94 ( m , 1h ) , 3.40 ( m , 2h ) , 3.49 ( t , j = 5.3 hz , 2h ) , 3.75 ( m , 12h ) , 4.17 ( s , 2h ) , 6.02 ( bs , 1h ) , 7.14 ( m , 2h ) , 7.25 ( t , j = 7.5 hz , 1h ) , 7.35 ( m , 3h ) , 7.40 ( t , j = 7.5 hz , 2h ) , 7.82 ( d , j = 7.5 hz , 1h ) . c nmr ( 125 mhz , cdcl3 ) 20.88 , 23.53 , 31.31 , 33.55 , 39.44 , 46.48 , 50.69 , 61.79 , 69.78 , 70.07 , 70.24 , 70.59 , 70.64 , 70.73 , 72.50 , 121.51 , 125.94 , 127.97 , 128.24 , 129.43 , 130.87 , 131.21 , 133.86 , 137.62 , 150.76 , 169.81 , 172.17 . hrms ( tof ) for c27h37n4o6s2 calcd , 577.2149 ; found , 577.2154 ( = 0.9 ppm ) . to a solution of succinic anhydride ( 16 ) ( 183 mg , 1.834 mmol ) and 1-amino-11-azido-3,6,9-trioxaundecane ( 400 g , 1.834 mmol ) in dichloromethane ( 3 ml ) was added triethylamine ( 25 l , 0.1834 mmol ) , and the mixture was allowed to react for 16 h at room temperature with stirring . purification of the crude product by column chromatography on silica gel with 2% ch3oh in ch2cl2 as eluent gave 17 ( 389 mg , 67% yield ) as a colorless liquid . h nmr ( 500 mhz , cdcl3 ) 2.54 ( m , 2h ) , 2.67 ( m , 2h ) , 3.40 ( m , 2h ) , 3.45 ( m , 2h ) , 3.55 ( t , j = 5.0 hz , 2h ) , 3.63 ( m , 2h ) , 3.68 ( m , 8h ) , 6.75 ( bs , 1h ) . c nmr ( 125 mhz , cdcl3 ) 30.35 , 30.99 , 39.51 , 50.68 , 69.63 , 69.93 , 70.14 , 70.44 , 70.57 , 70.65 , 172.71 , 175.29 . hrms ( tof ) for c12h23n4o6 calcd , 319.1612 ; found , 319.1622 ( = 3.2 ppm ) . to a solution of camptothecin ( 40 mg , 0.115 mmol ) , 17 ( 157 mg , 0.345 mmol ) , and dmap ( 105 mg , 0.115 mmol ) in ch2cl2 ( 6 ml ) was added dic ( 30 l , 0.127 mmol ) , and the mixture was allowed to react for 36 h at room temperature with stirring . the reaction mixture was cooled to 0 c , and the precipitated urea byproduct was removed by filtration . purification of the crude product by column chromatography on silica gel with 3% ch3oh in ch2cl2 as eluent gave 18 ( 53 mg , 72% yield ) as an off - white solid ; mp 100101 c . h nmr ( 500 mhz , cdcl3 ) 0.99 ( t , j = 7.5 hz , 3h ) , 2.20 ( m , 1h ) , 2.29 ( m , 1h ) , 2.51 ( m , 2h ) , 2.88 ( m , 2h ) , 3.363.45 ( m , 6h ) , 3.55 ( m , 2h ) , 3.60 ( m , 2h ) , 3.63 ( m , 6h ) , 5.28 ( m , 2h ) , 5.38 ( d , j = 17.1 hz , 1h ) , 5.68 ( d , j = 17.1 hz , 1h ) , 6.31 ( bs , 1h ) , 7.30 ( s , 1h ) , 7.66 ( t , j = 7.6 hz , 1h ) , 7.84 ( t , j = 7.8 hz , 1h ) , 7.93 ( d , j = 7.8 hz , 1h ) , 8.25 ( d , j = 8.6 hz , 1h ) , 8.42 ( s , 1h ) . c nmr ( 125 mhz , cdcl3 ) 7.58 , 29.40 , 30.67 , 37.70 , 39.31 , 49.90 , 50.63 , 66.99 , 69.67 , 69.97 , 70.16 , 70.49 , 70.54 , 70.62 , 76.12 , 96.39 , 119.90 , 128.00 , 128.13 , 128.42 , 129.66 , 130.64 , 131.11 , 146.08 , 146.19 , 148.82 , 152.34 , 157.37 , 176.54 , 170.78 , 171.93 . hrms ( tof ) for c32h37n6o9 calcd , 649.2617 ; found , 649.2622 ( = 0.8 ppm ) . hplc ( 2 ) : t = 10.8 min , purity > 98% . to a cooled solution of cyanuric chloride ( 19 ) ( 414 mg , 2.282 mmol ) in thf ( 20 ml ) at 0 c was added a solution of n - boc - ethylenediamine ( 366 mg , 2.282 mmol ) and dipea ( 0.6 ml , 3.432 mmol ) in thf ( 2 ml ) , and the mixture was stirred for 3 h at room temperature . then , a solution of 7 ( 954 mg , 2.282 mmol ) and dipea ( 0.6 ml , 3.423 mmol ) in thf ( 80 ml ) was added to the reaction mixture and refluxed for 16 h. the reaction mixture was concentrated in vacuo to afford a red oil . purification of the crude product by column chromatography on silica gel ( eluent : 7% ch3oh in ch2cl2 ) gave 20 ( 645 mg , 42% yield ) as a white solid ; mp 6365 c . h nmr ( 300 mhz , cd3od ) 1.34 ( dt , j = 6.6 , 12.6 hz , 2h ) , 1.41 ( s , 9h ) , 1.61 ( m , 4h ) , 2.21 ( t , j = 7.2 hz , 2h ) , 2.69 ( d , j = 12.6 hz , 1h ) , 2.91 ( dd , j = 5.0 , 12.6 hz , 1h ) , 3.20 ( m , 3h ) , 3.37 ( m , 4h ) , 3.54 ( m , 4h ) , 3.63 ( m , 10h ) , 4.29 ( dd , j = 4.4 , 8.0 hz , 1h ) , 4.48 ( dd , j = 4.4 , 8.0 hz , 1h ) . c nmr ( 100 mhz , cd3od ) 25.5 , 27.4 , 28.1 , 28.4 , 35.4 , 39.0 , 39.7 , 40.2 , 40.3 , 40.6 , 53.4 , 54.2 , 55.6 , 60.2 , 62.0 , 69.2 , 69.8 , 70.19 , 70.21 , 78.7 , 157.2 , 164.7 , 165.8 , 168.1 , 168.8 , 174.7 . hrms ( tof ) calcd for c28h49cln9o7s , 690.3159 ; found , 690.3157 ( = 0.3 ppm ) . hplc ( 2 ) : t = 10.0 min , purity > 98% . to a solution of 20 ( 794 mg , 1.151 mmol ) and dipea ( 0.4 ml , 2.302 mmol ) in thf ( 12 ml ) was added propargylamine ( 362 l , 6.600 mmol ) , and the mixture was stirred for 12 h at 50 c . reaction was monitored by esi - ms , and additional propargylamine was added as needed . the reaction mixture was cooled to 25 c and concentrated in vacuo to afford an orange oil . purification the crude product by column chromatography on silica gel ( eluent : 7% ch3oh in ch2cl2 ) gave 21 ( 560 mg , 69% yield ) as a light - yellow solid ; mp 6668 c . h nmr ( 300 mhz , cd3od ) 1.41 ( m , 2h ) , 1.42 ( s , 9h ) , 1.63 ( m , 4h ) , 2.20 ( t , j = 7.5 hz , 2h ) , 2.52 ( t , j = 2.4 hz , 1h ) , 2.69 ( d , j = 12.6 hz , 1h ) , 2.91 ( dd , j = 5.0 , 12.6 hz , 1h ) , 3.19 ( m , 3h ) , 3.36 ( t , j = 5.4 hz , 4h ) , 3.54 ( t , j = 5.4 hz , 4h ) , 3.64 ( m , 10h ) , 4.10 ( bs , 2h ) , 4.27 ( dd , j = 4.4 , 8.0 hz , 1h ) , 4.47 ( dd , j = 4.4 , 8.0 hz , 1h ) . c nmr ( 125 mhz , cd3od ) 27.01 , 28.94 , 29.64 , 29.93 , 30.94 , 36.89 , 40.55 , 41.21 , 41.50 , 41.56 , 57.16 , 61.77 , 63.52 , 70.74 , 71.19 , 71.40 , 71.44 , 71.73 , 71.76 , 158.76 , 166.27 , 176.30 . hrms ( tof ) calcd for c31h53n10o7s , 709.3814 ; found , 709.3814 ( = 0 ppm ) . hplc ( 2 ) : t = 10.0 min , purity > 98% . to a solution of 21 ( 400 mg , 0.564 mmol ) in ch2cl2 ( 6 ml ) was added tfa ( 0.5 ml , 6.57 mmol ) , and the mixture was stirred at 25 c for 48 h. the reaction mixture was concentrated in vacuo to afford a yellow oil , which was triturated with ether ( 20 ml ) and the crude product ( tfa salt ) crashed out . the resulting solid was washed with ether ( 20 ml 4 ) to afford 22 ( 408 mg , 100% yield ) as a white solid ; h nmr ( 400 mhz , cd3od ) 1.42 ( m , 2h ) , 1.63 ( m , 4h ) , 2.20 ( t , j = 7.2 hz , 2h ) , 2.54 ( m , 1h ) , 2.69 ( d , j = 12.6 hz , 1h ) , 2.92 ( dd , j = 5.1 , 12.6 hz , 1h ) , 3.36 ( t , j = 5.4 hz , 4h ) , 3.54 ( t , j = 5.4 hz , 4h ) , 3.64 ( m , 10h ) , 4.10 ( bs , 2h ) , 4.30 ( dd , j = 4.4 , 8.0 hz , 1h ) , 4.49 ( dd , j = 4.4 , 8.0 hz , 1h ) , 4.68 ( bs , 2h ) ; c nmr ( 100 mhz , cd3od ) 25.46 , 28.09 , 28.34 , 29.38 , 35.33 , 38.26 , 38.94 , 39.64 , 39.95 , 55.60 , 60.25 , 61.99 , 69.17 , 69.80 , 69.95 , 70.16 , 112.74 , 115.24 , 118.15 , 121.62 , 161.58 , 161.93 , 164.68 , 174.81 . ms ( esi ) m / z 609.3 ( m+h ) . a solution of 14 t ( 704 mg , 0.564 mmol ) and 22 ( 408 mg , 0.564 mmol ) in ch2cl2pyridine ( 4:1 ) ( 10 ml ) was allowed to react at 25 c for 36 h. the reaction was quenched by saturated nh4cl ( 10 ml ) , and the reaction mixture was extracted with ch2cl2 ( 20 ml 3 ) . the combined organic layers were washed with brine ( 5 ml 3 ) , dried over mgso4 , and concentrated in vacuo to afford a yellow oil . purification of the crude product by column chromatography on silica gel ( eluent : 8% ch3oh in ch2cl2 ) gave 23 ( 806 mg , 83% yield ) as a white solid ; mp 164166 c . h nmr ( 500 mhz , cd3od ) 0.86 ( m , 2h ) , 0.98 ( m , 2h ) , 1.17 ( s , 6h ) , 1.26 ( d , j = 6.8 hz , 3h ) , 1.41 ( s , 9h ) , 1.43 ( m , 2h ) , 1.63 ( m , 4h ) , 1.65 ( s , 3h ) , 1.70 ( m , 1h ) , 1.73 ( s , 3h ) , 1.76 ( s , 3h ) , 1.79 ( m , 1h ) , 1.87 ( m , 1h ) , 1.91 ( s , 3h ) , 2.20 ( t , j = 7.5 hz , 2h ) , 2.25 ( m , 4h ) , 2.38 ( s , 3h ) , 2.45 ( m , 2h ) , 2.65 ( bs , 1h ) , 2.69 ( d , j = 12.8 hz , 1h ) , 2.86 ( m , 1h ) , 2.90 ( dd , j = 5.0 , 12.8 hz , 1h ) , 3.18 ( m , 1h ) , 3.34 ( m , 2h ) , 3.35 ( t , j = 5.4 hz , 2h ) , 3.52 ( t , j = 5.4 hz , 2h ) , 3.54 ( m , 2h ) , 3.63 ( m , 12h ) , 3.84 ( d , j = 7.2 hz , 1h ) , 4.02 ( dd , j = 1.5 , 16.7 hz , 1h ) , 4.10 ( d , j = 16.7 hz , 1h ) , 4.17 ( m , 2h ) , 4.20 ( d , j = 8.4 hz , 2h ) , 4.28 ( dd , j = 4.4 , 8.0 , 1h ) , 4.30 ( dd , j = 6.5 , 10.6 hz , 1h ) , 4.47 ( dd , j = 4.4 , 8.0 hz , 1h ) , 4.84.9 ( m , 3h ) , 4.99 ( d , j = 8.9 hz , 1h ) , 5.26 ( bs , 1h ) , 5.67 ( d , j = 7.2 hz , 1h ) , 6.13 ( bt , j = 8.5 hz , 1h ) , 6.45 ( s , 1h ) , 7.26 ( m , 1h ) , 7.32 ( m , 2h ) , 7.50 ( t , j = 7.9 hz , 2h ) , 7.63 ( t , j = 7.5 hz , 1h ) , 7.78 ( m , 1h ) , 8.12 ( d , j = 7.5 hz , 2h ) . c nmr ( 125 mhz , cd3od ) 5.17 , 5.21 , 6.42 , 7.76 , 9.80 , 10.42 , 11.08 , 14.64 , 16.99 , 17.03 , 18.36 , 19.26 , 19.70 , 22.09 , 22.20 , 22.87 , 22.98 , 24.79 , 25.51 , 25.76 , 28.62 , 28.75 , 30.31 , 30.39 , 31.67 , 31.78 , 32.76 , 33.52 , 35.48 , 36.36 , 37.08 , 40.60 , 43.35 , 44.07 , 46.71 , 53.00 , 55.32 , 57.65 , 59.38 , 66.59 , 67.25 , 67.32 , 67.57 , 68.39 , 68.98 , 72.32 , 72.57 , 72.72 , 73.46 , 75.09 , 76.50 , 78.35 , 81.85 , 117.21 , 124.96 , 125.41 , 125.68 , 127.15 , 127.42 , 128.50 , 130.57 , 130.85 , 131.02 , 134.71 , 138.61 , 153.50 , 162.08 , 163.61 , 166.30 , 167.46 , 171.12 , 172.18 , 201.18 . hrms ( tof ) for c84h116n11o22s3 calcd , 1726.7453 ; found , 1726.7432 ( = 1.2 ppm ) . hplc ( 1 ) : t = 12.0 min , purity > 98% . to a solution of 23 ( 40 mg , 0.0232 mmol ) and ascorbic acid ( 4 mg , 0.0255 mmol ) in thf ( 1 ml ) was added 15c ( 19 mg , 0.0232 mmol ) in ch2cl2 ( 1 ml ) first , followed by an aqueous solution of cuso45h2o ( 6.4 mg , 0.0244 mmol ) ( 1 ml ) . the mixture was allowed react at room temperature for 14 h , and the reaction mixture was diluted with water ( 5 ml ) and extracted with ch2cl2 ( 10 ml 3 ) . the crude product was purified by column chromatography on silica gel ( eluent : 10% meoh in ch2cl2 ) to give 2 ( 54 mg , 92% yield ) as an off - white solid ; mp 138139 c . h nmr ( 500 mhz , cd3od ) 0.92 ( m , 5h ) , 1.03 ( m , 9h ) , 1.20 ( s , 6h ) , 1.31 ( m , 6h ) , 1.43 ( s , 9h ) , 1.63 ( m , 6h ) , 1.67 ( s , 3h ) , 1.76 ( s , 3h ) , 1.79 ( s , 3h ) , 1.94 ( s , 3h ) , 2.08 ( t , j = 7.5 hz , 1h ) , 2.24 ( m , 8h ) , 2.40 ( s , 3h ) , 2.47 ( m , 1h ) , 2.71 ( t , j = 12.8 hz , 2h ) , 2.78 ( m , 1h ) , 2.86 ( m , 1h ) , 2.91 ( dd , j = 5.5 , 12.8 hz , 1h ) , 3.19 ( m , 1h ) , 3.26 ( m , 1h ) , 3.37 ( m , 2h ) , 3.45 ( m , 2h ) , 3.63 ( m , 24h ) , 3.81 ( m , 6h ) , 4.03 ( m , 3h ) , 4.08 ( m , 2h ) , 4.10 ( d , j = 16.7 hz , 1h ) , 4.31 ( m , 3h ) , 4.52 ( m , 2h ) , 4.66 ( m , 2h ) , 5.00 ( t , j = 9.0 hz , 2h ) , 5.32 ( s , 2h ) , 5.35 ( s , 1h ) , 5.48 ( d , j = 16.7 hz , 1h ) , 5.60 ( d , j = 16.7 hz , 1h ) , 5.70 ( m , 1h ) , 6.15 ( bt , j = 8.5 hz , 1h ) , 6.47 ( s , 1h ) , 7.30 ( m , 6h ) , 7.41 ( d , j = 8.2 hz , 1h ) , 7.53 ( m , 2h ) , 7.65 ( m , 2h ) , 7.74 ( m , 3h ) , 7.92 ( t , j = 8.0 hz , 1h ) , 7.94 ( bs , 1h ) , 8.08 ( d , j = 8.0 hz , 1h ) , 8.14 ( d , j = 7.5 hz , 2h ) , 8.27 ( d , j = 8.5 hz , 1h ) , 8.61 ( s , 1h ) . c nmr ( 125 mhz , dmso - d6 ) 7.99 , 8.77 , 8.85 , 10.24 , 13.16 , 14.22 , 14.25 , 18.35 , 20.08 , 20.16 , 20.47 , 20.54 , 20.74 , 21.90 , 23.00 , 25.74 , 25.96 , 26.80 , 28.51 , 30.69 , 30.89 , 31.54 , 31.64 , 32.84 , 34.87 , 35.56 , 38.51 , 38.88 , 41.14 , 45.86 , 46.69 , 49.74 , 50.72 , 55.90 , 57.96 , 59.66 , 61.50 , 66.78 , 69.52 , 69.61 , 69.98 , 70.00 , 70.07 , 70.14 , 70.19 , 76.60 , 77.21 , 78.60 , 80.00 , 84.06 , 95.59 , 97.62 , 119.37 , 120.70 , 125.38 , 127.85 , 128.17 , 128.46 , 128.87 , 129.00 , 130.17 , 130.38 , 130.83 , 131.65 , 131.96 , 132.01 , 133.03 , 133.81 , 133.89 , 136.45 , 137.33 , 137.59 , 137.63 , 139.66 , 139.97 , 145.45 , 145.49 , 148.43 , 151.93 , 152.71 , 155.39 , 156.98 , 157.24 , 163.17 , 165.59 , 167.51 , 169.27 , 169.85 , 170.07 , 170.63 , 171.73 , 172.60 , 178.58 , 203.01 . hplc ( 1 ) : t = 11.3 min , purity > 97% . to a solution of 23 ( 12 mg , 6.95 mol ) , 18 ( 5 mg , 6.95 mol ) , and ascorbic acid ( 1.4 mg , 7.70 mol ) in thf ( 0.5 ml ) was added an aqueous solution of cuso45h2o ( 2 mg , 7.70 mol ) ( 0.1 ml ) . the mixture was allowed to react for 10 h at room temperature with stirring , and the reaction mixture was diluted with water ( 5 ml ) and extracted with ch2cl2 ( 5 ml 3 ) . the combined organic layers were concentrated in vacuo to afford a milky white solid . the crude product was purified by column chromatography on silica gel ( eluent : 10% meoh in ch2cl2 ) to afford 3 ( 12 mg , 54% yield ) as an off - white solid ; mp 143145 c . h nmr ( 500 mhz , cd3od ) 0.89 ( m , 2h ) , 0.98 ( m , 3h ) , 1.01 ( t , j = 7.5 hz , 3h ) , 1.07 ( m , 2h ) , 1.15 ( s , 3h ) , 1.16 ( s , 3h ) , 1.25 ( m , 6h ) , 1.40 ( m , 2h ) , 1.40 ( s , 9h ) , 1.60 ( m , 4h ) , 1.64 ( s , 3h ) , 1.73 ( s , 3h ) , 1.74 ( m , 3h ) , 1.75 ( s , 3h ) , 1.86 ( m , 2h ) , 1.90 ( m , 3h ) , 2.01 ( m , 1h ) , 2.20 ( m , 7h ) , 2.38 ( s , 3h ) , 2.42 ( m , 2h ) , 2.53 ( m , 1h ) , 2.68 ( d , j = 12.8 hz , 1h ) , 2.88 ( m , 3h ) , 3.15 ( m , 1h ) , 3.17 ( bs , 2h ) , 3.34 ( m , 2h ) , 3.39 ( bt , j = 5.4 hz , 2h ) , 3.43 ( m , 4h ) , 3.52 ( m , 6h ) , 3.62 ( m , 10h ) , 3.73 ( m , 2h ) , 3.80 ( m , 1h ) , 3.83 ( t , j = 7.3 hz , 2h ) , 4.02 ( d , j = 16.7 hz , 1h ) , 4.06 ( m , 1h ) , 4.20 ( m , 3h ) , 4.28 ( m , 2h ) , 4.38 ( m , 1h ) , 4.48 ( m , 3h ) , 4.91 ( bs , 2h ) , 4.98 ( t , j = 9.0 hz , 1h ) , 5.26 ( m , 1h ) , 5.31 ( m , 2h ) , 5.45 ( d , j = 16.7 hz , 1h ) , 5.58 ( d , j = 16.7 hz , 1h ) , 5.66 ( t , j = 6.7 hz , 1h ) , 6.12 ( bt , j = 8.5 hz,1h ) , 6.43 ( s , 1h ) , 6.45 ( bs , 1h ) , 7.22 ( m , 1h ) , 7.30 ( m , 2h ) , 7.40 ( s , 1h ) , 7.50 ( t , j = 8.2 hz , 2h ) , 7.63 ( t , j = 7.4 hz , 1h ) , 7.72 ( m , 2h ) , 7.79 ( m , 1h ) , 7.86 ( t , j = 7.4 hz , 1h ) , 7.93 ( bs , 1h ) , 8.06 ( d , j = 8.0 hz , 1h ) , 8.11 ( d , j = 7.8 hz , 2h ) , 8.18 ( d , j = 8.0 hz , 1h ) , 8.61 ( bs , 1h ) . c nmr ( 125 mhz , cd3od ) 6.70 , 7.77 , 7.82 , 9.02 , 12.41 , 17.25 , 19.61 , 20.95 , 21.88 , 24.70 , 25.09 , 25.48 , 25.58 , 27.39 , 28.09 , 28.33 , 28.33 , 28.76 , 30.75 , 31.22 , 35.34 , 36.12 , 38.04 , 38.94 , 43.20 , 46.67 , 50.26 , 57.94 , 60.24 , 61.97 , 66.32 , 67.46 , 69.03 , 69.17 , 69.84 , 69.92 , 69.95 , 70.03 , 70.20 , 71.03 , 73.25 , 73.41 , 74.90 , 75.30 , 75.59 , 76.06 , 76.41 , 77.68 , 78.07 , 79.10 , 80.95 , 84.41 , 87.85 , 88.10 , 96.77 , 119.15 , 119.81 , 127.87 , 128.00 , 128.28 , 128.41 , 128.60 , 129.74 , 130.01 , 130.62 , 133.16 , 137.31 , 146.26 , 146.96 , 148.27 , 157.59 , 166.18 , 167.97 , 168.90 , 170.05 , 170.13 , 170.92 , 171.06 , 172.63 , 173.67 , 173.72 , 203.80 . hrms ( tof ) calcd for c116h152n17o31s3 calcd , 2374.9997 ; found , 2374.9980 ( = 0.7 ppm ) . hplc ( 1 ) : t = 10.5 min , purity > 96% . to a solution of 23 ( 20 mg , 11.6 mol ) , 14p ( 6.7 mg , 11.6 mol ) , and ascorbic acid ( 2 mg , 12.7 mol ) in thf ( 1 ml ) was added an aqueous solution of cuso45h2o ( 3.2 mg , 12.7 mol ) ( 0.25 ml ) . the mixture was stirred at 25 c for 15 h , and the reaction mixture was lyophilized . purification of the lyophilized reaction mixture by column chromatography on silica gel ( eluent : 10% ch3oh in ch2cl2 ) gave 3 ( 21 mg , 79% yield ) as a colorless solid ; mp 125127 c . h nmr ( 500 mhz , cdcl3 ) 0.90 ( m , 3h ) , 1.02 ( bt , j = 8.0 hz , 2h ) , 1.18 ( s , 3h ) , 1.27 ( s , 3h ) , 1.33 ( d , j = 6.8 hz , 6h ) , 1.29 ( m , 2h ) , 1.37 ( s , 9h ) , 1.43 ( m , 2h ) , 1.49 ( m , 2h ) , 1.66 ( m , 4h ) , 1.69 ( s , 3h ) , 1.75 ( s , 3h ) , 1.78 ( m , 2h ) , 1.93 ( s , 3h ) , 2.17 ( t , j = 7.4 hz , 2h ) , 2.29 ( m , 1h ) , 2.39 ( s , 3h ) , 2.54 ( m , 2h ) , 2.73 ( d , j = 12.6 hz , 1h ) , 2.90 ( m , 2h ) , 2.92 ( dd , j = 5.0 , 12.6 hz , 1h ) , 3.15 ( m , 1h ) , 3.41 ( t , j = 5.0 hz , 2h ) , 3.44 ( t , j = 5.4 hz , 2h ) , 3.49 ( t , j = 5.4 hz , 2h ) , 3.69 ( m , 28h ) , 3.77 ( t , j = 5.0 hz , 1h ) , 3.83 ( m , 1h ) , 4.17 ( s , 2h ) , 4.20 ( d , j = 8.4 hz , 1h ) , 4.21 ( m , 2h ) , 4.31 ( d , j = 8.4 hz , 1h ) , 4.33 ( m , 1h ) , 4.42 ( m , 1h ) , 4.51 ( m , 1h ) , 5.00 ( m , 2h ) , 5.16 ( bs , 1h ) , 5.70 ( d , j = 7.2 hz , 1h ) , 6.06 ( s , 1h ) , 6.20 ( bt , j = 8.5,1h ) , 6.34 ( s , 1h ) , 7.14 ( d , j = 7.4 hz , 1h ) , 7.23 ( t , j = 7.4 hz , 1h ) , 7.34 ( m , 4h ) , 7.40 ( t , j = 8.0 hz , 2h ) , 7.50 ( t , j = 7.8 hz , 2h ) , 7.63 ( t , j = 7.4 hz , 1h ) , 7.81 ( bs , 1h ) , 7.83 ( d , j = 7.7 hz , 1h ) , 8.13 ( d , j = 7.7 hz , 2h ) . c nmr ( 125 mhz , cdcl3 ) 9.17 , 9.33 , 9.63 , 13.06 , 14.81 , 17.50 , 18.53 , 20.01 , 20.43 , 20.87 , 22.07 , 22.48 , 23.51 , 25.76 , 26.61 , 27.90 , 28.26 , 31.30 , 33.54 , 35.48 , 39.12 , 43.21 , 45.88 , 46.47 , 50.68 , 53.51 , 55.49 , 58.41 , 60.24 , 61.75 , 69.67 , 69.78 , 70.05 , 70.15 , 70.22 , 70.26 , 70.32 , 70.41 , 70.58 , 70.63 , 70.67 , 70.69 , 70.71 , 71.99 , 72.49 , 75.11 , 75.47 , 76.40 , 78.90 , 79.15 , 79.83 , 81.00 , 84.48 , 115.39 , 117.71 , 120.04 , 121.50 , 125.93 , 127.95 , 128.23 , 128.37 , 128.66 , 128.84 , 128.28 , 129.60 , 130.17 , 130.85 , 130.94 , 131.20 , 133.67 , 133.85 , 137.64 , 150.75 , 155.07 , 162.94 , 163.23 , 166.97 , 168.37 , 169.80 , 172.19 , 173.39 , 174.95 , 204.09 . hrms ( tof ) for c111h152n15o28s5 calcd , 2302.9529 ; found , 2302.9480 ( = 2.1 ppm ) . hplc ( 1 ) : t = 11.0 min , purity > 98% . a mixture of 22 ( 19 mg , 26.2 mol ) and 14p ( 12 mg , 26.2 mol ) in ch2cl2 ( 1 ml ) and pyridine ( 1 ml ) was stirred at room temperature for 10 h , and the reaction mixture was concentrated in vacuo to give a yellow oil . purification of the crude product by column chromatography on silica gel ( eluent : 4% ch3oh in ch2cl2 ) gave 24 ( 13 mg , 52% yield ) as a colorless oil . h nmr ( 500 mhz , cdcl3 ) 1.29 ( d , j = 6.8 hz , 3h ) , 1.43 ( t , j = 7.7 hz , 2h ) , 1.66 ( m , 4h ) , 1.84 ( m , 1h ) , 1.92 ( m , 1h ) , 2.22 ( m , 2h ) , 2.56 ( bs , 1h ) , 2.72 ( d , j = 12.8 hz , 1h ) , 2.90 ( m , 2h ) , 2.92 ( dd , j = 5.0 12.8 hz , 1h ) , 3.19 ( m , 1h ) , 3.38 ( t , j = 5.4 hz , 2h ) , 3.44 ( bs , 2h ) , 3.56 ( t , j = 5.4 hz , 2h ) , 3.66 ( m , 14h ) , 4.15 ( bs , 2h ) , 4.20 ( s , 2h ) , 4.30 ( dd , j = 4.4 , 8.0 hz , 1h ) , 4.49 ( dd , j = 4.4 , 8.0 hz , 1h ) , 7.14 ( d , j = 7.8 hz , 2h ) , 7.257.45 ( m , 6h ) , 7.83 ( d , j = 7.8 hz , 1h ) , 8.55 ( bs , 1h ) . c nmr ( 125 mhz , cdcl3 ) 19.57 , 25.47 , 28.09 , 28.39 , 31.27 , 32.97 , 35.35 , 38.74 , 39.00 , 39.68 , 46.00 , 55.62 , 60.22 , 61.97 , 69.18 , 69.55 , 69.84 , 69.89 , 70.18 , 70.21 , 120.99 , 125.64 , 127.67 , 128.05 , 128.27 , 129.95 , 130.34 , 130.75 , 131.21 , 131.60 , 134.01 , 137.38 , 148.66 , 150.95 , 164.73 , 170.23 , 174.75 . hrms ( tof ) for c45h63n10o8s3 calcd , 967.3987 ; found , 967.3991 ( = 0.4 ppm ) . to a solution of 24 ( 13 mg , 13.5 mol ) , 15c ( 11 mg , 13.5 mol ) , and ascorbic acid ( 2.6 g , 14.9 mol ) in thf ( 1 ml ) was added a solution of cuso45h2o ( 3.7 mg , 14.9 mmol ) in meoh the mixture was stirred at 25 c for 4 h , and the reaction mixture was concentrated in vacuo and extracted with ch2cl2 ( 10 ml 3 ) . the combined organic layers were dried over mgso4 , and concentrated in vacuo to give a yellow solid . purification of the crude product by column chromatography on silica gel ( eluent : 8% ch3oh in ch2cl2 ) gave conjugate 5 ( 13 mg , 54% yield ) as a white solid ; mp 127128 c . h nmr ( 500 mhz , dmso - d6 ) 0.90 ( t , j = 7.5 hz , 3h ) , 1.03 ( dd , j = 6.8 , 10.8 , 2h ) , 1.20 ( d , j = 6.8 hz , 3h ) , 1.24 ( s , 2h ) , 1.30 ( m , 2h ) , 1.49 ( m , 4h ) , 1.59 ( m , 1.59 ) , 1.71 ( m , 2h ) , 1.84 ( m , 1h ) , 2.07 ( m , 4h ) , 2.17 ( m , 4h ) , 2.57 ( d , j = 12.8 hz , 1h ) , 2.81 ( m , 2h ) , 2.95 ( m , 1h ) , 3.10 ( m , 1h ) , 3.14 ( m , 3h ) , 3.15 ( m , 4h ) , 3.25 ( m , 2h ) , 3.39 ( t , j = 5.4 hz , 4h ) , 3.433.49 ( m , 20h ) , 3.77 ( t , j = 5.0 hz , 2h ) , 4.06 ( m , 2h ) , 4.13 ( m , 1h ) , 4.17 ( m , 2h ) , 4.30 ( m , 1h ) , 4.46 ( m , 4h ) , 5.31 ( m , 2h ) , 5.45 ( d , j = 17.0 hz , 1h ) , 5.50 ( d , j = 17.0 hz , 1h ) , 6.37 ( s , 1h ) , 6.43 ( s , 1h ) , 7.09 ( s , 1h ) , 7.12 ( d , j = 7.6 hz , 2h ) , 7.247.44 ( m , 9h ) , 7.73 ( m , 3h ) , 7.88 ( m , 4h ) , 7.90 ( t , j = 8.1 hz , 1h ) , 8.14 ( d , j = 8.5 hz , 1h ) , 8.23 ( d , j = 8.5 hz , 1h ) , 8.69 ( s , 1h ) . c nmr ( 125 mhz , dmso - d6 ) 7.99 , 20.06 , 20.14 , 20.51 , 25.70 , 28.51 , 28.67 , 30.69 , 31.59 , 32.83 , 33.02 , 35.56 , 36.06 , 45.85 , 45.92 , 46.18 , 49.07 , 49.66 , 50.72 , 55.41 , 55.90 , 59.65 , 61.49 , 66.78 , 69.26 , 69.52 , 69.63 , 69.99 , 70.07 , 70.14 , 70.18 , 70.22 , 95.62 , 119.36 , 122.06 , 126.41 , 127.85 , 128.04 , 128.18 , 128.46 , 128.87 , 129.00 , 129.60 , 129.73 , 130.06 , 130.17 , 130.84 , 131.66 , 131.90 , 131.96 , 133.03 , 133.83 , 137.40 , 137.63 , 145.50 , 146.42 , 148.44 , 150.91 , 152.71 , 156.99 , 163.17 , 166.15 , 166.21 , 167.53 , 169.85 , 171.74 , 171.89 , 172.58 , 173.27 . hrms ( tof ) for c86h109n16o17s5 calcd , 1797.6755 ; found , 1797.6705 ( = 2.8 ppm ) . cells were cultured in rpmi-1640 cell culture medium ( gibco ) or dmem culture medium ( gibco ) , both supplemented with 5% ( v / v ) heat - inactivated fetal bovine serum ( fbs ) , 5% ( v / v ) nuserum , and 1% ( v / v ) penicillin and streptomycin ( penstrep ) at 37 c in a humidified atmosphere with 5% co2 . l1210 and l1210fr ( a gift from dr . lcc6-mdr , lcc6-wt , mcf-7 , mda mb 231 , mx-1 , and id8 cells were cultured as monolayers on 100 mm tissue culture dishes in a supplemented rpmi-1640 cell culture medium , and wi-38 as a monolayer in a supplemented dmem cell culture medium . cells were harvested , collected by centrifugation at 850 rpm for 5 min , and resuspended in fresh culture medium . cell cultures were routinely divided by treatment with trypsin ( tryple , gibco ) as needed every 24 days and collected by centrifugation at 850 rpm for 5 min , and then resuspended in fresh cell culture medium containing varying cell densities for subsequent biological experiments and analysis . cell suspensions ( 3 ml ) at 5 10 cells / ml were added to each individual well of 96-well plates and subsequently incubated overnight in the appropriate cell culture media . the cell culture media was replaced with 10 m solutions of 7 in cell culture media ( 3 ml ) . the cells were then incubated with 7 for 1 and 3 h at 37 c . in the case of leukemia cell lines ( l1210 and l1210fr ) , probe 7 ( 1 mm ) in dmso ( 30 m ) was added directly into fresh cell suspensions to give the final concentration of 10 m , and incubated for similar time intervals . after incubation , the cells were removed by treating with trypsin ( as needed ) , washed twice with pbs , collected by centrifugation , and resuspended in 150 l for imaging . flow cytometry analysis of the cells treated with probe 7 was performed with a flow cytometer , facscalibur , operating at a 488 nm excitation wavelength and detecting 530 nm emission wavelength with a 30 nm bandpass filter ( 515545 nm range ) . approximately 10000 cells were counted for each experiment using cellquest 3.3 software ( becton dickinson ) , and the distribution of fitc fluorescence was analyzed using winmdi 2.8 freeware ( joseph trotter , scripps research institute ) . cells treated as described above were resuspended in 150 l of pbs after each experiment and dropped onto an uncoated microslide with coverslip ( mattek corp . ) . confocal fluorescence microscopy ( cfm ) experiments were performed using a zeiss lsm 510 meta nlo two - photon laser scanning confocal microscope system , operating at a 488 nm excitation wavelength and at 527 23 nm detecting emission wavelength using a 505550 nm bandpass filter . images for conjugate 2 ( figure 6 ) were obtained using the camera mode with a filter set of 350 25 nm excitation wavelength and 420 nm long pass emission wavelength . the cytotoxicities of taxoid 1 and camptothecin were evaluated for single - agent administrations as well as for time - dependent administrations of equimolar combinations against various cancer cell lines by means of the standard quantitative colorimetric mtt assay . the inhibitory activity of each compound is represented by the ic50 value , which is defined as the concentration required for inhibiting 50% of the cell growth . cells were harvested , collected , and resuspended in 100 l of cell culture medium ( rpmi-1640 ) at a concentrations ranging from 0.5 to 1.5 10 cells per well in a 96-well plate . for adhesive cell types , cells were allowed to descend to the bottom of the wells overnight , and a fresh medium ( rpmi-1640 ) was added to each well upon removal of the old medium . for the mtt assay of the time - dependent administrations of equimolar amounts of taxoid 1 and camptothecin in a sequential manner , cells were resuspended in 200 l medium with 800010000 cells per well of a 96-well plate and incubated at 37 c for 24 h before drug treatment . two sets of serial dilutions of equimolar taxoid and camptothecin in sterile dmso were added using the cell culture medium . the residual medium in each well were aspirated , and the different drug solutions were added to each well of every column of the 96-well plate . after the addition of the first drug solution , the cells were incubated at 37 c for 24 h. then , the second drug solution was added , followed by incubation for additional 48 h at 37 c . the cytotoxicities ( ic50 , nm ) for conjugates 25 were evaluated in a similar manner . in dmso stock solutions , each conjugate was diluted to a series of concentrations in cell culture medium to prepare test solutions . after removing the old medium , these test solutions were added to the wells in the 96-well plate to give the final concentrations ranging from 0.5 to 5000 nm ( 100 l ) , and the cells were subsequently cultured at 37 c for 72 h. for the leukemia cell lines , cells were harvested , collected , and resuspended in the test solutions ranging from 0.5 to 5000 nm ( 100 l ) at 0.5 to 0.8 10 cells per well in a 96-well plate and subsequently incubated at 37 c for 72 h. in another series of experiments , cells were incubated with a conjugate at 37 c for 24 h and the drug medium was removed . then treated cells were washed with pbs , and gsh - oet ( 6 equiv to a conjugate ) in the cell culture medium ( 200 l ) was added to the wells . these cells were incubated at 37 c for additional 48 h , i.e. , the total incubation time was 72 h. for all experiments , after removing the test medium , the fresh solution of mtt in pbs ( 40 l of 0.5 mg mtt / ml ) was added to the wells , and the cells were incubated at 37 c for 3 h. the mtt solution was then removed , and the resulting insoluble violet formazan crystals were dissolved in 0.1 n hcl in 2-propanol with 10% triton x-100 ( 40 l ) to give a violet solution . the spectrophotometric absorbance measurement of each well in the 96-well plate was run at 570 nm using a labsystems multiskan ascent microplate reader .

novel tumor - targeting dual - warhead conjugates , 2 ( dw-1 ) and 3 ( dw-2 ) , which consist of a next - generation taxoid , 1 ( sb - t-1214 ) , and camptothecin as two warheads , self - immolative disulfide linkers for drug release , biotin as the tumor - targeting moiety , and 1,3,5-triazine as the tripod splitter module , were designed and synthesized . the potency of 2 was evaluated against mx-1 , mcf-7 , id8 , l1210fr ( br+ , biotin receptor overexpressed ) and wi38 ( br , normal ) cell lines in the absence and presence of glutathione ( gsh ) , which is an endogenous thiol that triggers drug release inside the cancer cells . with the gsh and resuspension protocol , 2 exhibited ic50 values of 3.229.80 nm against all br+ cancer cell lines , and 705 nm against wi38 . thus , there was a two orders of magnitude higher selectivity to cancer cells . also , a clear cooperative effect was observed for the taxoid camptothecin combination when two drugs were delivered to the cancer cells specifically in the form of a dual - warhead conjugate .

Introduction Results and Discussion Conclusion Experimental Section

in the past decade , significant advancement has been made on the development of tumor - targeted drug delivery systems ( ttddss ) , especially for monoclonal antibody drug conjugates ( adcs ) and small molecule drug conjugates ( smdcs ) . however , only one such tumor - targeting dual - drug conjugate has been reported for the combination of mitomycin c and desacetylvinblastine using folate as the tumor - targeting module . thus , there is an unmet need for the development of efficacious ttddss amenable to anticancer drug combinations . because a ttdds delivers anticancer drugs through receptor - mediated endocytosis ( rme ) , it should be able to circumvent multidrug resistance caused by abc transporter efflux pumps such as p - glycoprotein in the cancer cell membrane , which is one of the beneficial features of ttddss . for the selection of two drugs ( warheads ) for the novel dual - warhead molecular missiles , we focused on the combination of a microtubule - stabilizing agent ( taxanes ) and a topoisomerase i inhibitor ( camptothecin and its analogues ) in this study . for taxane anticancer agents , we have been developing highly potent next - generation taxoids which exhibit 23 orders of magnitude greater potency than paclitaxel and docetaxel against multidrug - resistant and paclitaxel - resistant cancer cell lines and tumors . we report here the design , synthesis , and biological evaluations of novel tumor - targeting dual - warhead conjugates , bearing a next - generation taxoid , 1 ( sb - t-1214 ) , and camptothecin as warheads , and biotin as the tumor - targeting module ( figure 1 ) . we designed a versatile platform , consisting of 1,3,5-triazine as the key tripod splitter module , self - immolative linkers with tetraethylene glycol diamine spacers to improve water solubility , and a propargylamine arm for the attachment of second warhead module . then , novel dual - warhead conjugate , 2 ( dw-1 ) , was synthesized based on this platform using click chemistry to attach the camptothecin module with a self - immolative linker and a tetraethylene glycol diamine spacer ( figure 2 ) . another dual - warhead conjugate , 3 ( dw-2 ) , bearing the camptothecin module with a simple ester linkage , was also synthesized to secure stepwise drug release ( figure 2 ) . in addition to these dual - warhead conjugates , we also designed two more drug conjugates using the same platform , wherein one of the warheads was replaced with a surrogate , i.e. thus , a conjugate with taxoid 1 and phenol , 4 ( sw - tax ) , and another with camptothecin and phenol , 5 ( sw - cpt ) , were synthesized in order to properly compare the efficacy of dual - warhead vs single - warhead in the same conjugate scaffold ( figure 3 ) . in addition to the fr , recent studies have disclosed that the biotin receptor ( br ) is overexpressed even more than the fr in a good number of cancer cell lines . thus , the br has emerged as a promising biomarker for tumor - targeted drug delivery , and we have successfully employed biotin as a tumor - targeting module for small - molecule - based as well as single - walled carbon nanotube - based drug conjugates . in the present study , we designed fluorescent probe 7 , which is more water - soluble than biotin - nhnh - fitc , for the assessment of the br overexpression level in several cancer cell lines to select appropriate cancer cell lines for evaluation of biotin drug conjugates , targeting the br . internalization of probe 7 into mx-1 ( breast ) , mcf-7 ( breast ) , id8 ( ovary ) , and wi38 ( lung fibroblast , normal ) cell lines via rme was monitored at 0 , 1 , and 3 h periods . previously studied murine leukemia cell lines l1210fr ( br+ ) and l1210 ( br ) were also used for comparison . internalization of probe 7 into various cell lines at 0 , 1 , and 3 h at 37 c . internalization of probe 7 monitered by flow cytometry and cfm : ( a ) flow cytometry analysis of 7 in id8 at 0 h ( red , control ) , 1 h ( green ) , and 3 h ( blue ) ; ( b , c ) cfm images of 7 in id8 at 1 and 3 h , respectively ; ( d ) flow cytometry analysis of 7 in mcf-7 at 0 h ( red , control ) , 1 h ( green ) , 3 h ( blue ) ; ( e , f ) cfm images of 7 in mcf-7 at 1 and 3 h , respectively . we also performed the assessment of the br expression level in additional human breast cancer cell lines and found that these cell lines also overexpress the receptor : bt-20 ( br ) , lcc6-wt ( br ) , lcc6-mdr ( br ) , mda - mb 231 ( br ) , and skbr3 ( br ) ( see supporting information ) . disulfide linkers , which can be conjugated to a cytotoxic warhead on one end and a tumor - targeting module on the other end . linkers consist of a 2-sulfhydrylphenylacetic acid component and a sulfanylalkanoic acid component , and the former is connected to a cytotoxic agent and the latter to a tumor - targeting module or a splitter unit linked to a tumor - targeting module ( see figure 2 ) . disulfide linker was designed to be self - immolative for releasing a cytotoxic warhead through disulfide thiol exchange reaction with endogenous thiols , represented by glutathione ( gsh ) , followed by facile benzothiolactonization . thus , when the tumor - targeting module navigates the drug - conjugate to the target biotin receptors on the tumor surface , the whole conjugate is internalized via receptor - mediated endocytosis ( rme ) . to investigate the effects of sequential drug release on the potency of dual warhead drug conjugates , cpt - linker construct 18 , bearing a simple ester linker , was designed to secure that the taxoid is released prior to cpt inside the cancer cell . among possible tripod splitter modules , we chose 1,3,5-triazine for novel dual - warhead conjugates with three arms . thus , dual - warhead conjugates , 2 and 3 , as well as their single - warhead surrogates , i.e. reactions with azides 15c , 18 , and 15p in the presence of cupric sulfate and ascorbic acid at 25 c to give the corresponding conjugates , 2 , 3 , and 4 , respectively , in 7392% yields . the effect of the combinations of taxoid 1 and camptothecin ( 1:1 ) on cytotoxicity was examined using three different timings for drug mixing against human breast carcinoma ( mx-1 and mcf-7 ) , murine ovarian ( id8 ) , and murine leukemia ( l1210fr ) cell lines . , ( a ) simultaneous exposure ( entry 3 ) , ( b ) taxoid first for 24 h , followed by camptothecin ( entry 4 ) , and ( c ) camptothecin first for 24 h , followed by taxoid ( entry 5 ) , exhibited a marked difference in the potency . when cancer cells were exposed to camptothecin first for 24 h , followed by equimolar concentrations of taxoid 1 ( entry 5 ) , drastic increases in the ic50 values as compared to those values for simultaneous exposure ( entry 3 ) were observed . in sharp contrast , when cancer cells were exposed to equimolar mixture of the two drugs ( entry 3 ) or to taxoid 1 first for 24 h , followed by equimolar concentrations of camptothecin ( entry 4 ) , ic50 values were essentially the same as those for taxoid 1 alone within errors ( entry 1 ) . confocal fluorescence microscopy images showing internalization of conjugate 2 in id8 ( left ) and mcf-7 ( right ) after incubation at 37 c for 10 h. cytotoxicities of novel dual - warhead conjugates , 2 and 3 , as well as their single - warhead surrogates , 4 and 5 , were evaluated in two ways against four br(+ ) cancer cell lines , mx-1 , mcf-7 , id8 and l1210fr , as well as normal human lung fibroblast cell line , wi38 , using the standard mtt assay . in the first experiment , mx-1 ( br ) , mcf7 ( br ) , id8 ( br ) , and l1210fr ( br ) cancer cells were incubated with conjugate 25 for 72 h , and the corresponding ic50 values were determined . as table 2 shows , the cytotoxicities of conjugates 2 and 3 based on their ic50 values were found to be in a range of 1965 nm ( entries 3 and 4 ) , which makes a sharp contrast with that against normal cell line wi38 ( ic50 742868 nm ) ( entries 3 and 4 ) . because we have confirmed that these four cancer cell lines overexpress br ( see figures 3 and 4 ) and also observed marked selectivity for br+ cancer cell lines as compared to normal wi38 cell line ( br ) , the internalization of conjugate 25 via rme should not be the major issue for these cancer cell lines . to clarify these points , glutathione ethyl ester ( gsh - oet ) ( 6 mol equiv to a conjugate ) was added to the resuspended cancer cells after the cells had been incubated with a conjugate for 24 h , followed by thorough washing with phosphate - buffered saline ( pbs ) , and further incubated for 48 h in the second experiment ( table 3 ) . conjugate 2 showed ic50 values of 3.29.8 nm against four br+ cancer cell lines , but that against wi38 was 742 nm ( entry 1 ) . thus , there is two order of magnitude difference in cytotoxicity between cancer cells and normal cells , which is unambiguously attributed to the highly efficient receptor - targeting by conjugate 2 . the results clearly indicate that there were synergistic or cooperative effects between taxoid 1 and camptothecin when these two drugs were delivered to cancer cells simultaneously and released inside cancer cells . it appears that all four br+ cancer cell lines are highly drug resistant to camptothecin and the potency was further decreased substantially when camptothecin and phenol were delivered to mx-1 , mcf-7 , and id8 cells via rme and released inside cancer cells ( table 3 , entry 4 ) as compared to that observed in the standard mtt assay of camptothecin ( table 2 , entry 2 ) . we have designed and successfully synthesized novel tumor - targeting dual - warhead conjugates , 2 and 3 , which consist of taxoid 1 and camptothecin as two warheads , self - immolative disulfide linkers for drug release , biotin as the tumor - targeting moiety , and 1,3,5-triazine as the tripod splitter module . for the introduction of the second warhead - linker module to the 1,3,5-triazine splitter module then , its acetylene moiety was used for click reaction , with the second drug - linker construct bearing an -azido group to install the second warhead module as the third arm . since biotin was used as the tumor - targeting moiety , we screened the expression levels of the biotin receptor ( br ) in various human breast cancer cell lines , mx-1 , mcf-7 , lcc6-wt , lcc6-mdr , mda - mb 231 , and skbr3 , which had not been reported previously in addition to the known br - overexpressing cancer cell lines , id8 ( human ovary ) and l1210fr ( murine leukemia ) , by means of flow cytometry and confocal fluorescence microscopy ( cfm ) . also , we confirmed that there was no appreciable expression of br in l1210 ( murine leukemia ) and wi38 ( normal , human lung fibroblast ) cell lines . the potencies of conjugates , 2 and 3 , were evaluated against mx-1 , mcf-7 , id8 , l1210fr , and wi38 . it was found that the amount of endogenous thiols such as glutathione ( gsh ) was insufficient to trigger the drug release via cleavage of the self - immolative disulfide linkers under in vitro assay conditions ( this is not an issue in vivo ) . then , the cells were further incubated for 48 h. thus , only the conjugate molecules internalized into the cancer cells in the first 24 h were treated with gsh - oet in this protocol . the addition of gsh - oet showed remarkable effect , as anticipated , and conjugate 2 exhibited ic50 values of 3.229.80 nm against all br+ cancer cell lines , while that against normal cell line , wi38 , was 705 nm . thus , there were two orders of magnitude difference in selectivity to cancer cells , which was impressive . also , the potency of 1 was 23 times as high as those of 3 and surrogate conjugate 4 , which clearly indicates the synergistic or cooperative effect of the taxoid it is noteworthy that this substantial enhancement in potency in this combination was observed only when these two drugs were internalized via rme . to a solution of 13p ( 154 mg , 0.410 mmol ) and hosu ( 0.141 g , 1.23 mmol ) in ch2cl2 ( 1.6 ml ) was added edchcl ( 94 mg , 0.493 mmol ) in ch2cl2 ( 1 ml ) , and the mixture was allowed to react for 16 h at room temperature with stirring . to a solution of 13p ( 149 mg , 0.396 mmol ) and 1-amino-11-azido-3,6,9-trioxaundecane ( 259 mg , 1.19 mmol ) in ch2cl2 ( 1 ml ) was added dic ( 67 l , 0.436 mmol ) , and the mixture was stirred for 10 h at room temperature . to a solution of succinic anhydride ( 16 ) ( 183 mg , 1.834 mmol ) and 1-amino-11-azido-3,6,9-trioxaundecane ( 400 g , 1.834 mmol ) in dichloromethane ( 3 ml ) was added triethylamine ( 25 l , 0.1834 mmol ) , and the mixture was allowed to react for 16 h at room temperature with stirring . then , a solution of 7 ( 954 mg , 2.282 mmol ) and dipea ( 0.6 ml , 3.423 mmol ) in thf ( 80 ml ) was added to the reaction mixture and refluxed for 16 h. the reaction mixture was concentrated in vacuo to afford a red oil . to a solution of 20 ( 794 mg , 1.151 mmol ) and dipea ( 0.4 ml , 2.302 mmol ) in thf ( 12 ml ) was added propargylamine ( 362 l , 6.600 mmol ) , and the mixture was stirred for 12 h at 50 c . to a solution of 21 ( 400 mg , 0.564 mmol ) in ch2cl2 ( 6 ml ) was added tfa ( 0.5 ml , 6.57 mmol ) , and the mixture was stirred at 25 c for 48 h. the reaction mixture was concentrated in vacuo to afford a yellow oil , which was triturated with ether ( 20 ml ) and the crude product ( tfa salt ) crashed out . lcc6-mdr , lcc6-wt , mcf-7 , mda mb 231 , mx-1 , and id8 cells were cultured as monolayers on 100 mm tissue culture dishes in a supplemented rpmi-1640 cell culture medium , and wi-38 as a monolayer in a supplemented dmem cell culture medium . in the case of leukemia cell lines ( l1210 and l1210fr ) , probe 7 ( 1 mm ) in dmso ( 30 m ) was added directly into fresh cell suspensions to give the final concentration of 10 m , and incubated for similar time intervals . after removing the old medium , these test solutions were added to the wells in the 96-well plate to give the final concentrations ranging from 0.5 to 5000 nm ( 100 l ) , and the cells were subsequently cultured at 37 c for 72 h. for the leukemia cell lines , cells were harvested , collected , and resuspended in the test solutions ranging from 0.5 to 5000 nm ( 100 l ) at 0.5 to 0.8 10 cells per well in a 96-well plate and subsequently incubated at 37 c for 72 h. in another series of experiments , cells were incubated with a conjugate at 37 c for 24 h and the drug medium was removed .

community - acquired pneumonia ( cap ) is commonly described as an acute infection of the lung parenchyma acquired in the community . it is most commonly bacterial in nature and is associated with clinical and/or radiological evidence of consolidation of part or parts of one or both lungs . cap is associated with a considerable burden of disease in most regions of the world [ 26 ] . it is one of the most important serious infectious diseases , accounting for a considerable number of hospital admissions , with an increasing incidence in many parts of the world and an increasing rate of serious complications . as part of the burden of respiratory infections , cap is well recognised to be a leading cause of death among the infectious diseases [ 6 , 8 ] . the reason that cap is so common relates to the very high prevalence of specific risk factors for this infection in patients worldwide . while a myriad of microorganisms may cause cap , in reality a relatively small number of pathogens predominate , in particular the bacteria , of which streptococcus pneumoniae ( pneumococcus ) is by far the most common . there is considerable concern about the emerging resistance among the usual cap pathogens to the most commonly used antimicrobial agents . there are a number of important decisions that need to be made with regard to the assessment and management of patients with cap , not least of which is an evaluation of the severity of the infection . a number of guidelines have been published worldwide , describing the optimal treatment of patients with cap , with the aim of improving patient outcomes . the remainder of this introductory overview of cap will focus on ( i ) burden of disease , ( ii ) risk factors , ( iii ) microbiology , ( iv ) antimicrobial chemotherapy , ( v ) antibiotic resistance , and ( vi ) assessment of severity of illness using clinical scoring systems and laboratory biomarkers individually and in combination . cap continues to be a cause of considerable morbidity and mortality in most parts of the world , being the most frequent infectious cause of death in patients in the usa and throughout europe . since aging is a significant risk factor for this infection and given that in many areas of the world , such as europe , the population is aging , an increase in incidence in the next decades is anticipated . the mechanism by which aging is associated with a risk for cap is multifactorial , not simply related to chronological age , but frequently associated with ( i ) underlying comorbid conditions that more commonly occur in the aging population ; ( ii ) a greater risk of being infected with antibiotic - resistant pathogens ; ( iii ) social factors ; and ( iv ) even place of residence . studies in the usa , europe , latin america , and the asia - pacific region attest to the fact that cap has a substantial clinical and economic burden , a high rate of antibiotic resistance among the pathogens , and a significant effect on both immediate and long - term prognosis , as well as on the quality of life of infected patients . given this high burden of disease , it is recommended that steps be taken to ensure appropriate treatment , ongoing surveillance for antimicrobial resistance among the common pathogens , and strategies , including vaccination , to prevent these infections . as shown in the list below , there is a considerable number of risk factors for cap that exist in populations all over the world , and most of these risk factors are associated with an impairment of the efficacy of host immune defence . in addition to aging , the common risk factors in adults are smoking , the presence of various underlying comorbid conditions , including chronic cardiorespiratory , renal and hepatic conditions , and , at least in some regions of the world , concomitant human immunodeficiency virus ( hiv ) infection . there is also some evidence that male patients and those of certain racial or ethnic groups may be at greater risk of pneumonia . risk factors for community - acquired pneumonia are as follows : extremes of age ( very young and the aging),male gender , certain populations ( various racial or ethnic groups),lifestyle factors ( excessive alcohol consumption and smoking),underlying comorbid conditions such aschronic cardiorespiratory illnesses , chronic renal disorders , hepatic conditions , diabetes mellitus , neoplastic diseases , human immunodeficiency virus infection , medications ( e.g. , inhaled corticosteroids , proton pump inhibitors),additional risk factors associated with pneumococcal infections in particular ( e.g. , myeloma , hypogammaglobulinemia ( such as igg2 deficiency ) , surgical asplenia , or functional asplenia ( such as in sickle cell disease ) . extremes of age ( very young and the aging ) , certain populations ( various racial or ethnic groups ) , lifestyle factors ( excessive alcohol consumption and smoking ) , underlying comorbid conditions such as chronic cardiorespiratory illnesses , chronic renal disorders , hepatic conditions , diabetes mellitus , neoplastic diseases , human immunodeficiency virus infection , chronic cardiorespiratory illnesses , chronic renal disorders , human immunodeficiency virus infection , medications ( e.g. , inhaled corticosteroids , proton pump inhibitors ) , additional risk factors associated with pneumococcal infections in particular ( e.g. , myeloma , hypogammaglobulinemia ( such as igg2 deficiency ) , surgical asplenia , or functional asplenia ( such as in sickle cell disease ) . smoking , both active and passive ( particularly in children ) , is a well - described risk factor for cap , particularly in hiv - infected persons , as well as for many other infectious diseases , and this has been comprehensively reviewed recently . the main mechanisms for this predisposition relate to the suppressive effect that smoking has on the protective actions of the airway mucociliary clearance mechanism , on the various components of the innate and adaptive immune systems of the host , as well as direct effects on microbial pathogens that promote their virulence , and possibly antibiotic resistance . several comorbid factors relate quite closely to the risk of cap and the possibility of more severe illness , as well as to the likelihood of a worse outcome . among the most common comorbid predisposing factors to cap , as mentioned above , are chronic obstructive pulmonary disease , congestive cardiac failure , diabetes mellitus , a high intake of alcohol , and smoking . however , various other conditions , including those of the neurological system , the liver , the kidney , and also neoplastic disease , represent important risk factors . more recently , there has been considerable interest in the fact that inhaled medication ( particularly inhaled corticosteroids ) appears to be a risk factor for cap and this has been reviewed elsewhere . in a number of regions of the world , such as in sub - saharan africa , concomitant infection with hiv represents a major risk factor for cap and has been extensively reviewed elsewhere [ 1214 ] . the spectrum of bacterial pathogens causing cap in hiv - infected patients is very similar to that in hiv - uninfected patients with streptococcus pneumoniae ( pneumococcus ) predominating . the clinical presentation of cap in hiv - infected persons , particularly pneumococcal cap , is similar to that among hiv - uninfected patients except that the patients are frequently younger , of the female sex , and have a greater frequency of respiratory symptoms [ 13 , 14 ] . furthermore , there is a similar spectrum of disease severities , with the commonly used severity of illness scores appearing to have an equivalent value in predicting outcome compared to hiv - uninfected individuals . however , when cases with bacteremic pneumococcal cap are stratified according to age and severity of illness , hiv - infected persons have been found to have a significantly higher mortality with an increasing trend as the cd4 cell count decreases . some investigators have therefore recommended that the decision to admit hiv - infected patients with cap to hospital should be based on both the cd4 cell count and the severity of illness , those with a cd4 cell count < 200/l blood always being admitted to hospital , and those with a higher cd4 cell count being admitted to hospital if warranted according to the severity of illness . recent studies have identified several mechanisms , specifically the presence of gene polymorphisms , including single nucleotide polymorphisms ( snps ) in genes encoding proteins of the innate immune system , which contribute not only to susceptibility for development of cap , but also to a worse outcome . for example , snps in the il-6 gene , specifically il-6 174 g / g , have been reported to protect patients with pneumococcal cap against development of ards , septic shock , and multiple organ dysfunction syndrome , resulting in less severe disease and lower mortality . in addition , investigation of the role of snps in the genes encoding the surfactant proteins ( sp ) a , b , c , and d revealed associations with both susceptibility for both development of cap and more severe disease [ 16 , 17 ] . similar , albeit statistically insignificant , findings were reported for sp - a and sp - d in patients with pneumococcal cap . the most prominent organism causing cap is the pneumococcus , and this remains true irrespective of the severity of infection across the spectrum of outpatients , inpatients not in the intensive care unit ( icu ) , and even cases with cap requiring icu admission [ 11 , 18 ] . studies in europe , the usa , latin america , the asia - pacific region , and elsewhere attest to the fact that the pneumococcus is consistently documented to be the most predominant pathogen [ 25 , 9 ] . interestingly , while it is well described that pneumococcal infections commonly complicate both seasonal and pandemic influenza infections , more recently it was documented that the pneumococcus was a common bacterial coinfection in patients with influenza a h1n1 infection who were admitted to hospital with cap [ 1921 ] . in the former two studies , the pneumococcus was the most common bacterial cause of bacterial co - infection , accounting for 62% and 54.8% of cases , respectively , and being associated with a greater risk of septic shock or need for vasopressors , as well as increased need for mechanical ventilation and a longer icu stay [ 20 , 21 ] . many recent studies of pneumococcal infection have focussed on the issue of pneumococcal serotypes causing disease , and the possible association of different serotypes with disease severity , particularly in relation to the release of the newer pneumococcal conjugate vaccines and the extended indication for their use in adults [ 2225 ] . while earlier studies suggested that host factors may be more important than isolate serotype in determining the severity and outcome of pneumococcal infections , suggesting that vaccination was unlikely to be associated with a change in these end - points , more recent studies suggest that ipd outcome is a serotype - related issue ; for example , serotype 3 is more commonly associated with septic shock [ 24 , 25 ] . when considering the likely benefit of vaccination , it also remains important to consider the serotypes implicated in nonbacteremic infections , which studies have suggested that due to greater serotype distribution are less comprehensively covered by currently available conjugate vaccines [ 26 , 27 ] . after the pneumococcus , the next most common pathogens are the so - called atypical pathogens , the respiratory viruses , and haemophilus influenzae . among the viruses , influenza predominates , but smaller numbers of various other respiratory viruses are also documented [ 11 , 28 , 29 ] . less commonly , additional pathogens are documented , particularly in cases with respiratory - related comorbid illnesses . these include staphylococcus aureus , pseudomonas aeruginosa , and the enterobacteriaceae . on presentation of adults with suspected cap , empiric antimicrobial chemotherapy is initiated according to the relevant national guidelines , with age , comorbidities , and disease severity being the primary determinants of the class of antibiotic(s ) and route of administration . in the case of outpatient therapy , previously healthy patients who had not received antibiotics during the 3-month period prior to presentation , monotherapy with a macrolide or doxycycline is recommended by the infectious diseases society of america ( idsa)/american thoracic society ( ats ) ; in those with comorbidities and/or prior recent use of antibiotics , the recommendation is the combination of an antipneumococcal -lactam and a macrolide , or alternatively , a respiratory fluoroquinolone . in the case of inpatient therapy in the non - icu setting , the guidelines advocate the combination of a -lactam and a macrolide , or , alternatively , monotherapy with a respiratory fluoroquinolone . in the case of patients admitted to intensive care , the combination of a -lactam with either a macrolide or a respiratory fluoroquinolone is recommended . clearly , these therapeutic strategies can be reevaluated on the basis of clinical response and acquisition of microbiological and other laboratory data . the rationale for implementation of combination therapy ( various combinations , but most frequently a -lactam with a macrolide ) in patients with severe cap was largely based on a series of observational studies , both retrospective and prospective , conducted between 1999 and 2010 , which demonstrated significantly lower in - hospital or icu mortality . although the microbiological mechanisms underpinning the apparent benefit of combination antibiotic therapy remain unknown , activity of macrolides / fluoroquinolones / tetracyclines against the atypical pathogens chlamydia pneumoniae , legionella pneumophila , and mycoplasma pneumoniae , as well as efficacy in eradicating polymicrobial sepsis , have been proposed . when considering these possible mechanisms , it is also important to note that the benefit of adding a macrolide to standard -lactam therapy is particularly evident in sicker patients , such as those with severe sepsis due to pneumonia , and in intubated patients [ 31 , 33 ] . benefit also extends to cases infected with macrolide - resistant pathogens ( e.g. , macrolide - resistant pneumococcal infections and even to cases with gram - negative infections ) . the aforementioned studies , as well as several others which failed to confirm a survival benefit when comparing monotherapy with combination therapy , have recently been extensively reviewed elsewhere [ 3437 ] . clearly , the issue of optimum antimicrobial chemotherapy of cap remains to be resolved and will be dependent on the acquisition of data from large , well - controlled multicentre , randomised clinical trials [ 3739 ] . one such trial , the cap - start study , is currently underway in holland [ 38 , 40 ] . multicentre , cluster randomised crossover trial involving seven dutch hospitals and 2100 hospitalised nonintensive care unit ( icu ) patients . it is designed to compare the therapeutic efficacy of ( i ) -lactam monotherapy ; ( ii ) -lactam / macrolide combination ; and ( iii ) fluoroquinolone monotherapy . the primary outcome is all - cause mortality 90 days after hospital admission [ 38 , 40 ] . there is a considerable body of literature devoted to the issue of antimicrobial resistance among the common pathogens causing cap , especially with regard to the pneumococcus [ 4144 ] . many studies describing the burden of disease caused by cap confirm that there is emerging and increasing resistance among many of the cap pathogens , a phenomenon that is occurring worldwide and which involves all classes of antimicrobial agents , to a greater or lesser extent [ 25 , 41 ] . accordingly , it has been recommended that the choice of empiric antimicrobial therapy for patients with cap must be based on prediction of the most likely infecting pathogens together with a full appreciation of the common antimicrobial susceptibility patterns of these pathogens in a given geographical area ( unit , ward , or practice ) . while it is clear that there is emerging resistance among the cap pathogens , and particularly the pneumococcus , it remains unclear whether the presence of antimicrobial resistance alone is associated with unfavourable treatment outcomes . there have been a number of studies and various reviews discussing the issue of antibiotic resistance in the management and outcome of pneumococcal cap [ 41 , 4549 ] . while there have been some studies that have purported to document a higher mortality rate in patients infected with penicillin - resistant compared with penicillin - susceptible pneumococcal pneumonia , most other studies or reviews have not reached similar conclusions [ 41 , 45 , 46 , 48 , 49 ] . in a large study of 844 hospitalised cases with bacteremic pneumococcal cap furthermore , these authors noted that discordant therapy ( receipt for the first 2 days after the positive blood culture of a single antibiotic that was inactive against the pneumococcus isolated ) with penicillin , cefotaxime , or ceftriaxone did not result in a higher mortality rate . others has confirmed that resistance to penicillins and third - generation cephalosporins have not been associated with significant increased mortality . one critical review of the literature documented only a single microbiological failure of a parenteral penicillin - class antibiotic in the treatment of a patient with pneumococcal pneumonia . some have suggested that the patients who are at risk for antibiotic - resistant pathogens ( due to conditions such as advanced age , or underlying comorbid conditions ) are those that may already have host risk factors for a higher mortality . it is , however , important to recognise that changes in the breakpoint definitions for the penicillins and third - generation cephalosporins have occurred since that time such that many of those pathogens previously described as penicillin - resistant would now be considered susceptible . nevertheless , the previous recommendations that therapy in these patients should continue with high - dose penicillins and broad - spectrum cephalosporins is still recommended in current guidelines [ 46 , 49 ] . the situation with macrolides and fluoroquinolones is less clear - cut [ 41 , 46 ] . macrolide resistance may be of low level , associated with an efflux mechanism ( mef gene ) , or of high level , associated with ribosomal target site mutations ( erm gene ) , and while the latter has clearly been associated with treatment failure , there have also been some cases of failure with the former , although relatively small in number [ 41 , 46 ] . for this reason , it has been recommended that awareness of pneumococcal macrolide resistance levels and patterns in a given region , as well as the risk factors in individual patients for macrolide resistance , clearly determine the utility of macrolide monotherapy in the management of pneumococcal cap . with the respiratory fluoroquinolones , it is clear that laboratory documented resistance is likely to be associated with clinical failure , but what is less well known is that organisms documented in the laboratory as being susceptible , sometimes harbour one - step mutations in their quinolone resistance - determining regions that may undergo further mutations on therapy that may render them resistant . clearly new options for the treatment of antibiotic resistant pneumococcal infections are desirable , and to this end several newer agents have recently been introduced which have enhanced activity against resistant pneumococcal infections . this topic has been reviewed elsewhere and includes a potential role for ceftaroline , linezolid , telavancin , and tigecycline . the severity of the infection dictates a number of important issues in the management of patients with cap . severity of illness determines the site of care ( in- or outpatient ) , the extent of the microbiological workup , and the choice of initial empiric antimicrobial therapy . increased severity of infection is associated with greater healthcare needs and costs . while to a large extent assessment of severity of infection is still based primarily on sound clinical judgment , researchers have been attempting to develop mechanisms by which severity may be objectively assessed , such as the use of clinical scoring systems , various biomarkers , or by measuring microbial load . a number of severity of illness scoring indices have been developed to assist in the evaluation of severity of pneumonia , of which the most commonly used are the pneumonia severity index ( psi ) and the curb-65 [ 6 , 7 , 52 , 53 ] . the psi uses 20 variables which include patient age , gender , presence or absence of comorbid conditions , and/or vital sign abnormalities , as well as various laboratory and radiographic parameters [ 6 , 54 ] . the curb-65 uses only 5 variables , namely , presence or absence of confusion , urea > 7 mmol / l , respiratory rate 30 breaths / minute , low blood pressure ( systolic < 90 mmhg or diastolic 60 mmhg ) , and age 65 years [ 6 , 54 ] . with both scoring systems , cases can be stratified into low- , moderate- , or high - risk groups . the psi was developed primarily to assist in identifying those patients who could safely be managed at home , whereas the curb-65 was developed to document patients that were more severely ill , including those who needed to be flagged for icu admission . one of the advantages of the curb-65 scoring system is that it is easy to use , but a limitation is that it may underestimate pneumonia severity in younger patients with comorbidities . the psi has been well validated and performs well in the assessment of low mortality risk patients , but it is complex to calculate and requires the evaluation of a number of laboratory parameters that potentially limits its use outside a hospital . a number of additional scoring systems have also been developed , including those designed for the assessment of more severely ill cases , particularly those cases flagged for icu admission , including the idsa / ats criteria , smart - cop , piro - cap , and scap , which appear to have better discriminatory values than psi or curb-65 in this situation [ 7 , 5456 ] . each of the scoring systems that have been developed has various strengths and potential weaknesses so that no system is ideal , being unable to identify all patients at risk , or to replace clinical judgment ; nonetheless , they are useful adjuncts for assessing cases [ 6 , 53 , 55 ] . also of concern is that the scoring systems have been developed for assessment of patients on admission only and there is clearly a need for evaluating patients during the course of their hospitalisation , particularly in the setting of clinical deterioration . more recently , a number of biomarkers has been tested to determine their ability to stratify risk in patients with cap , sometimes as an adjunct to clinical scoring systems [ 7 , 55 ] . among these are inflammatory markers , such as the white blood cell count , acute phase reactants , such as c - reactive protein ( crp ) , cytokines , such as interleukin-1 and tumour necrosis factor- , stress hormones , and various other molecules [ 6 , 53 , 55 ] . of these , c - reactive protein ( crp ) and procalcitonin ( pct ) have been particularly well studied and have been reported in many studies to be useful tools , sometimes with an accuracy similar to that of curb-65 or one of the other scoring systems , increasing the accuracy of severity assessment when used in combination with these scoring systems [ 7 , 55 , 57 ] . crp , for example , is universally available and in some studies has been shown to have value in site - of - care decisions in predicting 28-day mortality , and in the prediction of treatment failure [ 7 , 55 ] . pct has been shown in some studies to be more useful than crp in predicting severity and outcome in patients with cap but is not widely available in many healthcare systems , possibly because of its much higher cost [ 7 , 55 ] . it has been said that pct should be considered to be a prognostic indicator rather than a diagnostic factor , but there is evidence that it may safely help reduce the unnecessary use of antibiotics , reducing bacterial resistance and curbing healthcare costs without increasing mortality [ 5760 ] . other biomarkers include proatrial natriuretic peptide ( proanp ) , b - type natriuretic peptide ( bnp ) , provasopressin ( provp ) , adrenomedullin ( adm ) , proadrenomedullin ( proadm ) , arginine vasopresine ( avp ) , cortisol , d - dimers , copeptin , and soluble triggering receptor expressed on myeloid cells-1 ( trem-1 ) [ 7 , 53 , 55 ] . a number of recent studies has documented that the pneumococcal bacterial dna load correlates well with the severity of infection and has prognostic value , thus confirming a concept that has long been proposed , and which has become more accurate with the acquisition of more reliable assays [ 41 , 47 ] . it has also been suggested that repeated measurements of the bacterial dna load can accurately monitor treatment progress ; however , more studies are needed to confirm these findings . the evaluation of the clinical outcome of a patient with pneumonia is an important aspect of medical care and has been reviewed elsewhere . achievement of clinical stability dictates important management issues such as change from intravenous to oral therapy , the timing of hospital discharge , and assessment of likely patient outcome . a number of risk factors for clinical failure have been identified , but most do not recognize pneumococcal aetiology as an important issue . for example , in one study even the presence of pneumococcal bacteraemia did not increase the risk of poor outcome . respiratory tract infections , of which cap plays a large role , are a major cause of death worldwide . while many of the studies of cap mortality have concentrated on in - hospital or 30 day mortality , others have evaluated long - term mortality ( e.g. , over a one - year period ) . one such study in older patients noted a hospital mortality of 11% versus 5.5% in controls ( case controls matched for age , gender , race ) , while the one - year mortality was 40.9% versus 29.1% , respectively [ 52 , 63 ] . prior to that and subsequently , there has been a myriad of additional studies documenting high long - term mortality in patients with cap . one study documenting high long - term morbidity and mortality in cap patients noted that it occurred particularly in those cases with high initial psi scores . although many predisposing mechanisms have been documented , the presence of cardiovascular disease and other comorbid conditions , including hiv infection , the subsequent documentation of primary or secondary neoplasms , and alterations in immune function predominate . cardiovascular events are among the most studied , with a number of reports documenting an increased risk of cardiovascular events during and after serious infections , such as cap [ 64 , 6769 ] . the remaining sections of this review are focused on ( i ) the major virulence determinants of the pneumococcus , the most common cause of cap , and their involvement in triggering harmful inflammatory responses [ 70113 ] and ( ii ) control of these using pharmacological , anti - inflammatory strategies . the human airway employs numerous mechanisms to protect the airways from colonisation and invasive pneumococcal infection . in the case of innate immune defences , these include the cough reflex , the mucociliary escalator , and a range of pattern recognition receptors ( prrs ) . the prrs include toll - like receptors ( tlrs ) , nucleotide - binding oligomerisation domain receptors ( nod - like receptors , nlrs ) , rig-1-like receptors ( rlrs ) , and the manifold cytosolic dna sensors ( reviewed in ) . in addition , infected cells , or cells which are stressed , release host - derived molecules known as damage - associated molecular patterns ( damps ) or alarmins . during an infection , damps and pamps have been shown to synergize leading to synthesis and secretion of proinflammatory cytokines and chemokines , including il-1 and il-8 , as well as stimulating cell differentiation and cell death . although these mechanisms protect the airways from s. pneumoniae , antibody - mediated mechanisms , and cell - mediated immunity are critical in clearing the lower airways of this pathogen . cell - mediated resolution of nasopharyngeal colonisation involves both th1 and th17 responses [ 116 , 117 ] triggered by a variety of surface virulence determinants such as the bacterial capsule , pili , and other adhesins , as well as the toxin , pneumolysin . the major virulence determinants and mechanisms of subversion of host defences by s. pneumoniae are summarized in table 1 [ 70113 ] . the anti - phagocytic polysaccharide capsule is considered to be the main determinant of pneumococcal virulence and is essential for colonisation of the nasopharynx [ 71 , 72 , 84 ] . relative to the opaque variant , the transparent phenotype has decreased capsular thickness and expresses less pneumococcal surface protein a ( pspa ) in the setting of a higher level of expression of the major adhesin , choline - binding protein a ( cbpa ) , and the autolysin , lyta , favouring adhesion and colonisation . a further decrease in capsular thickness precedes the transition from colonisation to invasion of the epithelium [ 120 , 121 ] . this highly phagocytosis - resistant variant has increased capsular thickness and expression of pspa , in the setting of decreased expression of cbpa , favouring extrapulmonary dissemination . cell - wall fragments and capsular polysaccharides of s. pneumoniae are recognised and bound by antibodies which in turn bind complement , with c1q binding correlating closely with the deposition of c3b and c3bi on the bacterial surface . capsular serotypes differ with respect to invasiveness , due mainly to differences in complement deposition on the capsular polysaccharide , as well as binding of complement factor h . s. pneumoniae has been found to grow in chains of variable length and , in addition to capsular polysaccharide , longer chains appear to favour adherence and colonisation . these are only expressed by certain strains and enable the bacteria to survive in the lung and to bind to epithelial cells . pileated strains also induce a greater tnf - dependent inflammatory response , increasing the potential to produce lung injury and invade host tissue . although the ability of s. pneumoniae to grow and persist as biofilms does not appear to reflect virulence potential , it remains advantageous , as biofilm - encased bacteria show a reduced susceptibility to antimicrobial agents and resistance to immune recognition . domenesch and colleagues have shown that there is reduced phagocytosis of pneumococcal biofilms due to impaired deposition of c3b . biofilm formation by s. pneumoniae is also effective in preventing not only activation of the classical complement pathway due to reduced binding of c - reactive protein and the complement component c1q , but also by suppressing the pneumococcal surface protein c-(pspc- ) dependent activation of the alternative complement pathway . recent studies suggest that biofilms do not contribute to the development of invasive pneumococcal disease , but , rather appear to confer a quiescent mode of growth during colonisation . the role of the capsular polysaccharide in biofilm formation has not yet been determined conclusively , although the absence of the capsular polysaccharide was reported in one study to favour biofilm formation ; however , another study showed that decreased capsular polysaccharide formation was associated with decreased biofilm production . a recent study has shown that augmentation of pneumococcal biofilm formation due to cigarette smoking is likely to favour microbial colonisation and persistence . the pneumococcus is a major producer of h2o2 as a consequence of the activity of pyruvate oxidase , which is surprising , given that this catalase - negative pathogen is ill equipped to detoxify this oxidant . the pneumococcus not only utilises h2o2 as a virulence factor , causing significant damage to ciliated respiratory epithelium and impaired protective activity of the mucociliary escalator , but also to eliminate microbial competitors in the nasopharynx . in addition , pyruvate oxidase has been reported to act as a sensor of the oxygenation status of the microbial environment , regulating both nutritional capability and thickness of the anti - phagocytic capsule . the adherence and colonisation of host tissues by the pneumococcus is mediated by surface adhesins and enzymes [ 83 , 84 ] which are also important virulence factors of s. pneumoniae . however , excessive activity of autolysins results in the degradation of the cell - wall leading to cell lysis [ 86 , 87 ] . n - acetylmuramic acid l - alanine amidase , also known as lyta amidase , is the major autolysin of s. pneumoniae . the lysis of a portion of the bacterial cell - wall by lyta may increase the virulence of s. pneumoniae by promoting the release of potentially lethal toxins such as pneumolysin [ 84 , 86 ] . lytb , lytc , and choline binding protein e ( cbpe ) , all of which have been associated with nasopharyngeal colonisation . choline binding proteins ( cbp ) have a c - terminal binding module followed by a flexible proline - rich segment and a functional n - terminal module . cbpa binds to terminal choline residues of teichoic or lipoteichoic acids present on the surface of s. pneumoniae , anchoring the pathogens to human cell glycoconjugates , favouring the transition from colonisation to invasion . pneumococcal surface protein a ( pspa ) is also an important virulence factor of s. pneumoniae , inhibiting deposition of c3b on the bacterial surface , thereby interfering with complement activation and complement - dependent phagocytosis . pspa also binds to and interferes with lactoferrin , increasing the availability of free iron required for bacterial growth . the adhesins , pava and b of s. pneumoniae , promote the invasion of host cells and dissemination of the pneumococcus . pava has been shown to bind to the extracellular matrix component , fibronectin , while pavb binds both fibronectin and plasminogen . these adhesive effects are mediated by repetitive sequences designated streptococcal surface repeats ( ssure ) [ 95 , 96 ] . it has been suggested that pava may affect pneumococcal colonisation by modulating the expression or function of virulence factors of s. pneumoniae . three forms of neuraminidase have been identified in pneumococci , these being designated as nana [ 105 , 131 ] , nanb [ 104 , 131 ] , and nanc . neuraminidasea cleaves terminal sialic acid from cell surface glycans such as mucin , glycolipids , and glycoproteins , with resultant exposure of binding sites on the host cell surface contributing to pneumococcal adhesion and colonisation [ 83 , 104 , 105 , 132 ] . nanb is involved in the metabolic utilisation of sialic acid as a carbon and energy source by the pneumococcus , while nanc has a regulatory role [ 131 , 133 ] . hyaluronidase is secreted by pneumococci and breaks down the hyaluronic acid component of host connective tissue and extracellular matrix . increased epithelial permeability caused by the action of hyaluronidase favours the spread and colonisation of s. pneumoniae , especially when acting in concert with pneumolysin . the pneumococcal protein toxin , pneumolysin , is a member of the family of thiol - activated cytolysins and a critical virulence factor of the pathogen . the toxin binds to cholesterol in the cytoplasmic membrane of eukaryotic cells , followed by insertion into the membrane , leading to the formation of large pores and cytolysis [ 83 , 84 ] . in the early stages of infection in addition , the toxin has been shown to disrupt tight junctions thereby disrupting the integrity of the epithelial monolayer favouring invasiveness of the pathogen . at high , cytotoxic concentrations , pneumolysin may also inhibit the protective functions of cells of both the innate and adaptive immune systems , as well as maturation of dendritic cells . however , at lower noncytolytic concentrations , the toxin possesses proinflammatory activity as a consequence of sublytic pore formation and influx of ca into immune and inflammatory cells . this , in turn , causes hyperactivation of phagocytes , induction of proinflammatory cytokine production , and activation of the inflammasome , all of which are potentiated by the complement - activating properties of the toxin ( as described below ) . during pneumococcal cap , a high bacillary load , aggravated by the implementation of antimicrobial chemotherapy with bactericidal agents which promote disintegration of the pathogen , results in excessive release of pro - inflammatory bacterial cell - wall products , toxins , and dna . the consequence of these events is hyperactivation of host defence mechanisms , posing the potential threat of inflammation - mediated pulmonary damage and extrapulmonary spread of the pneumococcus . the major contributors to overexuberant inflammatory responses include ( i ) the pro - inflammatory , pore - forming interactions of pneumolysin with neutrophils , macrophages , and epithelial cells , potentiated by complement - activating properties of the toxin ; ( ii ) the interactions of pneumolysin , lipoteichoic acid , proteoglycan , and dna with prrs , especially on cells of the innate immune system and epithelial cells ; ( iii ) inappropriate induction of neutrophil extracellular trap ( net ) formation by pneumococcal -enolase and possibly pneumolysin and h2o2 ; and ( iv ) possible inappropriate oxidative activation of redox signalling mechanisms in immune and inflammatory cells by pneumococcal h2o2 . in addition to directly causing acute lung injury as a consequence of its cytotoxic effects on airway epithelium and endothelium , pneumolysin , via its complement - activating activities and sub - lytic , pore - forming interactions with neutrophils and macrophages , also potentiates the release of reactive oxygen species ( ros ) , granule proteases , leukotriene b4 , and prostaglandin e2 by promoting the movement of extracellular ca into the cells [ 137 , 138 ] . as a result of ca influx , several intracellular signalling these involve p38 and mitogen - activated protein kinases , transforming growth factor--activated kinase 1-mitogen - protein kinase 3/6-p38 / , ca - calcineurin , nuclear factor kappa b ( nfb ) , and activator protein 1 ( ap-1 ) [ 139142 ] . the consequence is increased production of il-8 and tnf , both of which promote neutrophil influx into the airways [ 140143 ] . in addition , pneumolysin also activates the nlrp3 inflammasome in dendritic cells , and presumably other immune and inflammatory cell types , thereby potentiating caspase-1-mediated conversion of pro - il-1 to the mature cytokine . the aforementioned direct cytolytic actions of pneumolysin , acting in concert with the indirect proinflammatory activities of the toxin , promote the epithelial / endothelial damage which favours dissemination of the pneumococcus . the interactions of the pneumococcus with the various prrs expressed by cells of the innate immune system , as well as epithelial cells , have been the subject of a recent review . pneumococcal cell wall components , pneumolysin , and dna have all been reported to interact with , and activate , several different types of prrs . in the case of the toll - like receptors ( tlrs ) , lipoteichoic acid , and , possibly proteoglycan , are tlr-2 ligands , pneumolysin has been reported to interact with and activate tlr-4 , while pneumococcal cpg - motif - containing dna is detected by tlr-9 . in each case , triggering of tlrs is accompanied by activation of nfb and synthesis of proinflammatory chemokines / cytokines , especially il-8 and tnf . in addition , pneumococcal proteoglycans are recognised by and activate the nucleotide oligomerisation domain - like receptor , nod2 , while microbial dna is detected by the abundant cytosolic sensors of pathogen - derived nucleic acid [ 146 , 147 ] . the consequence of these events is activation of nfb , as well as the interferon regulatory transcription factors 3 and 7 ( irf 3/7 ) . net formation is a postactivation strategy used mainly by dead and dying neutrophils and several other cell types including monocytes / macrophages and eosinophils , to isolate and kill microbial pathogens by trapping them in an extracellular matrix of citrullinated histones impregnated with antimicrobial granule proteins . in the case of the pneumococcus , pathogen - derived -enolase has been reported to induce net formation following exposure of neutrophils to the pathogen . although unproven , it seems probable that pneumolysin and pathogen - derived h2o2 are also potential inducers of net formation . however , as opposed to being an effective antipneumococcal host defence mechanism , the pneumococcus appears to be particularly adept at escaping from nets , apparently as a consequence of production of the endonuclease , end a , which mediates degradation of nets . in addition to subverting nets , the pneumococcus may also exploit poorly regulated net formation as a strategy to promote invasion and dissemination due to epithelial and endothelial cytotoxicity mediated by the histone components of nets . while its role in microbial virulence is well established , the involvement of pneumococcus - derived h2o2 in activating harmful inflammatory responses during cap and other infections remains to be established . this seems likely , however , as cell - permeable h2o2 is a potent activator of redox intracellular signalling mechanisms in many cell types , including those of the innate and adaptive immune systems [ 152 , 153 ] . these various mechanisms of hyperactivation of inflammatory responses operative during pneumococcal cap are summarised in table 2 . the primary objective of adjunctive pharmacological strategies in severe cap , especially severe pneumococcal disease , is to suppress overexuberant , harmful , pathogen - activated inflammatory responses , thereby attenuating inflammation - mediated pulmonary damage and dysfunction . in this setting , the three categories of anti - inflammatory agents which have attracted the greatest interest are macrolides , corticosteroids , and , more recently , statins [ 31 , 32 , 3439 ] . other categories of anti - inflammatory agent which remain largely untested , include the various types of 3-5-cyclic adenosine monophosphate- ( camp- ) elevating agents , as well as non - steroidal anti - inflammatory agents ( nsaids ) . despite the absence of irrefutable proof of efficacy , the inclusion of macrolides in the current guideline recommendations for the therapy of cap can be justified on several grounds . notwithstanding their primary antimicrobial activities , which complement those of -lactams in the treatment of cap , macrolides possess well - recognised anti - inflammatory properties . these are both pathogen- and host - directed and have recently been reviewed in detail elsewhere [ 34 , 35 ] . briefly , the pathogen - targeted anti - inflammatory activity of macrolides is achieved via the inhibitory effects of these agents on bacterial protein synthesis , thereby attenuating the production of pro - inflammatory toxins , such as pneumolysin in the case of the pneumococcus [ 35 , 154 ] . in addition , abrupt bacteriolysis and accompanying excessive inflammation due to release of cell - wall components and endotoxins , as may occur with bactericidal antibiotics , are also countered by the predominantly bacteriostatic activity of macrolides . the primary target of the secondary anti - inflammatory properties of macrolides , unrelated to antimicrobial activity , is neutrophil recruitment . macrolide - mediated inhibition of neutrophil mobilisation is achieved predominantly via inhibition of production of the neutrophil - mobilising cytokines / chemokines il-8 , il-17 , and tnf , not only by cells of the innate immune system , but also by various types of structural cells [ 35 , 155 ] . although not fully understood , these inhibitory effects of macrolides on the synthesis of pro - inflammatory cytokines / chemokines appear to be achieved at the level of gene transcription . this results from antagonism of transcription factors such as nuclear factor kappa b ( nfb ) , possibly via ( i ) interference with redox signalling mechanisms ; and ( ii ) enhancement of histone deacetylase activity [ 35 , 156158 ] . the beneficial therapeutic activities of these various pathogen- and host - directed anti - inflammatory activities of macrolides are evident in several chronic inflammatory diseases of the airways , particularly bronchiolitis obliterans , diffuse panbronchiolitis , and cystic fibrosis , and possibly chronic obstructive pulmonary disease ( reviewed in ) . although difficult to prove conclusively , several lines of evidence , both clinical and experimental , also support the involvement of the anti - inflammatory activities of macrolides in controlling severe sepsis and/or inflammation - mediated lung injury in acute bacterial infection . the first of these reported that the use of macrolides was associated with decreased mortality in patients with pneumonia and severe sepsis , even in patients infected with macrolide - resistant pathogens , such as gram - negative organisms . the second study , which was undertaken in patients with predominantly macrolide - resistant gram - negative sepsis and ventilator - associated pneumonia ( vap ) , reported that intravenous administration of clarithromycin ( 1 gram / daily ) for 3 days resulted in accelerated resolution of vap and earlier discontinuation of mechanical ventilation , as well as delaying , but not preventing , mortality . more recently , administration of a macrolide within 24 hours of trial entry , but not -lactam or fluoroquinolone antibiotics , to patients with acute lung injury secondary to pneumonia in most cases , was associated with significant decreases in both 180 day mortality and time to successful discontinuation of mechanical ventilation . the beneficial anti - inflammatory activities of macrolides and macrolide - like antimicrobial agents have also been demonstrated in various animal models of experimental chemotherapy of acute bacterial infection ( reviewed in ) . in one such study reported by karlstrm et al . , using a murine model of pneumococcal pneumonia secondary to influenza virus infection , treatment of animals with either azithromycin or clindamycin alone or in combination with ampicillin resulted in significantly improved survival compared to animals treated with ampicillin only . these beneficial effects of azithromycin / clindamycin were associated with decreased concentrations of airway pro - inflammatory cytokines and influx of inflammatory cells in the setting of less severe histopathological changes . because macrolides combine pathogen- and host - directed anti - inflammatory activities , therapy with these agents appears to be an ideal adjunctive strategy in severe cap , possibly in a subset of patients at risk for development of ali . nonetheless , widespread acceptance of an adjunctive role for macrolides in this clinical setting is dependent on the acquisition of compelling data from prospective , randomised , controlled clinical trials [ 3639 , 162 , 163 ] . corticosteroids ( cs ) are broad - spectrum anti - inflammatory agents , but unlike macrolides , they are less effective in targeting neutrophils . although an adjunctive role for systemic cs in the clinical management of adults with penicillin - susceptible pneumococcal meningitis is well recognised , their role in the adjunctive therapy of severe cap remains unproven . several relatively small prospective / retrospective trials conducted between 2005 and 2007 in hospitalised patients with severe cap , receiving systemic cs at doses varying from 40 to 200 mg / daily for periods of 3 days and longer , reported beneficial effects of these agents on , amongst others , duration of hospital stay , and mortality [ 166168 ] . however , this adjunctive promise of systemic cs was not confirmed in several more recent studies . in a large randomised , double - blinded , placebo - controlled trial , snijders et al . failed to detect beneficial effects of systemic administration of prednisone ( 40 mg / daily for 7 days ) on outcome in patients with cap , with the frequency of late failure ( > 72 hours after hospital admission ) being significantly ( p < 0.04 ) more common in cs - treated patients . , in a retrospective study covering an approximately 10.5 year period involving 3257 patients who received a mean systemic cs daily dose of 45 mg for varying periods of time , reported that administration of cs did not influence either clinical stability or mortality . somewhat worryingly , however , administration of cs significantly prolonged hospital stay ( 9 versus 6 days , p < 0.01 ) . in contrast , meijvis et al . reported that systemic administration of dexamethasone , at the comparatively low dose of 5 mg / daily for 4 days from the time of admission , to nonimmunocompromised patients who did not require icu admission , was associated with a significant decrease in length of hospital stay ( 7.5 versus 6.5 days p < 0.048 ) . these observations suggest that the dose of the systemic cs and the immune status of the patient are potential determinants of the success of adjunctive therapy with cs in patients with cap . in addition , the type of pathogen may also be a determinant of successful outcome of cs therapy , with infections caused by atypical pathogens , as opposed to those caused by s. pneumoniae , being more responsive to the beneficial actions of cs . however , as concluded in a recent meta - analysis , conclusive proof of the role , if any , of systemic cs in the adjunctive therapy of severe cap is dependent on the acquisition of compelling data from adequately powered randomised trials . three such studies are ongoing , one in the usa ( extended steroids in cape - escape , projected completion date january 2017 ) and two in europe . statins is the collective term for a group of pharmacological inhibitors of the enzyme , 3-hydroxy-3-methylglutaryl coenzyme a reductase , used to control hypercholesterolemia in the prevention of cardiovascular diseases and stroke . in addition to their cholesterol - lowering properties , statins have also been reported to possess significant anti - inflammatory activities which have been attributed to two major mechanisms . firstly , interference with the prenylation of the small g - proteins rac , ras , and rho , thereby attenuating g - protein - coupled receptor cellular signalling and activation in a variety of cell types , including immune and inflammatory cells [ 175177 ] . the consequence is decreased activation of nfb and resultant interference with the transcription of genes encoding various proinflammatory proteins such as inducible nitric oxide synthase , cyclooxygenase-2 and matrix metalloproteinase-9 [ 176 , 178 ] . secondly , via induction of heme oxygenase-1 expression , also resulting in attenuation of activation of nfb , as well as decreased production of ros in the setting of increased production of anti - inflammatory il-10 [ 178 , 179 ] . interestingly , a number of predominantly retrospective studies conducted between 2005 and 2011 have reported that statin use in the prevention of cardiovascular diseases is associated with improved outcome of patients with cap , possibly as a consequence of the anti - inflammatory activities of these agents . in the majority of these studies , which have recently been reviewed by corrales - medina and musher , in a retrospective study of patients ( n = 347 ) with pneumococcal pneumonia , who presented at a single medical center between 2000 and 2010 , reported that statin use , as opposed to administration of macrolides , was associated with decreased mortality 4 , 14 , 20 , and 30 days after admission . these findings suggest a mechanism in addition to those mentioned above by which statins may protect against invasive pneumococcal diseases . by decreasing plasma membrane concentrations of cholesterol in epithelial , endothelial , and immune / inflammatory cells , statins may restrict the binding of pneumolysin , thereby attenuating both the cytotoxic and pro - inflammatory activities of the toxin . although of considerable potential importance , the aforementioned studies have several significant limitations : ( i ) they do not address the adjunctive potential of statins administered at the time of presentation with cap ; ( ii ) concomitant use of statins may obscure the therapeutic potential of macrolides and cs ; and ( iii ) statin use may be associated with a healthy user effect , distinguishing a subgroup of patients who are likely to have a better outcome . once again , large randomised , prospective , controlled clinical trials are necessary to evaluate the use of statins as a potential adjunctive therapy in cap [ 36 , 39 ] . other largely untested adjunctive therapies in the clinical setting of cap include ( i ) pharmacological agents which increase intracellular concentrations of the cyclic nucleotide , camp , in immune and inflammatory cells and ( ii ) nsaids . the master regulator of innate immune cell function ; consequently pharmacological agents which increase the intracellular concentrations of this cyclic nucleotide possess broad - spectrum anti - inflammatory activities . agents falling into this category include camp - specific and nonspecific phosphodiesterase inhibitors , as well as agonists of 2-adrenoreceptors , adenosine a2a receptors , and the e - type prostaglandin receptors , ep2 and ep4 . although the potential of these agents is untested in cap , it is noteworthy that the 2-adrenoreceptor agonist , salbutamol , administered either intravenously or as an aerosol , has proved to be ineffective in the treatment of ali [ 181 , 182 ] . with respect to anti - inflammatory activity , salbutamol may not , however , be the most effective 2-agonist to use in this clinical setting . administration of the nsaid , naproxen , to healthy individuals has been reported to augment the bactericidal action of whole blood against a penicillin - resistant strain of s. pneumoniae ex vivo . these potentiating effects of the nsaid were associated with increased phagocytic activity of blood phagocytes , as well as increased generation of antimicrobial ros by these cells , due , presumably , to inhibition of production of immunosuppressive prostaglandin e2 and activation of adenylyl cyclase via ep2/ep4 receptors . somewhat paradoxically , however , these antipneumococcal actions of nsaids may predispose to inflammation - mediated tissue damage via interference with camp - mediated immunoregulatory activity . these and other types of adjunctive therapies , largely unsuccessful , have also been the subject of several recent reviews and include ( i ) intravenous gammaglobulin ( of unproven benefit ) ; ( ii ) monoclonal antibodies targeted against the il-1 or paf receptors , as well as tnf ; ( iii ) recombinant human activated protein c ( drotrecogin alfa ) ; and ( iv ) the recombinant tissue factor pathway inhibitor , tifacogin [ 34 , 162 ] . it is quite evident from a review of the scientific literature that cap , and in particular infection due to streptococcus pneumoniae , carries a considerable burden of disease among the world 's population . the pneumococcus is the most common microbial cause of cap , not only in mild infections , but also among patients requiring hospitalisation and even among critically ill cases . the reason that pneumococcal infection and cap remain so common throughout the world relates to the high prevalence of risk factors for this infection in the general population , which includes aging , lifestyle factors , and underlying comorbid illnesses and , at least in some parts of the world , concomitant hiv infection . pneumococcal pneumonia causes considerable morbidity and mortality and treatment of these infections is potentially being compromised by the emergence of resistance in this microorganism to the commonly used antibiotics . the pneumococcus expresses a large number of virulence factors , which not only render the microorganism very effective in causing infection , but also contribute to disease pathogenesis via their cytotoxic and proinflammatory activities . targeting these virulence factors additionally as part of overall therapy has the potential for improving the outcome of such infections .

community - acquired pneumonia ( cap ) remains a leading cause of morbidity and mortality among the infectious diseases . despite the implementation of national pneumococcal polyvalent vaccine - based immunisation strategies targeted at high - risk groups , streptococcus pneumoniae ( the pneumococcus ) remains the most common cause of cap . notwithstanding the hiv pandemic , major challenges confronting the control of cap include the range of bacterial and viral pathogens causing this condition , the ever - increasing problem of antibiotic resistance worldwide , and increased vulnerability associated with steadily aging populations in developed countries . these and other risk factors , as well as diagnostic strategies , are covered in the first section of this review . thereafter , the review is focused on the pneumococcus , specifically the major virulence factors of this microbial pathogen and their role in triggering overexuberant inflammatory responses which contribute to the immunopathogenesis of invasive disease . the final section of the review is devoted to a consideration of pharmacological , anti - inflammatory strategies with adjunctive potential in the antimicrobial chemotherapy of cap . this is focused on macrolides , corticosteroids , and statins with respect to their modes of anti - inflammatory action , current status , and limitations .

1. Overview of Community-Acquired Pneumonia 2. Virulence Determinants of 3. Harmful Effects of Excessive Activation of Antipneumococcal Host Defences 4. Adjunctive Anti-Inflammatory Therapeutic Strategies in Severe CAP 5. Conclusion

community - acquired pneumonia ( cap ) is commonly described as an acute infection of the lung parenchyma acquired in the community . as part of the burden of respiratory infections , cap is well recognised to be a leading cause of death among the infectious diseases [ 6 , 8 ] . while a myriad of microorganisms may cause cap , in reality a relatively small number of pathogens predominate , in particular the bacteria , of which streptococcus pneumoniae ( pneumococcus ) is by far the most common . there is considerable concern about the emerging resistance among the usual cap pathogens to the most commonly used antimicrobial agents . the remainder of this introductory overview of cap will focus on ( i ) burden of disease , ( ii ) risk factors , ( iii ) microbiology , ( iv ) antimicrobial chemotherapy , ( v ) antibiotic resistance , and ( vi ) assessment of severity of illness using clinical scoring systems and laboratory biomarkers individually and in combination . cap continues to be a cause of considerable morbidity and mortality in most parts of the world , being the most frequent infectious cause of death in patients in the usa and throughout europe . since aging is a significant risk factor for this infection and given that in many areas of the world , such as europe , the population is aging , an increase in incidence in the next decades is anticipated . studies in the usa , europe , latin america , and the asia - pacific region attest to the fact that cap has a substantial clinical and economic burden , a high rate of antibiotic resistance among the pathogens , and a significant effect on both immediate and long - term prognosis , as well as on the quality of life of infected patients . as shown in the list below , there is a considerable number of risk factors for cap that exist in populations all over the world , and most of these risk factors are associated with an impairment of the efficacy of host immune defence . in addition to aging , the common risk factors in adults are smoking , the presence of various underlying comorbid conditions , including chronic cardiorespiratory , renal and hepatic conditions , and , at least in some regions of the world , concomitant human immunodeficiency virus ( hiv ) infection . risk factors for community - acquired pneumonia are as follows : extremes of age ( very young and the aging),male gender , certain populations ( various racial or ethnic groups),lifestyle factors ( excessive alcohol consumption and smoking),underlying comorbid conditions such aschronic cardiorespiratory illnesses , chronic renal disorders , hepatic conditions , diabetes mellitus , neoplastic diseases , human immunodeficiency virus infection , medications ( e.g. , inhaled corticosteroids , proton pump inhibitors ) , additional risk factors associated with pneumococcal infections in particular ( e.g. smoking , both active and passive ( particularly in children ) , is a well - described risk factor for cap , particularly in hiv - infected persons , as well as for many other infectious diseases , and this has been comprehensively reviewed recently . the main mechanisms for this predisposition relate to the suppressive effect that smoking has on the protective actions of the airway mucociliary clearance mechanism , on the various components of the innate and adaptive immune systems of the host , as well as direct effects on microbial pathogens that promote their virulence , and possibly antibiotic resistance . several comorbid factors relate quite closely to the risk of cap and the possibility of more severe illness , as well as to the likelihood of a worse outcome . among the most common comorbid predisposing factors to cap , as mentioned above , are chronic obstructive pulmonary disease , congestive cardiac failure , diabetes mellitus , a high intake of alcohol , and smoking . however , various other conditions , including those of the neurological system , the liver , the kidney , and also neoplastic disease , represent important risk factors . the spectrum of bacterial pathogens causing cap in hiv - infected patients is very similar to that in hiv - uninfected patients with streptococcus pneumoniae ( pneumococcus ) predominating . recent studies have identified several mechanisms , specifically the presence of gene polymorphisms , including single nucleotide polymorphisms ( snps ) in genes encoding proteins of the innate immune system , which contribute not only to susceptibility for development of cap , but also to a worse outcome . for example , snps in the il-6 gene , specifically il-6 174 g / g , have been reported to protect patients with pneumococcal cap against development of ards , septic shock , and multiple organ dysfunction syndrome , resulting in less severe disease and lower mortality . in addition , investigation of the role of snps in the genes encoding the surfactant proteins ( sp ) a , b , c , and d revealed associations with both susceptibility for both development of cap and more severe disease [ 16 , 17 ] . the most prominent organism causing cap is the pneumococcus , and this remains true irrespective of the severity of infection across the spectrum of outpatients , inpatients not in the intensive care unit ( icu ) , and even cases with cap requiring icu admission [ 11 , 18 ] . studies in europe , the usa , latin america , the asia - pacific region , and elsewhere attest to the fact that the pneumococcus is consistently documented to be the most predominant pathogen [ 25 , 9 ] . in the former two studies , the pneumococcus was the most common bacterial cause of bacterial co - infection , accounting for 62% and 54.8% of cases , respectively , and being associated with a greater risk of septic shock or need for vasopressors , as well as increased need for mechanical ventilation and a longer icu stay [ 20 , 21 ] . many recent studies of pneumococcal infection have focussed on the issue of pneumococcal serotypes causing disease , and the possible association of different serotypes with disease severity , particularly in relation to the release of the newer pneumococcal conjugate vaccines and the extended indication for their use in adults [ 2225 ] . after the pneumococcus , the next most common pathogens are the so - called atypical pathogens , the respiratory viruses , and haemophilus influenzae . on presentation of adults with suspected cap , empiric antimicrobial chemotherapy is initiated according to the relevant national guidelines , with age , comorbidities , and disease severity being the primary determinants of the class of antibiotic(s ) and route of administration . in the case of outpatient therapy , previously healthy patients who had not received antibiotics during the 3-month period prior to presentation , monotherapy with a macrolide or doxycycline is recommended by the infectious diseases society of america ( idsa)/american thoracic society ( ats ) ; in those with comorbidities and/or prior recent use of antibiotics , the recommendation is the combination of an antipneumococcal -lactam and a macrolide , or alternatively , a respiratory fluoroquinolone . although the microbiological mechanisms underpinning the apparent benefit of combination antibiotic therapy remain unknown , activity of macrolides / fluoroquinolones / tetracyclines against the atypical pathogens chlamydia pneumoniae , legionella pneumophila , and mycoplasma pneumoniae , as well as efficacy in eradicating polymicrobial sepsis , have been proposed . clearly , the issue of optimum antimicrobial chemotherapy of cap remains to be resolved and will be dependent on the acquisition of data from large , well - controlled multicentre , randomised clinical trials [ 3739 ] . there is a considerable body of literature devoted to the issue of antimicrobial resistance among the common pathogens causing cap , especially with regard to the pneumococcus [ 4144 ] . while it is clear that there is emerging resistance among the cap pathogens , and particularly the pneumococcus , it remains unclear whether the presence of antimicrobial resistance alone is associated with unfavourable treatment outcomes . there have been a number of studies and various reviews discussing the issue of antibiotic resistance in the management and outcome of pneumococcal cap [ 41 , 4549 ] . for this reason , it has been recommended that awareness of pneumococcal macrolide resistance levels and patterns in a given region , as well as the risk factors in individual patients for macrolide resistance , clearly determine the utility of macrolide monotherapy in the management of pneumococcal cap . severity of illness determines the site of care ( in- or outpatient ) , the extent of the microbiological workup , and the choice of initial empiric antimicrobial therapy . the psi uses 20 variables which include patient age , gender , presence or absence of comorbid conditions , and/or vital sign abnormalities , as well as various laboratory and radiographic parameters [ 6 , 54 ] . with both scoring systems , cases can be stratified into low- , moderate- , or high - risk groups . although many predisposing mechanisms have been documented , the presence of cardiovascular disease and other comorbid conditions , including hiv infection , the subsequent documentation of primary or secondary neoplasms , and alterations in immune function predominate . the remaining sections of this review are focused on ( i ) the major virulence determinants of the pneumococcus , the most common cause of cap , and their involvement in triggering harmful inflammatory responses [ 70113 ] and ( ii ) control of these using pharmacological , anti - inflammatory strategies . in the case of innate immune defences , these include the cough reflex , the mucociliary escalator , and a range of pattern recognition receptors ( prrs ) . cell - mediated resolution of nasopharyngeal colonisation involves both th1 and th17 responses [ 116 , 117 ] triggered by a variety of surface virulence determinants such as the bacterial capsule , pili , and other adhesins , as well as the toxin , pneumolysin . relative to the opaque variant , the transparent phenotype has decreased capsular thickness and expresses less pneumococcal surface protein a ( pspa ) in the setting of a higher level of expression of the major adhesin , choline - binding protein a ( cbpa ) , and the autolysin , lyta , favouring adhesion and colonisation . capsular serotypes differ with respect to invasiveness , due mainly to differences in complement deposition on the capsular polysaccharide , as well as binding of complement factor h . the pneumococcus not only utilises h2o2 as a virulence factor , causing significant damage to ciliated respiratory epithelium and impaired protective activity of the mucociliary escalator , but also to eliminate microbial competitors in the nasopharynx . nanb is involved in the metabolic utilisation of sialic acid as a carbon and energy source by the pneumococcus , while nanc has a regulatory role [ 131 , 133 ] . in the early stages of infection in addition , the toxin has been shown to disrupt tight junctions thereby disrupting the integrity of the epithelial monolayer favouring invasiveness of the pathogen . at high , cytotoxic concentrations , pneumolysin may also inhibit the protective functions of cells of both the innate and adaptive immune systems , as well as maturation of dendritic cells . during pneumococcal cap , a high bacillary load , aggravated by the implementation of antimicrobial chemotherapy with bactericidal agents which promote disintegration of the pathogen , results in excessive release of pro - inflammatory bacterial cell - wall products , toxins , and dna . the major contributors to overexuberant inflammatory responses include ( i ) the pro - inflammatory , pore - forming interactions of pneumolysin with neutrophils , macrophages , and epithelial cells , potentiated by complement - activating properties of the toxin ; ( ii ) the interactions of pneumolysin , lipoteichoic acid , proteoglycan , and dna with prrs , especially on cells of the innate immune system and epithelial cells ; ( iii ) inappropriate induction of neutrophil extracellular trap ( net ) formation by pneumococcal -enolase and possibly pneumolysin and h2o2 ; and ( iv ) possible inappropriate oxidative activation of redox signalling mechanisms in immune and inflammatory cells by pneumococcal h2o2 . the interactions of the pneumococcus with the various prrs expressed by cells of the innate immune system , as well as epithelial cells , have been the subject of a recent review . in the case of the toll - like receptors ( tlrs ) , lipoteichoic acid , and , possibly proteoglycan , are tlr-2 ligands , pneumolysin has been reported to interact with and activate tlr-4 , while pneumococcal cpg - motif - containing dna is detected by tlr-9 . in the case of the pneumococcus , pathogen - derived -enolase has been reported to induce net formation following exposure of neutrophils to the pathogen . however , as opposed to being an effective antipneumococcal host defence mechanism , the pneumococcus appears to be particularly adept at escaping from nets , apparently as a consequence of production of the endonuclease , end a , which mediates degradation of nets . while its role in microbial virulence is well established , the involvement of pneumococcus - derived h2o2 in activating harmful inflammatory responses during cap and other infections remains to be established . in this setting , the three categories of anti - inflammatory agents which have attracted the greatest interest are macrolides , corticosteroids , and , more recently , statins [ 31 , 32 , 3439 ] . other categories of anti - inflammatory agent which remain largely untested , include the various types of 3-5-cyclic adenosine monophosphate- ( camp- ) elevating agents , as well as non - steroidal anti - inflammatory agents ( nsaids ) . despite the absence of irrefutable proof of efficacy , the inclusion of macrolides in the current guideline recommendations for the therapy of cap can be justified on several grounds . notwithstanding their primary antimicrobial activities , which complement those of -lactams in the treatment of cap , macrolides possess well - recognised anti - inflammatory properties . briefly , the pathogen - targeted anti - inflammatory activity of macrolides is achieved via the inhibitory effects of these agents on bacterial protein synthesis , thereby attenuating the production of pro - inflammatory toxins , such as pneumolysin in the case of the pneumococcus [ 35 , 154 ] . the primary target of the secondary anti - inflammatory properties of macrolides , unrelated to antimicrobial activity , is neutrophil recruitment . the beneficial therapeutic activities of these various pathogen- and host - directed anti - inflammatory activities of macrolides are evident in several chronic inflammatory diseases of the airways , particularly bronchiolitis obliterans , diffuse panbronchiolitis , and cystic fibrosis , and possibly chronic obstructive pulmonary disease ( reviewed in ) . although difficult to prove conclusively , several lines of evidence , both clinical and experimental , also support the involvement of the anti - inflammatory activities of macrolides in controlling severe sepsis and/or inflammation - mediated lung injury in acute bacterial infection . the second study , which was undertaken in patients with predominantly macrolide - resistant gram - negative sepsis and ventilator - associated pneumonia ( vap ) , reported that intravenous administration of clarithromycin ( 1 gram / daily ) for 3 days resulted in accelerated resolution of vap and earlier discontinuation of mechanical ventilation , as well as delaying , but not preventing , mortality . however , as concluded in a recent meta - analysis , conclusive proof of the role , if any , of systemic cs in the adjunctive therapy of severe cap is dependent on the acquisition of compelling data from adequately powered randomised trials . statins is the collective term for a group of pharmacological inhibitors of the enzyme , 3-hydroxy-3-methylglutaryl coenzyme a reductase , used to control hypercholesterolemia in the prevention of cardiovascular diseases and stroke . secondly , via induction of heme oxygenase-1 expression , also resulting in attenuation of activation of nfb , as well as decreased production of ros in the setting of increased production of anti - inflammatory il-10 [ 178 , 179 ] . interestingly , a number of predominantly retrospective studies conducted between 2005 and 2011 have reported that statin use in the prevention of cardiovascular diseases is associated with improved outcome of patients with cap , possibly as a consequence of the anti - inflammatory activities of these agents . in the majority of these studies , which have recently been reviewed by corrales - medina and musher , in a retrospective study of patients ( n = 347 ) with pneumococcal pneumonia , who presented at a single medical center between 2000 and 2010 , reported that statin use , as opposed to administration of macrolides , was associated with decreased mortality 4 , 14 , 20 , and 30 days after admission . although of considerable potential importance , the aforementioned studies have several significant limitations : ( i ) they do not address the adjunctive potential of statins administered at the time of presentation with cap ; ( ii ) concomitant use of statins may obscure the therapeutic potential of macrolides and cs ; and ( iii ) statin use may be associated with a healthy user effect , distinguishing a subgroup of patients who are likely to have a better outcome . other largely untested adjunctive therapies in the clinical setting of cap include ( i ) pharmacological agents which increase intracellular concentrations of the cyclic nucleotide , camp , in immune and inflammatory cells and ( ii ) nsaids . agents falling into this category include camp - specific and nonspecific phosphodiesterase inhibitors , as well as agonists of 2-adrenoreceptors , adenosine a2a receptors , and the e - type prostaglandin receptors , ep2 and ep4 . with respect to anti - inflammatory activity , salbutamol may not , however , be the most effective 2-agonist to use in this clinical setting . these potentiating effects of the nsaid were associated with increased phagocytic activity of blood phagocytes , as well as increased generation of antimicrobial ros by these cells , due , presumably , to inhibition of production of immunosuppressive prostaglandin e2 and activation of adenylyl cyclase via ep2/ep4 receptors . these and other types of adjunctive therapies , largely unsuccessful , have also been the subject of several recent reviews and include ( i ) intravenous gammaglobulin ( of unproven benefit ) ; ( ii ) monoclonal antibodies targeted against the il-1 or paf receptors , as well as tnf ; ( iii ) recombinant human activated protein c ( drotrecogin alfa ) ; and ( iv ) the recombinant tissue factor pathway inhibitor , tifacogin [ 34 , 162 ] . it is quite evident from a review of the scientific literature that cap , and in particular infection due to streptococcus pneumoniae , carries a considerable burden of disease among the world 's population . the pneumococcus is the most common microbial cause of cap , not only in mild infections , but also among patients requiring hospitalisation and even among critically ill cases . the reason that pneumococcal infection and cap remain so common throughout the world relates to the high prevalence of risk factors for this infection in the general population , which includes aging , lifestyle factors , and underlying comorbid illnesses and , at least in some parts of the world , concomitant hiv infection . the pneumococcus expresses a large number of virulence factors , which not only render the microorganism very effective in causing infection , but also contribute to disease pathogenesis via their cytotoxic and proinflammatory activities .

one prediction of the accordion model is that deletions in the tre could prevent the 3 template nucleotides from reaching the active site , so that primers paired in the alignment region ( nucleotides 4951 ) would give no reaction ( no reaction , fig . primers with sequences that aligned within the template might be able to reach the active site , especially with smaller tre deletions , but after one round of extension they would pair in the alignment region and undergo no further reaction . in general , the activities of different primers should reveal which template nucleotides are able to reach the active site in each mutant telomerase . to test this hypothesis each mutant ter was assembled into a telomerase rnp and tested in telomerase activity assays with primers representing each of the six permutations of telomeric sequence ( fig . for all double - nucleotide deletion mutants , rap was reduced compared to the activity of wild - type ( wt ) telomerase ( supplementary fig . 1b ) . deleting three sequential nucleotides had a dramatic effect , allowing robust addition of one repeat but very little rap ( fig . primer b , which anneals to the alignment region of the template , was essentially inactive even for the first round of extension , and primer c showed reduced activity . this primer usage profile was independent of the location of the deletion within the tre , suggesting that sequence is not as important as length . a similar observation was made for mutants with four sequential nucleotides deleted , but now primer c was also inactive ( fig . 2e ) , suggesting that in all four - nucleotide deletion mutants , template nucleotide 47 can no longer reach the active site . deletion of six nucleotides ( 5459 ) continued the trend , preventing ter nucleotide 46 from being used as template . the largest deletion tested ( 5259 ) was only able to efficiently use primer a , which base pairs with the penultimate nucleotide of the template ( fig . this eight - nucleotide deletion presumably acts as a molecular ruler , measuring the minimum distance between the pseudoknot and c43 in the active site . a second prediction of the accordion model is that flexibility in the single - stranded rna portion of the tbe , which is 5 of the template , is also required for template progression . insertions of nucleotides adjacent to the tbe are predicted to reinforce the misalignment of the template relative to the active site in the context of tre deletion mutants . we modified two tre deletion mutants ( 5659 , a four - nucleotide deletion , and 5459 , a six - nucleotide deletion ) by inserting two uridines between nucleotides 40 and 41 of the tbe , just 5 of the template ( fig . 3 ) . while the four - nucleotide tre deletion had only a modest ability to use primer d , the combination of the insertion of 2us in the tbe and the four - nucleotide tre deletion exacerbated this effect , resulting in a primer usage profile similar to that of the six - nucleotide tre deletion mutant . similarly , the six - nucleotide deletion mutant containing an insertion of two uridines 5 of the template had a weakened ability to use primer e , matching the primer usage profile of the eight - nucleotide deletion mutant . previous experiments with circularly permuted ( cp ) telomerase rnas demonstrated that discontinuities on either side of the template decreased rap . we interpret these results as showing that coordinated structural rearrangements on both sides of the template contribute to template movement . the previous experiments were performed in the absence of p65 , the telomerase - specific rna folding protein that is essential in vivo and has the ability to rescue many severely destabilizing tert and ter mutations in vitro . if nicked rnas were defective simply because of structural destabilization , they might likewise be suppressed in telomerase containing p65 . we therefore tested the activities of cprnas assembled into telomerase rnps in both the presence and absence of p65 ( supplementary fig . 2 ) . because the tbe is necessary for tert binding and proper telomerase function , we tested four circularly permuted rnas ( cprna ) within this region , and one in the tre . telomerase rnps containing the cprnas had essentially full first - round activity , but thereafter rap was severely reduced , with nicks in the tbe affecting the reaction similarly to the nick at position 59 in the tre . this limited rap even in the presence of p65 provides further support for the requirement for direct connectivity of the rna on both sides of the template . taken together , these biochemical experiments demonstrate that specific deletions and insertions , as well as the introduction of nicks , in the rna elements flanking the template evoke catalytic defects predicted by the accordion model . however , these biochemical assays can not directly probe rna structure ; therefore single - molecule frster resonance energy transfer ( smfret ) was employed to monitor rna conformational changes within individual telomerase - dna primer complexes at successive steps in the telomerase catalytic cycle ( fig . 3 ) . ideally , smfret experiments performed in the presence of dttp and dgtp could permit real - time observation of ter structural rearrangements throughout the telomerase catalytic cycle . to date , however , fret - based monitoring of telomerase activity has remained elusive . therefore , to simplify interpretation of our data , independent smfret experiments were performed with primers successively extended on the 3 end in the absence of dgtp and dttp . in this way , the primers used in separate experiments emulate the different conformational states that would be sampled during telomerase - catalyzed dna synthesis . surface - immobilized telomeric dna primers coupled to a donor dye ( cy3 ) were monitored in the presence of reconstituted telomerase enzymes harboring an acceptor dye ( cy5 ) at u63 in the tre ( fig . importantly , these modified dna primers and telomerase rnas support near wild - type levels of telomerase activity ( supplementary fig . 3 ) . to ensure telomerase enzymes bound the rna template in a single register , each primer used in our smfret experiments consisted of a 5-(tg)8 stretch followed by a single copy of one of the telomere repeat permutations ( supplementary fig . since fret efficiency is highly sensitive to the distance between the dyes , fret provides a direct readout of the intervening rna structure with high fret and low fret corresponding to compressed and extended rna , respectively . in this experiment , the binding of a cy3-labeled dna primer within the active site of a cy5-labeled telomerase enzyme is observed in real time as the onset of energy transfer between the two fret probes ( fig . 4b ) . for each experiment , histograms corresponding to the frequency of observed fret values were compiled and fit with gaussian functions to determine the center of the distribution ( fig . measurements from the bound primer to the tbe show a dramatic decrease in fret as the template - complementary sequence at the primer 3 end is extended ( fig . measurements of the distance from the primer to the tre show a smaller but appreciable rise in fret with the same series of primers . the larger fret change measured across the tbe region than across the tre has several possible explanations . first , the compression in the tbe may be greater in magnitude than the expansion in the tre . a second possibility is that the u63 labeling position may not be fixed in space throughout the catalytic cycle and further expansion may occur 3 of the tre . lastly , the observed asymmetry could be explained if the donor dye movement is out of plane with respect to the position of the acceptor dye . in other words , in three - dimensional space the relatively small fret change observed for the tre may in fact correspond to a significant rearrangement of the rna . many of the smfret distributions generated in our experiments are broader than would be expected for a single fret distribution . in particular , we observed a gradual broadening of the fret distributions as the length of the dna primer was increased ( supplementary fig . analysis of smfret trajectories revealed the broadening to be derived from anti - correlated donor and acceptor fluctuations in individual traces over a narrow range of fret values ( ~0.15 fret ) ( supplementary fig . is not known at present , we opted to treat the majority of the data by fitting to a single gaussian function to extract the average fret value for each primer . one exception to this approach was for data obtained with the tre ( u63 ) label and the primer corresponding to a complete telomeric repeat ( primer b ) . in this experiment , we observed a substantial number of binding events exhibiting a long - lived 0.73 fret state ( fig . 4d , asterisk ; supplementary fig . 6 , primer b ) which is likely due to an alternative primer conformation . these data were therefore fit with two gaussian functions ( centered at 0.57 and 0.73 ) in order to avoid the artificially large apparent increase in fret resulting from a single - gaussian fit . notably , transient binding events corresponding to a similar high fret state were also observed for the shorter primers ( d , e , f , a ) ( supplementary fig . 7 , tre ( u63)-labeled enzyme ) , but their short durations resulted in a negligible contribution to the fret histograms . given that primer b is the only primer that shows this stable alternative high fret state and that primer b represents the substrate for translocation during rap , it is possible that the high fret state represents a post - translocated state of the enzyme that is preferentially stabilized for telomeric primers terminating in a complete telomere repeat sequence . importantly , our conclusion that there is increased fret between the tre ( u63 ) and the most extended primer holds if we consider either of the two individual fret states ( 0.57 and 0.73 ) or the average of the two . one prediction of the accordion model is that deletions in the tre could prevent the 3 template nucleotides from reaching the active site , so that primers paired in the alignment region ( nucleotides 4951 ) would give no reaction ( no reaction , fig . primers with sequences that aligned within the template might be able to reach the active site , especially with smaller tre deletions , but after one round of extension they would pair in the alignment region and undergo no further reaction . in general , the activities of different primers should reveal which template nucleotides are able to reach the active site in each mutant telomerase . to test this hypothesis each mutant ter was assembled into a telomerase rnp and tested in telomerase activity assays with primers representing each of the six permutations of telomeric sequence ( fig . for all double - nucleotide deletion mutants , rap was reduced compared to the activity of wild - type ( wt ) telomerase ( supplementary fig . 1b ) . deleting three sequential nucleotides had a dramatic effect , allowing robust addition of one repeat but very little rap ( fig . primer b , which anneals to the alignment region of the template , was essentially inactive even for the first round of extension , and primer c showed reduced activity . this primer usage profile was independent of the location of the deletion within the tre , suggesting that sequence is not as important as length . a similar observation was made for mutants with four sequential nucleotides deleted , but now primer c was also inactive ( fig . 2e ) , suggesting that in all four - nucleotide deletion mutants , template nucleotide 47 can no longer reach the active site . deletion of six nucleotides ( 5459 ) continued the trend , preventing ter nucleotide 46 from being used as template . the largest deletion tested ( 5259 ) was only able to efficiently use primer a , which base pairs with the penultimate nucleotide of the template ( fig . this eight - nucleotide deletion presumably acts as a molecular ruler , measuring the minimum distance between the pseudoknot and c43 in the active site . a second prediction of the accordion model is that flexibility in the single - stranded rna portion of the tbe , which is 5 of the template , is also required for template progression . insertions of nucleotides adjacent to the tbe are predicted to reinforce the misalignment of the template relative to the active site in the context of tre deletion mutants . we modified two tre deletion mutants ( 5659 , a four - nucleotide deletion , and 5459 , a six - nucleotide deletion ) by inserting two uridines between nucleotides 40 and 41 of the tbe , just 5 of the template ( fig . 3 ) . while the four - nucleotide tre deletion had only a modest ability to use primer d , the combination of the insertion of 2us in the tbe and the four - nucleotide tre deletion exacerbated this effect , resulting in a primer usage profile similar to that of the six - nucleotide tre deletion mutant . similarly , the six - nucleotide deletion mutant containing an insertion of two uridines 5 of the template had a weakened ability to use primer e , matching the primer usage profile of the eight - nucleotide deletion mutant . previous experiments with circularly permuted ( cp ) telomerase rnas demonstrated that discontinuities on either side of the template decreased rap . we interpret these results as showing that coordinated structural rearrangements on both sides of the template contribute to template movement . the previous experiments were performed in the absence of p65 , the telomerase - specific rna folding protein that is essential in vivo and has the ability to rescue many severely destabilizing tert and ter mutations in vitro . if nicked rnas were defective simply because of structural destabilization , they might likewise be suppressed in telomerase containing p65 . we therefore tested the activities of cprnas assembled into telomerase rnps in both the presence and absence of p65 ( supplementary fig . because the tbe is necessary for tert binding and proper telomerase function , we tested four circularly permuted rnas ( cprna ) within this region , and one in the tre . telomerase rnps containing the cprnas had essentially full first - round activity , but thereafter rap was severely reduced , with nicks in the tbe affecting the reaction similarly to the nick at position 59 in the tre . this limited rap even in the presence of p65 provides further support for the requirement for direct connectivity of the rna on both sides of the template . taken together , these biochemical experiments demonstrate that specific deletions and insertions , as well as the introduction of nicks , in the rna elements flanking the template evoke catalytic defects predicted by the accordion model . however , these biochemical assays can not directly probe rna structure ; therefore single - molecule frster resonance energy transfer ( smfret ) was employed to monitor rna conformational changes within individual telomerase - dna primer complexes at successive steps in the telomerase catalytic cycle ( fig . 3 ) . ideally , smfret experiments performed in the presence of dttp and dgtp could permit real - time observation of ter structural rearrangements throughout the telomerase catalytic cycle . to date therefore , to simplify interpretation of our data , independent smfret experiments were performed with primers successively extended on the 3 end in the absence of dgtp and dttp . in this way , the primers used in separate experiments emulate the different conformational states that would be sampled during telomerase - catalyzed dna synthesis . surface - immobilized telomeric dna primers coupled to a donor dye ( cy3 ) were monitored in the presence of reconstituted telomerase enzymes harboring an acceptor dye ( cy5 ) at u63 in the tre ( fig . importantly , these modified dna primers and telomerase rnas support near wild - type levels of telomerase activity ( supplementary fig . 3 ) . to ensure telomerase enzymes bound the rna template in a single register , each primer used in our smfret experiments consisted of a 5-(tg)8 stretch followed by a single copy of one of the telomere repeat permutations ( supplementary fig . since fret efficiency is highly sensitive to the distance between the dyes , fret provides a direct readout of the intervening rna structure with high fret and low fret corresponding to compressed and extended rna , respectively . in this experiment , the binding of a cy3-labeled dna primer within the active site of a cy5-labeled telomerase enzyme is observed in real time as the onset of energy transfer between the two fret probes ( fig . 4b ) . for each experiment , histograms corresponding to the frequency of observed fret values were compiled and fit with gaussian functions to determine the center of the distribution ( fig . measurements from the bound primer to the tbe show a dramatic decrease in fret as the template - complementary sequence at the primer 3 end is extended ( fig . measurements of the distance from the primer to the tre show a smaller but appreciable rise in fret with the same series of primers . the larger fret change measured across the tbe region than across the tre has several possible explanations . first , the compression in the tbe may be greater in magnitude than the expansion in the tre . a second possibility is that the u63 labeling position may not be fixed in space throughout the catalytic cycle and further expansion may occur 3 of the tre . lastly , the observed asymmetry could be explained if the donor dye movement is out of plane with respect to the position of the acceptor dye . in other words , in three - dimensional space the relatively small fret change observed for the tre may in fact correspond to a significant rearrangement of the rna . many of the smfret distributions generated in our experiments are broader than would be expected for a single fret distribution . in particular , we observed a gradual broadening of the fret distributions as the length of the dna primer was increased ( supplementary fig . 4 , primers f , a , and b ) . analysis of smfret trajectories revealed the broadening to be derived from anti - correlated donor and acceptor fluctuations in individual traces over a narrow range of fret values ( ~0.15 fret ) ( supplementary fig . 5 and 6 ) . since the physical basis for these fret states is not known at present , we opted to treat the majority of the data by fitting to a single gaussian function to extract the average fret value for each primer . one exception to this approach was for data obtained with the tre ( u63 ) label and the primer corresponding to a complete telomeric repeat ( primer b ) . in this experiment , we observed a substantial number of binding events exhibiting a long - lived 0.73 fret state ( fig . . 6 , primer b ) which is likely due to an alternative primer conformation . these data were therefore fit with two gaussian functions ( centered at 0.57 and 0.73 ) in order to avoid the artificially large apparent increase in fret resulting from a single - gaussian fit . notably , transient binding events corresponding to a similar high fret state were also observed for the shorter primers ( d , e , f , a ) ( supplementary fig . 7 , tre ( u63)-labeled enzyme ) , but their short durations resulted in a negligible contribution to the fret histograms . given that primer b is the only primer that shows this stable alternative high fret state and that primer b represents the substrate for translocation during rap , it is possible that the high fret state represents a post - translocated state of the enzyme that is preferentially stabilized for telomeric primers terminating in a complete telomere repeat sequence . importantly , our conclusion that there is increased fret between the tre ( u63 ) and the most extended primer holds if we consider either of the two individual fret states ( 0.57 and 0.73 ) or the average of the two . because tert is a reverse transcriptase related to retroviral and retrotransposon reverse transcriptases , it is thought to use a similar metal - ion - catalyzed mechanism for nucleotide addition . in contrast , the mechanism by which it achieves its unique activities -- template translocation and repeat addition processivity --remains unknown . here , the predictable effects of nucleotide deletions on primer use and measurements of sequential primer position using single - molecule fret both support an rna accordion model for rap . we conclude that the rna elements flanking the template serve to position the template for nucleotide addition and for rap . the requirement for connectivity of the rna on the 5 side of the template might be a consequence of its interaction with the rna - binding domain of tert , which might be disrupted by nicking . this is difficult to address in the absence of a detailed structure of the telomerase rnp . in addition , a small rna stem - loop might form within the tre during telomere synthesis , and this stem - loop could melt for translocation . this alternative model has been tested with deletion and point mutations predicted to stabilize or destabilize the putative hairpin . if hairpin formation and melting contributed to translocation , one would expect a large effect on rap in the context of these mutants , similar to the effect on rap observed with nicked rnas or with rnas containing deletions of three or more nucleotides in the tre . however , in the presence of mutants that either stabilized or destabilized the putative hairpin , only a modest impact on rap was observed ; rap decreased by less than fifty percent as compared to wt for all mutants tested ( supplementary fig . while our data do not directly address the energetics of translocation , the disruption of rap by rna nicking observed by collins and in our supplementary fig . more specifically , upon synthesis of a complete telomere dna repeat , the tre region of the rna may exist in a high - energy state . for example , this single - stranded region of rna could become compressed , or energetically unfavorable ter - tert interactions could be produced . polymerases move along their templates without external energy ; the energy provided by nucleotide incorporation and pyrophosphate release can be converted directly into directional movement or it can be stored within scrunched nucleic acid that can later be manifested as translocation . in rna polymerases , for example , the dna template bunches near the active site during rna synthesis initiation , resulting in a higher energy state that likely provides the energy for promoter clearance . the conformational changes observed within telomerase ter are reminiscent of this mechanism . here , however , rather than the primer dna storing energy , perhaps the rna downstream of the template bunches up as the template is copied , potentially storing energy during repeat synthesis , and releasing it to drive template recycling . the molecular basis for compaction could also involve base stacking and rna - tert protein interactions . in addition , it is possible that a ratchet might be involved in translocation ; the pawl for this ratchet would eliminate backtracking of the template until the end is reached . a ratchet mechanism is compatible with rna conformational changes being necessary for translocation , and thus could represent an elaboration of the accordion model . further understanding of the translocation mechanism awaits detailed structural information concerning the rna - protein interactions in the vicinity of the active site and their dynamics during processive extension of telomeric dna .

telomerase is a ribonucleoprotein ( rnp ) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers . this rnp minimally contains a reverse transcriptase protein subunit ( tert ) that catalyzes telomeric dna synthesis and an rna subunit ( ter ) that has templating , architectural and protein - scaffolding roles . telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3 end of a primer following a single binding event , a process known as repeat addition processivity ( rap ) . using biochemical assays and single - molecule frster resonance energy transfer ( smfret ) experiments on tetrahymena thermophila telomerase , we now directly demonstrate that ter contributes to template positioning within the active site and to the template translocation required for rap . we propose that the single - stranded rna elements flanking the template act as a molecular accordion , undergoing reciprocal extension and compaction during telomerase translocation .

RESULTS Deleting TRE nucleotides reduces RAP in a predictable manner Insertions in the TBE reinforce template misalignment Discontinuities flanking the RNA template eliminate RAP Single-molecule FRET reveals RNA movement as predicted DISCUSSION Supplementary Material

one prediction of the accordion model is that deletions in the tre could prevent the 3 template nucleotides from reaching the active site , so that primers paired in the alignment region ( nucleotides 4951 ) would give no reaction ( no reaction , fig . primers with sequences that aligned within the template might be able to reach the active site , especially with smaller tre deletions , but after one round of extension they would pair in the alignment region and undergo no further reaction . in general , the activities of different primers should reveal which template nucleotides are able to reach the active site in each mutant telomerase . to test this hypothesis each mutant ter was assembled into a telomerase rnp and tested in telomerase activity assays with primers representing each of the six permutations of telomeric sequence ( fig . for all double - nucleotide deletion mutants , rap was reduced compared to the activity of wild - type ( wt ) telomerase ( supplementary fig . primer b , which anneals to the alignment region of the template , was essentially inactive even for the first round of extension , and primer c showed reduced activity . this primer usage profile was independent of the location of the deletion within the tre , suggesting that sequence is not as important as length . 2e ) , suggesting that in all four - nucleotide deletion mutants , template nucleotide 47 can no longer reach the active site . the largest deletion tested ( 5259 ) was only able to efficiently use primer a , which base pairs with the penultimate nucleotide of the template ( fig . this eight - nucleotide deletion presumably acts as a molecular ruler , measuring the minimum distance between the pseudoknot and c43 in the active site . a second prediction of the accordion model is that flexibility in the single - stranded rna portion of the tbe , which is 5 of the template , is also required for template progression . insertions of nucleotides adjacent to the tbe are predicted to reinforce the misalignment of the template relative to the active site in the context of tre deletion mutants . we modified two tre deletion mutants ( 5659 , a four - nucleotide deletion , and 5459 , a six - nucleotide deletion ) by inserting two uridines between nucleotides 40 and 41 of the tbe , just 5 of the template ( fig . while the four - nucleotide tre deletion had only a modest ability to use primer d , the combination of the insertion of 2us in the tbe and the four - nucleotide tre deletion exacerbated this effect , resulting in a primer usage profile similar to that of the six - nucleotide tre deletion mutant . similarly , the six - nucleotide deletion mutant containing an insertion of two uridines 5 of the template had a weakened ability to use primer e , matching the primer usage profile of the eight - nucleotide deletion mutant . previous experiments with circularly permuted ( cp ) telomerase rnas demonstrated that discontinuities on either side of the template decreased rap . we interpret these results as showing that coordinated structural rearrangements on both sides of the template contribute to template movement . we therefore tested the activities of cprnas assembled into telomerase rnps in both the presence and absence of p65 ( supplementary fig . 2 ) . because the tbe is necessary for tert binding and proper telomerase function , we tested four circularly permuted rnas ( cprna ) within this region , and one in the tre . telomerase rnps containing the cprnas had essentially full first - round activity , but thereafter rap was severely reduced , with nicks in the tbe affecting the reaction similarly to the nick at position 59 in the tre . this limited rap even in the presence of p65 provides further support for the requirement for direct connectivity of the rna on both sides of the template . taken together , these biochemical experiments demonstrate that specific deletions and insertions , as well as the introduction of nicks , in the rna elements flanking the template evoke catalytic defects predicted by the accordion model . however , these biochemical assays can not directly probe rna structure ; therefore single - molecule frster resonance energy transfer ( smfret ) was employed to monitor rna conformational changes within individual telomerase - dna primer complexes at successive steps in the telomerase catalytic cycle ( fig . to date , however , fret - based monitoring of telomerase activity has remained elusive . therefore , to simplify interpretation of our data , independent smfret experiments were performed with primers successively extended on the 3 end in the absence of dgtp and dttp . in this way , the primers used in separate experiments emulate the different conformational states that would be sampled during telomerase - catalyzed dna synthesis . surface - immobilized telomeric dna primers coupled to a donor dye ( cy3 ) were monitored in the presence of reconstituted telomerase enzymes harboring an acceptor dye ( cy5 ) at u63 in the tre ( fig . importantly , these modified dna primers and telomerase rnas support near wild - type levels of telomerase activity ( supplementary fig . to ensure telomerase enzymes bound the rna template in a single register , each primer used in our smfret experiments consisted of a 5-(tg)8 stretch followed by a single copy of one of the telomere repeat permutations ( supplementary fig . since fret efficiency is highly sensitive to the distance between the dyes , fret provides a direct readout of the intervening rna structure with high fret and low fret corresponding to compressed and extended rna , respectively . in this experiment , the binding of a cy3-labeled dna primer within the active site of a cy5-labeled telomerase enzyme is observed in real time as the onset of energy transfer between the two fret probes ( fig . 4b ) . for each experiment , histograms corresponding to the frequency of observed fret values were compiled and fit with gaussian functions to determine the center of the distribution ( fig . measurements from the bound primer to the tbe show a dramatic decrease in fret as the template - complementary sequence at the primer 3 end is extended ( fig . measurements of the distance from the primer to the tre show a smaller but appreciable rise in fret with the same series of primers . a second possibility is that the u63 labeling position may not be fixed in space throughout the catalytic cycle and further expansion may occur 3 of the tre . lastly , the observed asymmetry could be explained if the donor dye movement is out of plane with respect to the position of the acceptor dye . in other words , in three - dimensional space the relatively small fret change observed for the tre may in fact correspond to a significant rearrangement of the rna . many of the smfret distributions generated in our experiments are broader than would be expected for a single fret distribution . in particular , we observed a gradual broadening of the fret distributions as the length of the dna primer was increased ( supplementary fig . is not known at present , we opted to treat the majority of the data by fitting to a single gaussian function to extract the average fret value for each primer . in this experiment , we observed a substantial number of binding events exhibiting a long - lived 0.73 fret state ( fig . these data were therefore fit with two gaussian functions ( centered at 0.57 and 0.73 ) in order to avoid the artificially large apparent increase in fret resulting from a single - gaussian fit . notably , transient binding events corresponding to a similar high fret state were also observed for the shorter primers ( d , e , f , a ) ( supplementary fig . 7 , tre ( u63)-labeled enzyme ) , but their short durations resulted in a negligible contribution to the fret histograms . given that primer b is the only primer that shows this stable alternative high fret state and that primer b represents the substrate for translocation during rap , it is possible that the high fret state represents a post - translocated state of the enzyme that is preferentially stabilized for telomeric primers terminating in a complete telomere repeat sequence . importantly , our conclusion that there is increased fret between the tre ( u63 ) and the most extended primer holds if we consider either of the two individual fret states ( 0.57 and 0.73 ) or the average of the two . one prediction of the accordion model is that deletions in the tre could prevent the 3 template nucleotides from reaching the active site , so that primers paired in the alignment region ( nucleotides 4951 ) would give no reaction ( no reaction , fig . primers with sequences that aligned within the template might be able to reach the active site , especially with smaller tre deletions , but after one round of extension they would pair in the alignment region and undergo no further reaction . in general , the activities of different primers should reveal which template nucleotides are able to reach the active site in each mutant telomerase . to test this hypothesis each mutant ter was assembled into a telomerase rnp and tested in telomerase activity assays with primers representing each of the six permutations of telomeric sequence ( fig . for all double - nucleotide deletion mutants , rap was reduced compared to the activity of wild - type ( wt ) telomerase ( supplementary fig . primer b , which anneals to the alignment region of the template , was essentially inactive even for the first round of extension , and primer c showed reduced activity . this primer usage profile was independent of the location of the deletion within the tre , suggesting that sequence is not as important as length . 2e ) , suggesting that in all four - nucleotide deletion mutants , template nucleotide 47 can no longer reach the active site . the largest deletion tested ( 5259 ) was only able to efficiently use primer a , which base pairs with the penultimate nucleotide of the template ( fig . this eight - nucleotide deletion presumably acts as a molecular ruler , measuring the minimum distance between the pseudoknot and c43 in the active site . a second prediction of the accordion model is that flexibility in the single - stranded rna portion of the tbe , which is 5 of the template , is also required for template progression . insertions of nucleotides adjacent to the tbe are predicted to reinforce the misalignment of the template relative to the active site in the context of tre deletion mutants . we modified two tre deletion mutants ( 5659 , a four - nucleotide deletion , and 5459 , a six - nucleotide deletion ) by inserting two uridines between nucleotides 40 and 41 of the tbe , just 5 of the template ( fig . while the four - nucleotide tre deletion had only a modest ability to use primer d , the combination of the insertion of 2us in the tbe and the four - nucleotide tre deletion exacerbated this effect , resulting in a primer usage profile similar to that of the six - nucleotide tre deletion mutant . similarly , the six - nucleotide deletion mutant containing an insertion of two uridines 5 of the template had a weakened ability to use primer e , matching the primer usage profile of the eight - nucleotide deletion mutant . previous experiments with circularly permuted ( cp ) telomerase rnas demonstrated that discontinuities on either side of the template decreased rap . we interpret these results as showing that coordinated structural rearrangements on both sides of the template contribute to template movement . if nicked rnas were defective simply because of structural destabilization , they might likewise be suppressed in telomerase containing p65 . because the tbe is necessary for tert binding and proper telomerase function , we tested four circularly permuted rnas ( cprna ) within this region , and one in the tre . telomerase rnps containing the cprnas had essentially full first - round activity , but thereafter rap was severely reduced , with nicks in the tbe affecting the reaction similarly to the nick at position 59 in the tre . this limited rap even in the presence of p65 provides further support for the requirement for direct connectivity of the rna on both sides of the template . taken together , these biochemical experiments demonstrate that specific deletions and insertions , as well as the introduction of nicks , in the rna elements flanking the template evoke catalytic defects predicted by the accordion model . however , these biochemical assays can not directly probe rna structure ; therefore single - molecule frster resonance energy transfer ( smfret ) was employed to monitor rna conformational changes within individual telomerase - dna primer complexes at successive steps in the telomerase catalytic cycle ( fig . 3 ) . to date therefore , to simplify interpretation of our data , independent smfret experiments were performed with primers successively extended on the 3 end in the absence of dgtp and dttp . in this way , the primers used in separate experiments emulate the different conformational states that would be sampled during telomerase - catalyzed dna synthesis . surface - immobilized telomeric dna primers coupled to a donor dye ( cy3 ) were monitored in the presence of reconstituted telomerase enzymes harboring an acceptor dye ( cy5 ) at u63 in the tre ( fig . importantly , these modified dna primers and telomerase rnas support near wild - type levels of telomerase activity ( supplementary fig . to ensure telomerase enzymes bound the rna template in a single register , each primer used in our smfret experiments consisted of a 5-(tg)8 stretch followed by a single copy of one of the telomere repeat permutations ( supplementary fig . since fret efficiency is highly sensitive to the distance between the dyes , fret provides a direct readout of the intervening rna structure with high fret and low fret corresponding to compressed and extended rna , respectively . in this experiment , the binding of a cy3-labeled dna primer within the active site of a cy5-labeled telomerase enzyme is observed in real time as the onset of energy transfer between the two fret probes ( fig . 4b ) . for each experiment , histograms corresponding to the frequency of observed fret values were compiled and fit with gaussian functions to determine the center of the distribution ( fig . measurements from the bound primer to the tbe show a dramatic decrease in fret as the template - complementary sequence at the primer 3 end is extended ( fig . measurements of the distance from the primer to the tre show a smaller but appreciable rise in fret with the same series of primers . a second possibility is that the u63 labeling position may not be fixed in space throughout the catalytic cycle and further expansion may occur 3 of the tre . lastly , the observed asymmetry could be explained if the donor dye movement is out of plane with respect to the position of the acceptor dye . in other words , in three - dimensional space the relatively small fret change observed for the tre may in fact correspond to a significant rearrangement of the rna . many of the smfret distributions generated in our experiments are broader than would be expected for a single fret distribution . in particular , we observed a gradual broadening of the fret distributions as the length of the dna primer was increased ( supplementary fig . 4 , primers f , a , and b ) . since the physical basis for these fret states is not known at present , we opted to treat the majority of the data by fitting to a single gaussian function to extract the average fret value for each primer . in this experiment , we observed a substantial number of binding events exhibiting a long - lived 0.73 fret state ( fig . these data were therefore fit with two gaussian functions ( centered at 0.57 and 0.73 ) in order to avoid the artificially large apparent increase in fret resulting from a single - gaussian fit . notably , transient binding events corresponding to a similar high fret state were also observed for the shorter primers ( d , e , f , a ) ( supplementary fig . 7 , tre ( u63)-labeled enzyme ) , but their short durations resulted in a negligible contribution to the fret histograms . given that primer b is the only primer that shows this stable alternative high fret state and that primer b represents the substrate for translocation during rap , it is possible that the high fret state represents a post - translocated state of the enzyme that is preferentially stabilized for telomeric primers terminating in a complete telomere repeat sequence . importantly , our conclusion that there is increased fret between the tre ( u63 ) and the most extended primer holds if we consider either of the two individual fret states ( 0.57 and 0.73 ) or the average of the two . because tert is a reverse transcriptase related to retroviral and retrotransposon reverse transcriptases , it is thought to use a similar metal - ion - catalyzed mechanism for nucleotide addition . in contrast , the mechanism by which it achieves its unique activities -- template translocation and repeat addition processivity --remains unknown . here , the predictable effects of nucleotide deletions on primer use and measurements of sequential primer position using single - molecule fret both support an rna accordion model for rap . we conclude that the rna elements flanking the template serve to position the template for nucleotide addition and for rap . the requirement for connectivity of the rna on the 5 side of the template might be a consequence of its interaction with the rna - binding domain of tert , which might be disrupted by nicking . this is difficult to address in the absence of a detailed structure of the telomerase rnp . in addition , a small rna stem - loop might form within the tre during telomere synthesis , and this stem - loop could melt for translocation . if hairpin formation and melting contributed to translocation , one would expect a large effect on rap in the context of these mutants , similar to the effect on rap observed with nicked rnas or with rnas containing deletions of three or more nucleotides in the tre . however , in the presence of mutants that either stabilized or destabilized the putative hairpin , only a modest impact on rap was observed ; rap decreased by less than fifty percent as compared to wt for all mutants tested ( supplementary fig . more specifically , upon synthesis of a complete telomere dna repeat , the tre region of the rna may exist in a high - energy state . for example , this single - stranded region of rna could become compressed , or energetically unfavorable ter - tert interactions could be produced . polymerases move along their templates without external energy ; the energy provided by nucleotide incorporation and pyrophosphate release can be converted directly into directional movement or it can be stored within scrunched nucleic acid that can later be manifested as translocation . in rna polymerases , for example , the dna template bunches near the active site during rna synthesis initiation , resulting in a higher energy state that likely provides the energy for promoter clearance . here , however , rather than the primer dna storing energy , perhaps the rna downstream of the template bunches up as the template is copied , potentially storing energy during repeat synthesis , and releasing it to drive template recycling . the molecular basis for compaction could also involve base stacking and rna - tert protein interactions . in addition , it is possible that a ratchet might be involved in translocation ; the pawl for this ratchet would eliminate backtracking of the template until the end is reached . a ratchet mechanism is compatible with rna conformational changes being necessary for translocation , and thus could represent an elaboration of the accordion model . further understanding of the translocation mechanism awaits detailed structural information concerning the rna - protein interactions in the vicinity of the active site and their dynamics during processive extension of telomeric dna .

obesity is associated with elevated free fatty acid levels , leading to the development of insulin resistance , diabetes , and cardiovascular disease ( cvd ) [ 13 ] . the development of cvd is characterised by impaired nitric oxide ( no ) release from vascular endothelium and decreased blood flow to insulin target tissues resulting in insulin resistance , termed as endothelial dysfunction . however , the mechanisms by which obesity causes both insulin resistance and vascular dysfunction are not fully understood . in this respect , increasing attention has been paid to the direct vascular effects of adipose tissue ( at ) derived factors termed a few of these adipokines are characterised by their favourable effects to maintain the body 's energy and vascular homeostasis ; however , adipokines have also been implicated in the pathogenesis of obesity - related disorders , such as atherosclerosis , specifically , by increasing the expression of proangiogenic / proatherogenic factors like endothelial gelatinases ( matrix metalloproteinases 2 and 9 ) and vascular endothelial growth factor ( vegf ) . leptin , adiponectin , monocyte chemoattractant protein- ( mcp- ) 1 , plasminogen activator inhibitor- ( pai- ) 1 , tumour necrosis factor ( tnf ) , interleukin- ( il- ) 6 , and resistin are a few of these adipokines implicated in endothelial dysfunction . endothelial dysfunction in obesity is characterised by increased generation of oxygen - derived free radicals ( ros ) . this is contributed by vascular cells and inflamed hypertrophied adipocytes as a result of endoplasmic reticulum ( er ) stress and mitochondrial dysfunction . enzymes of mitochondrial electron transport chain , xanthine oxidase , cyclooxygenases , lipoxygenases , myeloperoxidases , cytochrome p450 monooxygenase , heme oxygenases , peroxidases , and nad(p)h oxidases contribute to endothelial dysfunction . uncoupling of endothelial nitric oxide synthase ( enos ) is a major contributor to ros production . this results in decreased no ( nitric oxide ) bioavailability , increased o2 production , and formation of peroxynitrite ( onoo ) , a key mediator of lipid peroxidation and foam cell formation in atherosclerotic lesions . additionally , ros accumulation results in activation of signalling cascades that regulate transcription factors , including nf- ( nuclear factor kappa beta ) adhesion molecules , chemotactic factors , antioxidant enzymes , and vasoactive substances promoting adhesion and migration of circulating monocytes initiating atherosclerotic lesions . dysregulated adipokine production leading to increased ros generation forms a major feedback loop in initiation , maintenance , and progression of endothelial dysfunction . adiponectin and leptin are the two widely studied most - abundant , circulating adipokines . in this review , we discuss the diverse roles of leptin and adiponectin in endothelial dysfunction with emphasis on proangiogenic / proatherogenic factors in the endothelial cells . adiponectin , the most abundantly secreted adipokine ( 220 g / ml in circulating plasma ) , was first identified as acrp30adipose complement - related protein of 30 kda because of its high similarity to complement protein . adiponectin exerts its insulin - sensitizing effects by increasing -oxidation of fatty acids , in the process reducing serum triglyceride and levels of free - fatty acids , and thus indirectly improving insulin sensitivity of the liver . in addition to its metabolic actions , adiponectin is also reported to possess antiatherogenic and anti - inflammatory properties . circulating low adiponectin levels ( hypoadiponectinemia ) adiponectin is a 247-amino acid protein with four domains , an amino - terminal signal sequence , a variable region , a collagenous domain , and a carboxy - terminal globular domain [ 13 , 1719 ] , and undergoes posttranslational modifications within the adipocytes into multimeric forms including trimers , hexamers , and high - molecular - weight ( hmw ) oligomers . more importantly , cleavage of globular domain of full - length adiponectin ( fad ) by activated monocytes has been reported to conversely affect the protective role of fad . both globular adiponectin ( gad ) and fad exert their effects via transmembrane g - protein coupled receptors , adiponectin receptor 1 ( adipor1 ) , and adiponectin receptor 2 ( adipor2 ) . these receptors have been described as structurally related integral plasma membrane proteins with seven transmembrane domains having their extracellular c terminus and intracellular n terminus regions [ depicted in figure 1 ] . adipor1 is abundantly synthesised and expressed in skeletal muscle , whereas adipor2 is found predominantly in liver . both receptors have also been described in pancreatic -cells , macrophages , endothelial cells , and smooth muscle cells within atherosclerotic plaques [ 23 , 24 ] . c - terminus of adipor1 possesses high affinity for gad , whereas adipor2 exhibits intermediate affinity for both the gad and fad . overexpression and gene knockout experiments in rodents have demonstrated the ability of these receptors to activate amp - activated protein kinase ( ampk ) , p38 mitogen activated protein kinase ( p38 mapk ) , and peroxisome - proliferator - activated receptor- ( ppar- ) and to stimulate fatty acid oxidation and glucose uptake in murine hepatocytes and c2c12 myocytes . globular , trimeric , and high - molecular - weight ( hmw ) adiponectin forms activate different signal transduction pathways . additionally , osmotin , a plant protein with structural similarities to mammalian globular adiponectin , binds to adiponectin receptors and activates ampk in c2c12 myocytes . exercise training increases adipor1/r2 mrna expression in human skeletal muscle , whereas no significant change has been reported in human subcutaneous adipose tissue during calorie restriction . the expression levels of adipor1 and adipor2 in skeletal muscle , as well as plasma adiponectin concentrations , have been described to be lower in individuals with a family history of type 2 diabetes mellitus ( t2 dm ) than in those with no family history . the expression level of both receptors correlated positively with insulin sensitivity . a study by zhang et al . , 2010 , had demonstrated significant reduction in expression of adipor2 in both coronary arterioles and aortas of diabetic mice , with no changes in adipor1 expression levels . another adiponectin - binding protein with a preference for hmw adiponectin multimers and no affinity for the trimeric adiponectin has been identified as t - cadherin . since this protein is a glycosylphosphatidylinositol - anchored extracellular protein devoid of any intracellular domain , the mechanism explaining its role in adiponectin intracellular signalling has not been fully clarified . leptin , a 16 kda protein , is an adipose - tissue specific adipokine involved in regulation of food intake and energy haemostasis . leptin also has multiple roles in carbohydrate and lipid metabolism , reproductive system , and inflammatory and immune reactions . leptin has been shown to exert atherogenic , thrombotic , and angiogenic actions on the vasculature [ 4850 ] . it has been linked extensively with obesity leading to cvds including atherosclerosis , myocardial infarction , and stroke [ 51 , 52 ] . leptin acts on target cells through plasma membrane receptors ( figure 2 ) and exists in at least six isoforms , ob - ra through ob - rf , signalling predominantly via jak / stat ( janus kinases / signal transducers , and activators of transcription ) pathway . functional leptin receptors ( both short and long forms of ob - r ) have been identified on endothelial cells [ 38 , 54 ] , and numerous studies link the possible mechanisms responsible for leptin - induced endothelial dysfunction . jak-2/irs-2/pi3-k / akt pathways and nuclear translocation of stat ( signal transducer and activator of transcription ) proteins have been implicated to play a pivotal role in leptin - mediated effects in endothelial cells [ 53 , 55 ] . interestingly , in states of obesity despite a paradoxical impairment of the satiety response , leptin resistance does not extend to leptin mediated endothelial dysfunction [ 5659 ] . recent studies have demonstrated increased expression and modulation of astrocytic leptin receptor subtypes ( ob - r ) in adult - onset obesity facilitating increased leptin transport via the human brain endothelial cell barrier . although this selective response of leptin has gained much attention , the molecular basis remains poorly understood . decreased production of no by the endothelial cells is considered as the hallmark of endothelial dysfunction . adiponectin has been reported to increase no production in endothelial cells by the activation of phosphotidylinositol-3 ( pi-3 ) kinase / akt signalling pathway . furthermore , the involvement of ampk and protein kinase a [ pka , or cyclic amp- ( camp- ) dependent protein kinase ] signalling have also been implicated to play a major role in both adiponectin induced no production and suppression of endothelial ros generation , inhibiting endothelial nf- ( nuclear factor kappa beta ) signalling [ 32 , 6265 ] . animal studies conducted in adiponectin knockout ( ko ) mice have shown a significant reduction in endothelium - dependent vasodilatation . adiponectin and lectin - like oxidized ldl ( ox - ldl ) receptor- ( lox- ) 1 have been demonstrated to exhibit a reciprocal pattern in states of endothelial dysfunction and inflammatory insults . adiponectin administration in apolipoprotein e ( apoe ) knockout ( ko ) mice restored no - mediated endothelium - dependent vasorelaxation and decreased aortic lox-1 expression , implicating a key biological function of adiponectin in reducing systemic oxidative mediators and ox - ldl uptake . more importantly , studies in t2 dm mice have elucidated a similar reciprocal regulation between adiponectin and tnf- affecting the regulation of both coronary and aortic endothelial function . additionally , in vivo studies have indicated the critical role of adiponectin in alleviating sepsis - induced microvascular dysfunction leading to blood brain barrier ( bbb ) dysfunction in the mouse brain via modulation of e - selectin expression . clinical studies have demonstrated impaired production of enos in the vasculature consequently leading to decreased endothelium - dependent vasorelaxation in subjects with decreased adiponectin levels . furthermore , adiponectin drastically improves oxidized ldl induced decrease in enos activity [ 36 , 70 ] . more importantly , to simulate pathological states of obesity and diabetes , the role of adiponectin in hyperglycaemic / hyperinsulinaemic environments has been studied . xiao et al . have demonstrated a protective action of gad in alleviating endothelial dysfunction caused due to intermittent hyperglycaemia , implicating the involvement of akt , ampk , and enos signalling pathways . the deleterious effects of hyperglycaemia in obese and diabetic subjects extend to a concomitant decrease in circulating endothelial progenitor cells ( epcs ) leading to impaired endothelial repair . studies have indicated that gad promoted epc migration and tube formation and dose - dependently upregulated phosphorylation of enos , akt , and augmented no production . additionally , in vivo results have revealed that gad rescued high glucose induced impairment of epc functions by restoration of enos activity and vasculogenesis . it is interesting to note that adiponectin induced activation of enos as well as increased production of no by the endothelial cells is crucial in mediating its anti - inflammatory effects . in this context , in vivo studies have demonstrated that pharmacological blockade of enos leads to decreased protective effect imparted by adiponectin , leading to increased leukocyte adhesion by tnf- . endothelial dysfunction includes the activation of endothelial adhesion molecular cascade critical in facilitating the entry of macrophages into the vessel wall . circulating low adiponectin levels in metabolic diseases like obesity and diabetes have been linked to the triggering of an inflammatory signalling cascade , leading to the early development of atherosclerosis . the development of a similar scenario in adipoq/ ( adiponectin knock - out mice ) mice further strengthens the association between adiponectin and cvd . furthermore , adiponectin has been shown to inhibit the vascular inflammatory response of endothelium to tnf- induced activation of nf- and increased expression of adhesion molecules vascular cell adhesion molecule ( vcam-1 ) , intercellular adhesion molecule ( icam-1 ) , and endothelial selectin ( e - selectin ) . functional effects induced by mediators of systemic inflammation like tnf- and subsequent interactions with adipokines have a significant influence in either promoting or downregulating vascular insult . adiponectin has been shown to induce in vitro angiogenesis in endothelial cells via ampk - enos pathway . studies conducted in adiponectin - overexpressed mice brain ( following transfection with adenoassociated viral vector ( aav ) containing adiponectin gene ) have shown a significant benefit induced by adiponectin following ischemic insult . this protective action was related to adiponectin induced focal angiogenesis involving vegf and ampk pathways . on the other hand , other groups have demonstrated the potent inhibition of endothelial angiogenic events like migration and proliferation by adiponectin , involving mapk and camp - pka pathways . similar antiangiogenic effects of adiponectin have been studied in tumour growth suppression involving rho kinase / ifn - inducible protein 10 and matrix metalloproteinase 9 ( mmp-9 ) . numerous studies have implicated the vasoprotective actions of fad by reducing the expression of endothelial adhesion molecules and inhibiting tnf- induced cytokine production from macrophages via nf-/camp - dependent pathway [ 32 , 7880 ] . animal studies in ad ( mice completely lacking adiponectin ) and ad ( adiponectin - hemizygous mice ) mice showed an increased expression of e - selectins and vcams . adiponectin has been demonstrated to suppress vegf - stimulated hcaec migration via camp / pka - dependent signalling . clinical studies in patients with acute coronary syndromes have shown a negative correlation between circulating adiponectin levels and mmp-9/timp-1 ratio , an independent predictor of atherosclerotic plaque stability . however , three independent studies have demonstrated that gad activates nf- leading to activation of the proinflammatory adhesion cascade , proliferation , and increased procoagulability in endothelial cells and cardiac fibroblasts [ 39 , 40 , 81 ] . furthermore , studies by hattori et al . have indicated the suppression of cytokine induced inflammatory cascade via nf- by gad , albeit with a prolonged response time . the authors attribute this to desensitisation of the receptor , seen in instances of cytokine overload . more recently , colocalization studies conducted by xu et al . have demonstrated that adiponectin induces interaction between lymphotoxin- ( lt- ) b receptor ( ltbr ) and human adipor1 , subsequently resulting in inhibition of the nf- pathway . studies have demonstrated the ability of leukocyte elastase secreted by activated monocytes and neutrophils to cleave the globular domain of adiponectin . this local generation of gad at sites of inflammation , namely , in atherosclerotic lesions , could be having pathophysiological relevance given the differential actions of multimeric forms of adiponectin . in a study comparing the differential effects of fad and gad in human aortic endothelial cells ( haec ) , both peptides upregulated no production by ampk - dependent pathways . however , in contrast to fad , gad activated nf- and p38 mapk signalling pathways , resulting in cyclooxygenase-2 ( cox-2 ) production and subsequently prostacyclin 2 [ pgi ] release . this study further demonstrated that monocyte - endothelial adhesion enhanced by gad remained unaffected with either abrogation of adipor1 [ sirna ] signalling or cox-2 [ sirna ] downregulation , thereby suggesting independent mechanisms governing actions of fad and gad . the obvious discrepancies between the experimental outcomes could be due to the differences in the forms of adiponectin used ( table 1 ) . recently , we undertook a study to investigate the effect of gad and fad ( figure 3 ) on endothelial cell proliferation as well as in vitro migration and angiogenesis in relation to the induction of endothelial angiogenic factors , specifically , mmp-2 , mmp-9 , and vegf ; furthermore , we examined the involvement of the adiponectin receptors , that is , adiponectin receptor 1 ( adipor1 ) and adiponectin receptor 2 ( adipor2 ) , within this context . more importantly , given the connection between the coexistence of hyperglycaemia and systemic inflammation with vascular disease in pathological states such as diabetes mellitus , we also studied the interaction between glucose and c - reactive protein ( crp ) [ a potent proinflammatory protein ] , respectively , with gad and fad . finally , since amp - activated protein kinase ( ampk ) , a stress - activated protein kinase , and akt have been implicated as critical mediators of adiponectin induced angiogenesis in both normoxic and ischemic tissues we found that gad led to a significant increase in in vitro endothelial proliferation , migration , and angiogenesis with concomitant increase in mmp-2 , mmp-9 , and vegf gene and protein production , as well as mmp-2 and mmp-9 activation . the effect of gad on vegf appears to be mediated by adipor1 whereas the effect of gad on mmp-2 and mmp-9 appears to be mediated by adipor1 and adipor2 . on the other hand , only endothelial cell proliferation was significantly increased by fad and appears to be mediated by adipor2 ; no significant effects on mmp-2 , mmp-9 , and vegf were observed . 2004 had reported that mouse fad stimulates in vitro migration and angiogenesis and suggested that this effect may be beneficial in line with the report by shibata et al . , 2004 , who demonstrated that adiponectin promotes ischemia - mediated revascularization in adiponectin - knockout mice . it is important to note that although in vitro angiogenic assays have been merited as useful reporters in deciphering specific steps , they however lack the complex interplay of multiple factors vital for in vivo processes [ 31 , 33 ] . taken together , it remains unclear as to whether our observations reflect on balance a beneficial or detrimental effect of adiponectin . thus it seems imperative to study the local effects of various multimers of adiponectin in situ , for instance , in atherosclerotic plaques , to ascertain the potential pro / anti - inflammatory actions of this adipokine . leptin has multiple proinflammatory and immune mediated effects on the vasculature . on engagement with leptin receptors expressed on vascular cell walls , leptin induces oxidative stress responses , increases mcp-1 , tnf- , il-6 , and endothelin-1 , and potentiates proliferation , along with the expression of other endothelial cell adhesion molecules , mmps , vegf , and impaired smooth - muscle cell function , resulting in impaired endothelium - dependent vasodilatation promoting hypertension and atherosclerosis . clinical studies have reported a positive correlation between circulating leptin , plasma thrombomodulin , and vcam-1 levels . endothelium dependent leptin induced vasorelaxation observed in rat arterial rings was promptly inhibited by increasing extracellular calcium and inhibition of no synthase . intra - arterial administration of leptin showed a similar vasoactive response independent of no in humans . additionally , a direct vasorelaxive effect of leptin on smooth muscle cells independent of endothelium was also observed in both rat and human arterial samples [ 88 , 89 ] . acute hyperleptinemia induced vasodilatory effects and this seemingly contradicts the coexisting hypertension and increased leptin levels on obesity . a plausible explanation for this could be attributed to the acute and chronic effects of leptin on the vasculature . recent in vivo studies have revealed additional induction of endothelial nnos ( neuronal nitric oxide synthase ) expression by leptin as a compensatory mechanism to induce endothelium - dependent relaxation in enos ( / ) mice . more importantly , hyperleptinemia induced endothelial dysfunction may play a crucial role in the differential actions of leptin . experiments by naseem have indicated that leptin initiated upregulation of inducible no synthase ( inos ) , which may or may not lead to net increased no production and paradoxically impairs endothelial function by inducing oxidative stress . furthermore , a significant vasodilatory response induced by leptin in lean zucker rats failed to do so in obese hyperleptinemic zucker rats . as mentioned , leptin has been shown to induce oxidative stress by increasing the formation of reactive oxygen species ( ros ) , a key mediator of endothelial dysfunction [ 42 , 93 ] . this generated ros has potent peroxidant effects and thereby reduces the bioavailability of no in aortic endothelial cells , vascular smooth muscle cells , and macrophages . additionally , ros further contributes to endothelial dysfunction by upregulating proinflammatory cascades including adhesion and chemotactic pathways in endothelial cells . it is interesting to note that genetically modified ( ob / ob- leptin knock out ) mice maintain a relative hypotensive status in comparison with their wild types . this could be attributed to the disturbance in the fine balance between the sympathetic nervous system and endothelial cell mediated regulation of vasomotor tone . with respect to the regulation of leptin receptors and endothelial dysfunction , a study by park et al . 2012 has revealed that leptin receptors in coronary arterioles are downregulated in high - fat fed sedentary mice leading to endothelial dysfunction . however , when subjected to exercise , the expression of leptin receptors in coronary arterioles was restored along with maintenance of enos phosphorylation , leptin sensitivity , and redox balance . as mentioned previously , leptin - mediated actions in endothelial cells , including angiogenesis , it is interesting to note the increased expressions of both ob - r and mmps in atherosclerotic plaques , particularly the endothelial lining of neointimal regions , suggesting the role of leptin in mediating aberrant angiogenesis . both in vivo and in vitro studies have demonstrated the activation of endothelial ob - r by leptin , leading to capillary tube formation , a prerequisite for angiogenesis . showed that leptin induced activation of mitogen - activated protein kinase family erk1/2 leads to an increase in endothelial cell viability in serum - free media . leptin has been shown to upregulate key proangiogenic molecules like the gelatinases ( mmps , mmp-2/-9 ) and timps . additionally , leptin has been shown to upregulate and act synergistically with the key angiogenic mediators like fgf-2 , vegf , and its receptor vegfr1 , stimulating vascular permeability , consequently resulting in functional angiogenesis . it is important to note that wound healing disorder ( due to deficient angiogenesis ) in ob / ob mice is corrected by both topical and systemic leptin administration but not in fa / fa zucker rats ( rats with a recessive trait of the leptin receptor ) , due to the absence of functional leptin receptors . in a recent study involving an obese nzo ( mice with phosphatidylcholine transfer protein mutation leading to abnormal lipid homeostasis ) mice model , the angiogenic potential of leptin was found to be insignificant , perhaps due to the relative inactivity of its receptor in these mice . studies in huvecs have implicated the involvement of a functional endothelial p38 ( mapk)/akt / cox-2 signalling axis for leptin 's proangiogenic effects and more importantly this signalling pathway is regulated upstream by obrb - dependent activation of vegfr2 receptor . in vivo findings have implicated increased mobilisation of vascular progenitor cells mobilized from the bone marrow in response to leptin stimulation leading to angiogenesis . these effects of leptin seem to be mediated via ob - r induced activation of nox2 and mmp9 . additional studies have evidenced the importance of an obr - src kinase - alpha v beta 5 cross talk in leptin mediated functional effects in enhancing the angiogenic potential of circulating angiogenic cells ( cacs ) . more importantly , cacs derived from obese , hyperleptinemic individuals were associated with relative insensitivity to the angiogenic effects of leptin . leptin induced epcs and no production has been shown to play critical roles in melanoma tumour growth induction . extending these findings to tumour angiogenesis , recent studies have implicated intratumoral leptin to exert proangiogenic effects stimulating tube formation and proliferation of endothelial cells . more importantly , the authors have also demonstrated the therapeutic potential of a peptide obr antagonist in inhibiting these proangiogenic effects of leptin via the vegf pathway . interestingly , leptin induced proliferation / migration as well as expression of proangiogenic molecules in breast cancer has been recently demonstrated to involve extensive crosstalk between notch and interleukin-1 ( nilco ) pathways . leptin has been shown to upregulate various mediators of vascular inflammation like tnf- , il-2 , il-6 , mcp-1 , ros , th1-type cytokines , and tgf- from endothelial cells and pbmcs [ 56 , 108110 ] . in vitro studies have demonstrated leptin induced increases in tissue factor ( tf ) and cellular adhesion molecules ( cams ) expression in human coronary endothelial cells ( hcaecs ) via nf- leading to increased procoagulant activity and leukocyte adhesion . additional molecules pivotal in vascular inflammation including pai-1 ( plasminogen activator inhibitor-1 ) and p - selectin have been documented to be induced upon leptin treatment [ 112 , 113 ] . clinical studies have shown a positive correlation with pai-1 , vwf , tpa , and plasma fibrinogen levels and an inverse relationship with protein c and tissue factor pathway inhibitor . these findings clearly demonstrate a strong link with circulating leptin and increased platelet activity observed in the metabolic syndrome [ 114117 ] . it is therefore not surprising to note the decreased incidence of atherosclerosis in hyperlipidaemic mice ( ob / ob ; apoe ) . as discussed previously , converse actions of leptin and adiponectin in the vascular system thereby , the leptin to adiponectin ratio ( l : a ) is higher in these subjects . various clinical studies have been conducted to elucidate the relationship between l : a ratio and markers of atherosclerotic disease including carotid intima media thickness ( cimt ) and pulse wave velocity [ 119121 ] . in yet another clinical study , l : a ratio has been demonstrated as a useful biomarker for the prevalence of metabolic syndrome , in comparison with either leptin or adiponectin levels on their own . additionally , visceral fat mass and cardiorespiratory fitness levels have been documented to influence this ratio . subjects with enos polymorphisms with or without hyperinsulinemia have a higher l : a ratio and are more prone for cardiovascular events , suggesting a genetic link in the associated risk factors . have demonstrated that high serum l / a ratio is positively correlated with serum triglyceride levels , serving as surrogate markers of vascular inflammation in at - risk young severely obese individuals , which is independent of waist circumference ( wc ) and bmi . additionally , l : a ratio represented a powerful independent predictor of intima media thickness ( imt ) , correlating with anthropometric , metabolic , and clinical parameters . furthermore , components of the metabolic syndrome were correlated positively with leptin / hmw adiponectin ratio , independent of parameters including age , smoking status , exercise , low - density lipoprotein ( ldl ) cholesterol , and bmi . however , in contrary to the above mentioned findings , l : a ratio failed to establish any significant differences in disease parameters , in a study conducted in patients with severe coronary heart disease . it is noteworthy to mention that these above mentioned studies do differ in patient characteristics and pathological parameters leading to opposing results . adipose tissue secreted factors or adipokines have been implicated in facilitating communication between adipose tissue and vasculature comprising the adipovascular axis . both proinflammatory and anti - inflammatory activities of these secreted adipokines seem to be crucial in creating a homeostatic response which remains disturbed in states of adipose tissue expansion . in addition to alterations in the circulating levels , the local ( i.e. , tissue concentration ) availability of the activated forms of these adipokines has a significant bearing in influencing vascular function . for example , it is important to consider the actions of locally available gad fragment of adiponectin , which could potentially drive leptin - induced effects . in - depth understanding of the mechanisms and properties of adipokine - receptor interactions and downstream signalling cascades studies investigating the vascular effects of various multimeric / cleaved forms of adipokines will help in developing novel therapeutic strategies and targets in counteracting obesity - related metabolic and cvds . additionally large multicentric clinical studies with strict inclusion - exclusion metabolic criteria need to be performed .

obesity is a major health burden with an increased risk of cardiovascular morbidity and mortality . endothelial dysfunction is pivotal to the development of cardiovascular disease ( cvd ) . in relation to this , adipose tissue secreted factors termed adipokines have been reported to modulate endothelial dysfunction . in this review , we focus on two of the most abundant circulating adipokines , that is , leptin and adiponectin , in the development of endothelial dysfunction . leptin has been documented to influence a multitude of organ systems , that is , central nervous system ( appetite regulation , satiety factor ) and cardiovascular system ( endothelial dysfunction leading to atherosclerosis ) . adiponectin , circulating at a much higher concentration , exists in different molecular weight forms , essentially made up of the collagenous fraction and a globular domain , the latter being investigated minimally for its involvement in proinflammatory processes including activation of nf- and endothelial adhesion molecules . the opposing actions of the two forms of adiponectin in endothelial cells have been recently demonstrated . additionally , a local and systemic change to multimeric forms of adiponectin has gained importance . thus detailed investigations on the potential interplay between these adipokines would likely result in better understanding of the missing links connecting cvd , adipokines , and obesity .

1. Introduction 2. Obesity and Molecular Aspects of Endothelial Dysfunction 3. Adiponectin 4. Leptin 5. Conclusions/Future Directions

obesity is associated with elevated free fatty acid levels , leading to the development of insulin resistance , diabetes , and cardiovascular disease ( cvd ) [ 13 ] . the development of cvd is characterised by impaired nitric oxide ( no ) release from vascular endothelium and decreased blood flow to insulin target tissues resulting in insulin resistance , termed as endothelial dysfunction . in this respect , increasing attention has been paid to the direct vascular effects of adipose tissue ( at ) derived factors termed a few of these adipokines are characterised by their favourable effects to maintain the body 's energy and vascular homeostasis ; however , adipokines have also been implicated in the pathogenesis of obesity - related disorders , such as atherosclerosis , specifically , by increasing the expression of proangiogenic / proatherogenic factors like endothelial gelatinases ( matrix metalloproteinases 2 and 9 ) and vascular endothelial growth factor ( vegf ) . leptin , adiponectin , monocyte chemoattractant protein- ( mcp- ) 1 , plasminogen activator inhibitor- ( pai- ) 1 , tumour necrosis factor ( tnf ) , interleukin- ( il- ) 6 , and resistin are a few of these adipokines implicated in endothelial dysfunction . endothelial dysfunction in obesity is characterised by increased generation of oxygen - derived free radicals ( ros ) . enzymes of mitochondrial electron transport chain , xanthine oxidase , cyclooxygenases , lipoxygenases , myeloperoxidases , cytochrome p450 monooxygenase , heme oxygenases , peroxidases , and nad(p)h oxidases contribute to endothelial dysfunction . uncoupling of endothelial nitric oxide synthase ( enos ) is a major contributor to ros production . this results in decreased no ( nitric oxide ) bioavailability , increased o2 production , and formation of peroxynitrite ( onoo ) , a key mediator of lipid peroxidation and foam cell formation in atherosclerotic lesions . additionally , ros accumulation results in activation of signalling cascades that regulate transcription factors , including nf- ( nuclear factor kappa beta ) adhesion molecules , chemotactic factors , antioxidant enzymes , and vasoactive substances promoting adhesion and migration of circulating monocytes initiating atherosclerotic lesions . dysregulated adipokine production leading to increased ros generation forms a major feedback loop in initiation , maintenance , and progression of endothelial dysfunction . adiponectin and leptin are the two widely studied most - abundant , circulating adipokines . in this review , we discuss the diverse roles of leptin and adiponectin in endothelial dysfunction with emphasis on proangiogenic / proatherogenic factors in the endothelial cells . adiponectin , the most abundantly secreted adipokine ( 220 g / ml in circulating plasma ) , was first identified as acrp30adipose complement - related protein of 30 kda because of its high similarity to complement protein . adiponectin exerts its insulin - sensitizing effects by increasing -oxidation of fatty acids , in the process reducing serum triglyceride and levels of free - fatty acids , and thus indirectly improving insulin sensitivity of the liver . circulating low adiponectin levels ( hypoadiponectinemia ) adiponectin is a 247-amino acid protein with four domains , an amino - terminal signal sequence , a variable region , a collagenous domain , and a carboxy - terminal globular domain [ 13 , 1719 ] , and undergoes posttranslational modifications within the adipocytes into multimeric forms including trimers , hexamers , and high - molecular - weight ( hmw ) oligomers . more importantly , cleavage of globular domain of full - length adiponectin ( fad ) by activated monocytes has been reported to conversely affect the protective role of fad . both globular adiponectin ( gad ) and fad exert their effects via transmembrane g - protein coupled receptors , adiponectin receptor 1 ( adipor1 ) , and adiponectin receptor 2 ( adipor2 ) . overexpression and gene knockout experiments in rodents have demonstrated the ability of these receptors to activate amp - activated protein kinase ( ampk ) , p38 mitogen activated protein kinase ( p38 mapk ) , and peroxisome - proliferator - activated receptor- ( ppar- ) and to stimulate fatty acid oxidation and glucose uptake in murine hepatocytes and c2c12 myocytes . globular , trimeric , and high - molecular - weight ( hmw ) adiponectin forms activate different signal transduction pathways . additionally , osmotin , a plant protein with structural similarities to mammalian globular adiponectin , binds to adiponectin receptors and activates ampk in c2c12 myocytes . exercise training increases adipor1/r2 mrna expression in human skeletal muscle , whereas no significant change has been reported in human subcutaneous adipose tissue during calorie restriction . another adiponectin - binding protein with a preference for hmw adiponectin multimers and no affinity for the trimeric adiponectin has been identified as t - cadherin . since this protein is a glycosylphosphatidylinositol - anchored extracellular protein devoid of any intracellular domain , the mechanism explaining its role in adiponectin intracellular signalling has not been fully clarified . leptin has been shown to exert atherogenic , thrombotic , and angiogenic actions on the vasculature [ 4850 ] . it has been linked extensively with obesity leading to cvds including atherosclerosis , myocardial infarction , and stroke [ 51 , 52 ] . leptin acts on target cells through plasma membrane receptors ( figure 2 ) and exists in at least six isoforms , ob - ra through ob - rf , signalling predominantly via jak / stat ( janus kinases / signal transducers , and activators of transcription ) pathway . functional leptin receptors ( both short and long forms of ob - r ) have been identified on endothelial cells [ 38 , 54 ] , and numerous studies link the possible mechanisms responsible for leptin - induced endothelial dysfunction . jak-2/irs-2/pi3-k / akt pathways and nuclear translocation of stat ( signal transducer and activator of transcription ) proteins have been implicated to play a pivotal role in leptin - mediated effects in endothelial cells [ 53 , 55 ] . interestingly , in states of obesity despite a paradoxical impairment of the satiety response , leptin resistance does not extend to leptin mediated endothelial dysfunction [ 5659 ] . although this selective response of leptin has gained much attention , the molecular basis remains poorly understood . decreased production of no by the endothelial cells is considered as the hallmark of endothelial dysfunction . adiponectin has been reported to increase no production in endothelial cells by the activation of phosphotidylinositol-3 ( pi-3 ) kinase / akt signalling pathway . furthermore , the involvement of ampk and protein kinase a [ pka , or cyclic amp- ( camp- ) dependent protein kinase ] signalling have also been implicated to play a major role in both adiponectin induced no production and suppression of endothelial ros generation , inhibiting endothelial nf- ( nuclear factor kappa beta ) signalling [ 32 , 6265 ] . adiponectin and lectin - like oxidized ldl ( ox - ldl ) receptor- ( lox- ) 1 have been demonstrated to exhibit a reciprocal pattern in states of endothelial dysfunction and inflammatory insults . additionally , in vivo studies have indicated the critical role of adiponectin in alleviating sepsis - induced microvascular dysfunction leading to blood brain barrier ( bbb ) dysfunction in the mouse brain via modulation of e - selectin expression . more importantly , to simulate pathological states of obesity and diabetes , the role of adiponectin in hyperglycaemic / hyperinsulinaemic environments has been studied . it is interesting to note that adiponectin induced activation of enos as well as increased production of no by the endothelial cells is crucial in mediating its anti - inflammatory effects . in this context , in vivo studies have demonstrated that pharmacological blockade of enos leads to decreased protective effect imparted by adiponectin , leading to increased leukocyte adhesion by tnf- . endothelial dysfunction includes the activation of endothelial adhesion molecular cascade critical in facilitating the entry of macrophages into the vessel wall . circulating low adiponectin levels in metabolic diseases like obesity and diabetes have been linked to the triggering of an inflammatory signalling cascade , leading to the early development of atherosclerosis . the development of a similar scenario in adipoq/ ( adiponectin knock - out mice ) mice further strengthens the association between adiponectin and cvd . furthermore , adiponectin has been shown to inhibit the vascular inflammatory response of endothelium to tnf- induced activation of nf- and increased expression of adhesion molecules vascular cell adhesion molecule ( vcam-1 ) , intercellular adhesion molecule ( icam-1 ) , and endothelial selectin ( e - selectin ) . adiponectin has been shown to induce in vitro angiogenesis in endothelial cells via ampk - enos pathway . on the other hand , other groups have demonstrated the potent inhibition of endothelial angiogenic events like migration and proliferation by adiponectin , involving mapk and camp - pka pathways . numerous studies have implicated the vasoprotective actions of fad by reducing the expression of endothelial adhesion molecules and inhibiting tnf- induced cytokine production from macrophages via nf-/camp - dependent pathway [ 32 , 7880 ] . animal studies in ad ( mice completely lacking adiponectin ) and ad ( adiponectin - hemizygous mice ) mice showed an increased expression of e - selectins and vcams . adiponectin has been demonstrated to suppress vegf - stimulated hcaec migration via camp / pka - dependent signalling . however , three independent studies have demonstrated that gad activates nf- leading to activation of the proinflammatory adhesion cascade , proliferation , and increased procoagulability in endothelial cells and cardiac fibroblasts [ 39 , 40 , 81 ] . have demonstrated that adiponectin induces interaction between lymphotoxin- ( lt- ) b receptor ( ltbr ) and human adipor1 , subsequently resulting in inhibition of the nf- pathway . this local generation of gad at sites of inflammation , namely , in atherosclerotic lesions , could be having pathophysiological relevance given the differential actions of multimeric forms of adiponectin . however , in contrast to fad , gad activated nf- and p38 mapk signalling pathways , resulting in cyclooxygenase-2 ( cox-2 ) production and subsequently prostacyclin 2 [ pgi ] release . this study further demonstrated that monocyte - endothelial adhesion enhanced by gad remained unaffected with either abrogation of adipor1 [ sirna ] signalling or cox-2 [ sirna ] downregulation , thereby suggesting independent mechanisms governing actions of fad and gad . the obvious discrepancies between the experimental outcomes could be due to the differences in the forms of adiponectin used ( table 1 ) . recently , we undertook a study to investigate the effect of gad and fad ( figure 3 ) on endothelial cell proliferation as well as in vitro migration and angiogenesis in relation to the induction of endothelial angiogenic factors , specifically , mmp-2 , mmp-9 , and vegf ; furthermore , we examined the involvement of the adiponectin receptors , that is , adiponectin receptor 1 ( adipor1 ) and adiponectin receptor 2 ( adipor2 ) , within this context . more importantly , given the connection between the coexistence of hyperglycaemia and systemic inflammation with vascular disease in pathological states such as diabetes mellitus , we also studied the interaction between glucose and c - reactive protein ( crp ) [ a potent proinflammatory protein ] , respectively , with gad and fad . finally , since amp - activated protein kinase ( ampk ) , a stress - activated protein kinase , and akt have been implicated as critical mediators of adiponectin induced angiogenesis in both normoxic and ischemic tissues we found that gad led to a significant increase in in vitro endothelial proliferation , migration , and angiogenesis with concomitant increase in mmp-2 , mmp-9 , and vegf gene and protein production , as well as mmp-2 and mmp-9 activation . on the other hand , only endothelial cell proliferation was significantly increased by fad and appears to be mediated by adipor2 ; no significant effects on mmp-2 , mmp-9 , and vegf were observed . thus it seems imperative to study the local effects of various multimers of adiponectin in situ , for instance , in atherosclerotic plaques , to ascertain the potential pro / anti - inflammatory actions of this adipokine . on engagement with leptin receptors expressed on vascular cell walls , leptin induces oxidative stress responses , increases mcp-1 , tnf- , il-6 , and endothelin-1 , and potentiates proliferation , along with the expression of other endothelial cell adhesion molecules , mmps , vegf , and impaired smooth - muscle cell function , resulting in impaired endothelium - dependent vasodilatation promoting hypertension and atherosclerosis . a plausible explanation for this could be attributed to the acute and chronic effects of leptin on the vasculature . more importantly , hyperleptinemia induced endothelial dysfunction may play a crucial role in the differential actions of leptin . as mentioned , leptin has been shown to induce oxidative stress by increasing the formation of reactive oxygen species ( ros ) , a key mediator of endothelial dysfunction [ 42 , 93 ] . this generated ros has potent peroxidant effects and thereby reduces the bioavailability of no in aortic endothelial cells , vascular smooth muscle cells , and macrophages . additionally , ros further contributes to endothelial dysfunction by upregulating proinflammatory cascades including adhesion and chemotactic pathways in endothelial cells . this could be attributed to the disturbance in the fine balance between the sympathetic nervous system and endothelial cell mediated regulation of vasomotor tone . with respect to the regulation of leptin receptors and endothelial dysfunction , a study by park et al . 2012 has revealed that leptin receptors in coronary arterioles are downregulated in high - fat fed sedentary mice leading to endothelial dysfunction . however , when subjected to exercise , the expression of leptin receptors in coronary arterioles was restored along with maintenance of enos phosphorylation , leptin sensitivity , and redox balance . as mentioned previously , leptin - mediated actions in endothelial cells , including angiogenesis , it is interesting to note the increased expressions of both ob - r and mmps in atherosclerotic plaques , particularly the endothelial lining of neointimal regions , suggesting the role of leptin in mediating aberrant angiogenesis . both in vivo and in vitro studies have demonstrated the activation of endothelial ob - r by leptin , leading to capillary tube formation , a prerequisite for angiogenesis . showed that leptin induced activation of mitogen - activated protein kinase family erk1/2 leads to an increase in endothelial cell viability in serum - free media . leptin has been shown to upregulate key proangiogenic molecules like the gelatinases ( mmps , mmp-2/-9 ) and timps . additionally , leptin has been shown to upregulate and act synergistically with the key angiogenic mediators like fgf-2 , vegf , and its receptor vegfr1 , stimulating vascular permeability , consequently resulting in functional angiogenesis . it is important to note that wound healing disorder ( due to deficient angiogenesis ) in ob / ob mice is corrected by both topical and systemic leptin administration but not in fa / fa zucker rats ( rats with a recessive trait of the leptin receptor ) , due to the absence of functional leptin receptors . in a recent study involving an obese nzo ( mice with phosphatidylcholine transfer protein mutation leading to abnormal lipid homeostasis ) mice model , the angiogenic potential of leptin was found to be insignificant , perhaps due to the relative inactivity of its receptor in these mice . extending these findings to tumour angiogenesis , recent studies have implicated intratumoral leptin to exert proangiogenic effects stimulating tube formation and proliferation of endothelial cells . interestingly , leptin induced proliferation / migration as well as expression of proangiogenic molecules in breast cancer has been recently demonstrated to involve extensive crosstalk between notch and interleukin-1 ( nilco ) pathways . leptin has been shown to upregulate various mediators of vascular inflammation like tnf- , il-2 , il-6 , mcp-1 , ros , th1-type cytokines , and tgf- from endothelial cells and pbmcs [ 56 , 108110 ] . in vitro studies have demonstrated leptin induced increases in tissue factor ( tf ) and cellular adhesion molecules ( cams ) expression in human coronary endothelial cells ( hcaecs ) via nf- leading to increased procoagulant activity and leukocyte adhesion . additional molecules pivotal in vascular inflammation including pai-1 ( plasminogen activator inhibitor-1 ) and p - selectin have been documented to be induced upon leptin treatment [ 112 , 113 ] . these findings clearly demonstrate a strong link with circulating leptin and increased platelet activity observed in the metabolic syndrome [ 114117 ] . as discussed previously , converse actions of leptin and adiponectin in the vascular system thereby , the leptin to adiponectin ratio ( l : a ) is higher in these subjects . in yet another clinical study , l : a ratio has been demonstrated as a useful biomarker for the prevalence of metabolic syndrome , in comparison with either leptin or adiponectin levels on their own . additionally , visceral fat mass and cardiorespiratory fitness levels have been documented to influence this ratio . furthermore , components of the metabolic syndrome were correlated positively with leptin / hmw adiponectin ratio , independent of parameters including age , smoking status , exercise , low - density lipoprotein ( ldl ) cholesterol , and bmi . adipose tissue secreted factors or adipokines have been implicated in facilitating communication between adipose tissue and vasculature comprising the adipovascular axis . , tissue concentration ) availability of the activated forms of these adipokines has a significant bearing in influencing vascular function . for example , it is important to consider the actions of locally available gad fragment of adiponectin , which could potentially drive leptin - induced effects . in - depth understanding of the mechanisms and properties of adipokine - receptor interactions and downstream signalling cascades studies investigating the vascular effects of various multimeric / cleaved forms of adipokines will help in developing novel therapeutic strategies and targets in counteracting obesity - related metabolic and cvds .

semiconducting conjugated polymer materials offer a wealth of opportunity for the development of organic - based optoelectronics with several advantages including light weight , flexibility , low toxicity , and inexpensive fabrication for applications such as photovoltaic cells and light - emitting diodes . there remains a challenge however to achieve sufficient power conversion efficiencies and durability for viable devices . one of the key reasons behind this is the important role of material morphology and conformation within such materials . while it is understood that many of the desirable properties , such as the control over solubility allowing solution processing and low bulk modulus providing for flexibility , arise due to the polymeric nature of the constituent molecules and the inherently statistical nature of their mixing , it is often the case that this same principle can lead to the existence of phenomena such as deep - tail trap - states which can inhibit the conductivity . this is primarily due to the delicate physics of both intramolecular conjugation - its sensitivity to local distortions along the backbone of a polymer and the effect this has on the resulting optical absorption and emission dynamics - and the intermolecular excitation transfer dynamics - the interplay of alignment and separation of conjugated segments or chromophores and their spectral overlaps with the frster - type transfer of excitons and polarons . these questions of the interplay of the dynamics and statistical mechanics of conjugated polymer - based materials suggest the application of molecular dynamics ( md ) to understand and predict macroscopic properties based on detailed knowledge on an atomic scale . in such simulations , in lieu of an explicit quantum mechanical treatment , the classical dynamics of a molecule are generated using force - fields , which appropriately describe the averaged effect of the molecular electrons on the covalent bonding and van der waal type forces . this significantly reduces the computational expense of analyzing questions of conformational properties and , when coupled with modern computer power , allows for the simulation of reasonably long chains . it is imperative that the force - fields used to describe molecular motion are of sufficiently high accuracy to reproduce observed experimental behavior . historically , md methods are often designed with biochemical simulations in mind , e.g. dynamical simulations of large proteins , and , as such , there exists a number of available force - fields - each parametrized in order to optimize their efficacy . while these force - fields contain many parameters which are transferable to the polymer system , certain aspects require a careful reparametrization due to the conjugated nature of the molecular physics involved . the challenge is to utilize as many transferable parameters as is possible while identifying which parameters require further attention and reparametrization . arguably , the most important term to be considered is the energetic profile governing the dihedral dynamics between monomers in a conjugated system . accurate modeling of the dihedral profile and energy barriers between conformers is of utmost importance as the excited - state landscape of conjugated molecules is governed by the dihedral angles , and , as such , the optical properties of the materials are crucially dependent on these torsions . another key element to be considered is how interatomic electrostatic interactions , which arise due to local deviations in electronic charge densities , are described . while there exist a number of such fitting schemes , the resp scheme is generally considered to be one of the more robust and accurate procedures for this task . the majority of methodological approaches for generating parameters for md simulations of conjugated polymers give strong weight to the importance of the acquisition of appropriate intermonomer dihedral angle profiles and partial charges in order to obtain accurate predictions from the resulting md . however , many of these sources provide conflicting viewpoints on the appropriate levels of quantum chemical theory required . furthermore , there is often a lack of systematic parameter development and benchmarking of given methodology , and , as such , there is a large degree of ambiguity in the accuracy of a given method as well as whether or not a prescribed computational route could be replaced by a significantly less computationally expensive one . given the wide range of organic molecules which are of interest for applications the computational cost and complexity of obtaining parameters is a constraining barrier to progress through the exploitation of md . a further point that has , to date , been neglected is how sensitive a given parametrization scheme is to variation in length of molecules and lengths of their associated alkyl side chains . a typical solution - cast mixture of conjugated polymers requires the attachment of branched alkyl side chains to provide solubility . it has also been shown that these side chains and their interference with the intrinsic molecular motion of the conjugated backbone of the molecule have a key role in effects such as the inhibition of excitonic diffusion as well as the emergence of exotic bulk behavior such as the well - studied -phase of polyfluorene . in light of the above , the work presented here establishes a systematic approach to the force - field parametrization . in addition , our methodology also determines the domain of applicability of a given set of parameters for molecules of varying length and varying length of side chain . we also highlight cases in which the universality of a given parameter set to these variations is broken . in doing so , we put forward a parametrization protocol which conforms sufficiently to established benchmarks of accuracy ; avoids unnecessarily computationally intensive calculation methods ; and may be applied , in extension , to any type of conjugated system with alkyl side chains . a minimal set of force - field parameters contains parameters of five types : three describing the energetics due to covalent bonding between atoms and two describing noncovalent interactions . the three covalent terms quantify bond - stretching , changes in the angle between three atoms ( angle - bending ) , and changes in the dihedral angles between four atoms . these are modeled by functions ranging from quadratic ( particularly for two - atom vibrations ) to fourier - based functions for the angular types . noncovalent interactions are of the form of lennard - jones and coulomb potentials which account for london dispersion , pauli repulsion , and , in the case of the coulomb potentials , electrostatic interactions between local variations in electronic density . we choose the opls force - field parameter scheme ( as implemented in gromacs 4.6.5 ) as our starting point due to its provision of parameters for many atoms in a multitude of different molecular frameworks as well as its use in previous works to parametrize conjugated polymers . to parametrize a given molecule , the first step is to build on the appropriate parameters found within opls for a monomer . for example , to build a molecule containing two connected units of thiophene ( hereafter referred to as a 2mer and , for molecules with x connected units , as an xmer ) , the opls parameters for each atom of an individual thiophene unit are implemented for each unit . this leaves only the bonds , angles , and dihedrals associated with the linking bond between the two units and partial charges to be determined . schematic highlighting the required new parameters in going from a parameter set for thiophene to dithiophene . the missing force - field parameters are the bond - stretching term between the two carbons circled in red ; the angle - bending term between each combination of three blue - circled atoms ( two from one unit and one from the other ) ; and the dihedral angle term for each combination of four blue - circled atoms ( two from one unit and two from the other ) . we expect that bond - stretching and angle - bending within the monomer are reliably parametrized by the opls force - field terms thus the intermonomer dihedral profile is the primary unknown from the covalent terms . in the case of a nonbonding interaction , we can use existing lennard - jones terms without any modifications and focus on partial charge specification and validation . subsequent sections deal with the details of dihedral energy profile calculation ( section 3 ) , partial charge generation ( section 4 ) , implementation of modifications ( section 5 ) , and preliminary md calculations performed with our obtained parameter sets ( section 6 ) . we also provide a further example of how our scheme might be applied for the copolymer f8bt in section 7 . in order to generate sufficiently accurate dihedral potentials at low computational expense , we utilize the method of performing geometry optimizations over the span of a dihedral rotation ( scan ) using computationally inexpensive techniques and refine these results with a further single - point energy ( sp ) calculation using more accurate methods . we find that the dihedral potentials for all lengths of conjugated backbone are accurately described by those of their corresponding 2mer . we generate partial charges using the resp method with input electronic densities calculated using the above scan - sp approach . with respect to molecules of varying backbone length , it is possible to generate a set of charges which are generalizable to any length of molecule . by considering the progression with length of the net charges on the internal monomer units , we can determinine the length at which the net charges go to zero . this indicates converged charge distributions which can be built into a general parameter set . with respect to variations of side - chains , there appears to be no means of generalizing such variations beyond recalculation . once the appropriate partial charges have been determined , these are directly implemented into the force - field . the dihedral profile , on the other hand , must be implemented by means of a so as to ensure that energetic terms already described by the force - field and partial charges are not double - counted . this is , in spirit , performing an analogous scan using the force - field parameters ( without the required dihedral ) in order to obtain the contribution already accounted for by the force - field and subtracting this from the calculated dft profile . we choose fluorene and thiophene oligomers as our model systems with and without their alkyl side - chains ( see figure 2 ) . these molecules are experimentally well - characterized conjugated systems against which to test our methodology . schematic of ( a ) fluorene and ( b ) thiophene 2mers with the associated dihedral angle , , highlighted in each case . to obtain a reliable dihedral potential , the ethos of the scan - sp approach is to use lower levels of theory such as dft for obtaining geometries and higher levels of theory , for example , local methods ( mp2 , ccsd , ccsd(t ) ) or dft with a larger basis set for sp energy calculations . the first step , the scan , involves determining , by relaxed scan geometry optimization , a series of molecular geometries for different values of dihedral angle between the units of a 2mer . geometry optimization can become a computationally cumbersome process , especially when large or exotic molecules are considered . however , in the case of many conjugated molecules , the calculation of reliable geometries for molecules of considerable length has been shown to be practically possible using moderate levels of theory . while it is possible to obtain accurate geometries in the scan step , as we will go on to show , the energies obtained from this step are often quite inaccurate . this is why the second step , the sp step , using higher levels of theory in order to obtain accurate energetic profiles is necessary . for all dft calculations , we have determined that this initial choice performs comparably well to a number of dft functionals as well as more involved methodology , such as mp2 , ccsd(t ) , and density - fitted and local versions of each . details of these tests and comparisons are provided in the supporting information ( section 1.1 ) . to account for the possible contributions from dispersion interactions not natively incorporated in dft methods , we have also repeated our dft calculations utilizing the gd3bj dispersion correction functional of grimme et al . in cases with small ( up to ethyl ) side - chains , we find that dispersion effects are negligible . as such , the following results do not include these correction terms , and a more thorough breakdown of our gd3bj results is given in the supporting information ( section 1.3 ) . following our scan - sp approach , we have calculated 2mer dihedral profiles using various basis sets from the 6 - 31 g family with added polarization and diffuse functions for scans and the cc - pvtz basis set for sp calculations . our results are also compared against the benchmark cbs - limit ccsd(t ) calculations of bloom et al . for thiophene . this first step allows us to ascertain the appropriate level of theory required for further extending our investigation . throughout this work we utilize the polymer convention for dihedral angle labeling this convention casts the trans conformation at 0 and the cis conformation at 180. dihedral potentials taken from other works have been transformed so as to fit with this convention . a full breakdown of the various methodological choices made is given in the supporting information ( section 1.2 ) . for both fluorene and thiophene ( figure 3(a ) and ( b ) respectively ) , comparison of our choices is made against a scan performed using cc - pvtz for both scan and sp ( herein referred to as the cc - pvtz result ) , and further comparison is made to benchmark results , obtained by bloom et al . , performed with mp2/aug - cc - pvtz for the scan and ccsd(t ) in the complete basis set ( cbs ) limit for the sp ( herein referred to as the ccsd(t)/cbs result ) . it can be seen from figure 3(b ) that the cc - pvtz result of thiophene is in good agreement with the ccsd(t)/cbs result . as performing such a high - level calculation as that of bloom et al . for as large a molecule as fluorene is computationally prohibitive , we use thecc - pvtz result for fluorene as a benchmark for our results . dihedral profiles from geometry scans for cam - b3lyp with various basis sets in ( a ) fluorene and ( b ) thiophene 2mers . those with additional cc - pvtz single - point calculations full geometry optimizations using cc - pvtz are also included and in ( b ) a comparison is made to the ccsd(t)/cbs thiophene result of bloom et al . from the results for fluorene ( figure 3(a ) ) , it is observed that the energetics of the geometry optimization alone are quite inaccurate when compared to the cc - pvtz result : those without diffuse functions considerably overestimate ( 2 kj / mol ) both the barriers at the planar conformations and the barrier at 90 ; and those with diffuse functions overestimate the planar barrier but considerably underestimate the 90 barrier to a similar degree . for thiophene ( figure 3(b ) ) , those which include diffuse functions in the basis set provide considerably less accurate results than those without . using the diffuse functions , the energetics of the conjugation barrier ( 90 ) appear to be underestimated when compared to the cc - pvtz and ccsd(t)/cbs results . this difference is 23 kj / mol which at 2030% of the barrier itself is a significant discrepancy . however , it is also observed that none of the 6 - 31g - type basis sets exhibit suitable accuracy as both overestimate the height of the 180 energy barrier in thiophene by 2 kj / mol . that while there exists significant deviation in the energetic barrier heights due to the inclusion of diffuse functions , upon performing sp calculations with cc - pvtz this difference becomes negligible . this implies that all discussed choices of the basis set provide very similar geometries and that the final accuracy of the dihedral profile is more dependent on the choice of the basis set for the following sp calculation . it follows from this that the appropriate choice for geometry optimization is 6 - 31 g * due to it being considerably less computationally expensive than the others . utilizing a further sp calculation is found to offer significant improvement to the resulting potentials . in thiophene , it is observed that the error in the sp curves is 50% of that of the initial 6 - 31 g * calculation when compared to the ccsd(t ) curve . for the largest deviation , at the 180 barrier , this translates to a reduction of the absolute error to < 1 kj / mol from 2 kj / mol . when compared to the 6 - 31+g * * curve , this error reduction is considerably greater . comparison is also made to the results of a full optimization using cc - pvtz ( i.e. , performing the scan step with cc - pvtz without any further sp calculation ) . this comparison shows that the results of our scan - sp approach are almost identical to that of using cc - pvtz for geometry optimization . this lends further credence to the idea that reliable geometries are obtained from basis set choices as low as 6 - 31g*. with the above in mind , our choice of cam - b3lyp/6 - 31 g * scans and cam - b3lyp / cc - pvtz sp s is well justified . now that a computational methodology is established , we examine to what extent the dihedral profile is sensitive to the length of the conjugated backbone . figure 4 demonstrates the invariance of the calculated dihedral profile of the end - most intermonomer junction for both molecules over a backbone length range of 210 units . in fluorene , there is almost no dependence on chain length , while in thiophene there is a very slight deviation of < 1 kj / mol in the energetic barrier heights . we attribute this deviation to the dispersive contribution of the additional neighboring thiophene . as the thiophene units are considerably smaller than those of fluorene , there is a far smaller separation between the end dihedral and the third unit which , in turn , leads to long - range dispersive forces between the third unit and the first unit which are strongest at the 90 position . dihedral profiles for various backbone lengths of ( a ) fluorene and ( b ) thiophene . we also demonstrated that the dihedral energetic profile is invariant to its position along a chain longer than a 2mer and that a profile is invariant to the value of the neighboring dihedral angle . these are shown and discussed in detail in the supporting information ( section 1.4 ) . thus , we have shown that we can utilize the same dihedral energy profile for all intermonomer dihedrals within any length of molecule . we now turn to the influence of alkyl side chains on the dihedral energy profiles . scans have been performed for side chains of lengths varying from 1 to 10 units . in the case of fluorene , initial geometries with side chains on opposing sides of the molecule have been chosen . there are two reasons for this : first , the inclusion of dispersion interactions in dft calculations of this type often lead to convergence problems as the relatively high flexibility of the side chains allows for many possible local minimal conformations . due to this , the calculations both become increasingly computationally expensive and generate results which are strongly dependent on initial conditions , resulting in ambiguity in their translation into force - field parameters . second , by effectively removing the side chain side chain interactions , what is observed is the effect of the presence of the side chains on the electronic properties of the backbone . for the purposes of generating good force - fields for md calculations , this is the crucial point as this determines whether or not , in principle , the bare dihedrals along the molecular backbone require modification due to the presence of the side chain . with the above considered , figure 5(a ) gives the resulting dihedral profiles calculated for fluorene with various lengths of side - chain . we observe that increasing side - chain length has very little effect on the energetics of the dihedral . however , in the case of thiophene ( figure 5(b ) ) , the problem becomes considerably more complex due to the close proximity of the side - chain to the dihedral itself . in this case fluorene has side - chains much further from the center of dihedral rotation than thiophene . as such , the notion of separating long - range interactions from those due to the electronic conjugation is considerably more complicated for thiophene , while it does not pose any issues in fluorene . dihedral profiles for 2mers of ( a ) fluorene , ( b ) thiophene , and ( c ) the side - chain(s ) are labeled by a chemical formula e.g. h1 and c5h11 refer to no side - chain(s ) and pentyl side - chain(s ) , respectively . schematic highlighting the difference in the location of the alkyl side - chains in 2mers of fluorene and thiophene . the blue area represents the location of the intermonomer dihedral ; the green represents the alkyl side - chain ; and , in thiophene , the red area highlights the steric conflict involved . the issue of this unavoidable steric repulsion creates a problem in interpreting the dihedral profile . as our parametrization is built on the ability to transfer parameters for long - range dispersive forces , the steric contribution to the dihedral profile due to the side - chains is captured by the existing terms in the force - field . as such , we wish to determine the effect of the side - chain on the covalently bound component of the dihedral rotation i.e. the effect it has on orbital conjugation as it is this component which comprises the required parameters in the force - field . in light of this ( see schematics in figure 5 . ) in the mirrored 2mer , the side - chain of one unit is moved from the 3 carbon position to the 2 carbon position so as to remove the steric conflict . the nature of the approximation is that the effect of the side - chain on the conjugation - dependent component of the dihedral energetics is the same in both cases . given the nature of the delocalized orbitals across the molecule as well as evidence suggesting that side - chains do not have a strong effect on conjugated phenomena ( which can be seen in the agreement of the fundamental transition energies and transition densities shown in section 1.5 of the supporting information ) , we conclude that this approximation is valid . figure 5(c ) depicts the energetic profile of the mirrored case , and it is found there is only a slight deviation ( 1 kj / mol ) due to the presence of side - chains . we attribute this deviation , in a similar manner to those seen in figure 4 , to small remaining dispersive forces between the mirrored side - chain and the neighboring unit . these results strongly suggest that any variance in the dihedral energetics on account of the inclusion of side - chains is mediated entirely by known long - range , noncovalent interactions . this suggests that the dihedral profile of a force - field for these molecules is invariant to the addition of side - chains though this statement is analyzed quantitatively in section 5 . we obtain partial charge by utilizing the resp scheme using the antechamber program within the ambertools 14 suite . our choice of this scheme is based both on consistency with the recommended methodology for opls parametrization as well as the robustness of the method to slight perturbations in geometry as opposed to , for example , esp charges . not only does this result in more accurate partial charges for utilization within an md force - field , the robustness of the method also allows us to accurately determine variations due to the changes in molecular environment that we are interested in . input electronic densities for the resp calculations are obtained from sp calculations with cam - b3lyp / cc - pvtz using geometries taken from cam - b3lyp/6 - 31 g * optimization . when generating parameters for md simulations of repeating structures of many units , such as polymers or oligomers , partial charges are often implemented using a three - residue model in which three sets of charges are built : one for each end unit and one for the central unit . in order to build such a model that is fully scalable to a variety of lengths , a first prerequisite is the requirement that the net charge for each individual central residue and the sum of the end residues have a net zero charge . this ensures the overall charge neutrality of the molecule and that this neutrality will remain when generating longer molecules by inserting extra central units . as it is possible for the end residues to have a nonzero net charge , if one wishes for a model with which scaling to many lengths is possible , it is necessary to determine the length of molecule at which the above criteria are met . only for molecule lengths greater than this length the remainder of this section provides an overview of the main results from our partial charge calculations and their implications . a more exhaustive breakdown of our partial charge analyses ( such as the progression of total monomer charges and individual partial charges with varying backbone and side - chain length ) is given in the supporting information ( section 2 ) . as an example , figure 7 presents the total charge of each monomer unit for 9mer of fluorene , 9,9-dioctylfluorene , thiophene , and 3-hexylthiophene . for the first three of these , the total charge for each monomer is close to zero across the entire molecule , whereas for hexylthiophene , there is a small but notable residual charge on the two end units . the key feature that distinguishes hexylthiophene from the other molecules is the breaking of reflection symmetry between each end of the molecule . at one end of the hexylthiophene molecule , the hexyl side - chain is on the side of the thiophene nearest the neighboring unit , while , at the other end , the side - chain is on the side further from the neighboring unit . this asymmetry of molecular structure forms an intrinsic asymmetry in the charge distribution of each end of the molecule i.e. a dipole exists . given the small net charge difference ( 0.1 e ) between each end , the dipole itself is not strong ; however , the distribution of charges on each of the end units due to this asymmetry is notably distinct from other units . ( see supporting information figure s14 . ) thus , if one wishes to build a three - residue model for hexylthiophene with accurate partial charges , the end residues must encompass two units of the molecule . in doing so , these two end residues will each have a nonzero net charge which cancel each other . total charge on each monomer unit of 9mers of fluorene , dioctylfluorene , thiophene , and hexylthiophene . the end monomers of hexylthiophene retain a considerably larger net charge than those of the other molecules . to generate a model which is generalizable to all lengths of side - chain , two requirements must be met : first , there must exist a length of side - chain beyond which the charge distribution of the conjugated backbone is invariant ; and , second , it must be possible to generate a standard methylene ( ch2 ) group and a methyl ( ch3 ) group which can be added in repeatedly to create longer structures . to analyze whether these requirements may be met , the charge distributions of a monomer with varying lengths of side chain have also been calculated . figure 8 displays the convergence of the total charge on the main conjugated component excluding the side chain . it is observed in both cases that such a convergence exists , and this convergence also implies that the charges on this part of the molecule are invariant . as such , the first requirement is satisfied . analyzing the charge distributions of the side - chains in this case , it is observed that generating a standardized methylene group is not possible at the lengths of side chain we have considered ( up to 10 carbons ) . this is because , while there are convergences in the end - most and innermost groups on a given side - chain , those in the middle of a long chain vary considerably as side - chain length is varied . it is seen here that the methylene group taken from the middle of the side - chain still fluctuates greatly in charge , primarily on the carbon atoms , even up to a 10 carbon long side - chain , while the two end groups have stabilized in charge . this is symptomatic of the strong asymmetry between each end of the side - chain - one is connected to a large molecule while the other is terminated only by a hydrogen . as such , if it were to be possible to generate a sufficiently accurate standard methylene group charges for subsequent additions , a much longer side - chain would be required . as lengths of side chain greater than 12 are not often used in practical settings , it remains that if one wishes to include partial charges for use with side - chains , it is necessary to compute these charges for each length of side - chain required . relationship of the total charge , excluding side - chains , of monomers of dialkylfluorene and 3-alkylthiophene of various lengths of an alkyl chain . each displays a convergence to near zero at around 6 carbons side - chain length . sample of beginning , middle , and end side - chain charges for various lengths of a side - chain in a monomer of alkylthiophene . it is observed that , in contrast to the end groups , the charge of the carbon on the inner methylene group ( yellow triangles ) does not converge with increased side - chain length . each molecular configuration is generated using pre - existing bond - stretching and angle - bending terms from the opls force - field . for both fluorene and thiophene , full sets exist for individual monomers , while only fluorene has parameters for the bonds and angles around the intermonomer junction . as they are lacking for oligo - thiophenes , the fluorene intermonomer parameters are used for the thiophene intermonomer junctions . each molecule has improper dihedral terms at junctions with one heavy ( i.e. , non - hydrogen ) atom joined to three other heavy atoms to re - enforce planarity across the monomer units as well as at the intermonomer junction . these may be more clearly observed from the gromacs topology ( .top ) files provided along with the supporting information . partial charges are implemented in the force - field by means of generating a three - residue model as described in the previous section and inputting these into the force - field parameter set . if one were to directly implement a dihedral potential , the presence of these interactions would lead to a double - counting of terms already included within the quantum chemistry calculation . in order to avoid this double - counting , it is necessary to isolate the effect of the extra interactions and subtract these from the dihedral profile . the required force - field contribution to the dihedral potential is isolated by performing scans over intervals of 10 from 0 to 180 in a manner analogous to that of the scans performed using dft . as we wish to isolate all interactions relevant in the dihedral rotation which are not the covalent interaction and also wish to restrain the dihedral at each value in the scan , the four covalent energetic functions at each intermonomer juncture are used to impose restraints . in order to generate an effective restraint at a given angle , 0 , each of the four dihedral terms are placed under the influence of a periodic potential , vr , given by1 care must be taken in choosing the value of kc so as to find a balance between forming an effective restraint without inducing any unwanted distortion to the molecule . for molecules with methyl or no side - chains , the choice of kc = 5 10 kj / mol is suitable . in the case of ethylthiophene , a large reduction is necessary kc = 10 kj / mol which we attribute to the prevalence of large forces in the side - chain - dihedral area . from this , the geometry is then optimized in vacuum using the conjugate - gradients minimization algorithm within gromacs 4.6.5 , and the total energy of each point along the scan is calculated to form the corresponding profile . with the force - field ( ff ) contribution isolated , the required dihedral profile is obtained by subtracting the ff contribution from the dft scan . the resulting subtracted profile is then fitted to a fifth - order ryckaert - bellmans function2 the fit function described in eq 2 yields two sets of parameters . this results from the difference of 180 between one pair of dihedral angles and another of the four used . for example , for a dihedral angle of 0 in the polymer convention , the dihedral angle of the two pairs of four atoms in the trans position is , while the two pairs in the cis position have a ( + 180) dihedral angle . as such , the function cosine terms in eq 2 must be modified to cos( + 180 ) = cos( ) in order to yield the appropriate energy . figure 10 provides examples of the curves obtained in the subtraction process for 2mers of fluorene , thiophene , and ethylthiophene . in the cases with no side - chains ( figure 10(a ) and ( b ) ) , it can be seen that this procedure and the fitting with the ryckaert - bellemans function result in a force - field which quantitatively mimics the dft dihedral potential to a very high degree of accuracy . subtraction profiles for ( a ) fluorene , ( b ) thiophene , and ( c ) ethylthiophene . each figure displays the calculated dft profile ; the profile obtained from the ff scan ; the resulting subtracted profile ; the fit of the subtracted profile to a fifth - order ryckaert - bellmans function ; and the resulting effective profile given by the addition of the ff scan profile and the fitted profile . ( legend applies to all graphs . ) it is noted that this procedure results in a far less smooth fit for ethylthiophene ( figure 10(c ) ) . as discussed with respect to the dft scans , the introduction of side - chains is a potential source of inconsistency in the calculations due to the relative freedom of side - chains . to minimize this inconsistency , the starting geometries are taken from the dft calculations so as to reproduce the side - chain conformations as well as possible . however , this does not , in all cases , lead to a perfect correspondence in the side - chain conformations between the dft and ff scans . as is shown in figure 10 , the subtraction procedure leads to an overall dihedral potential from the force - field which corresponds closely to that of the dft . to further test this correspondence , we have calculated the difference in energy , em , between the two local - minima ( one in the first quadrant of the dihedral and one in the second ) of each molecule from dft and our force - field and compare them in table 1 . in all instances , we find agreement to within 0.2 kj / mol ( 0.08 rt at room temperature ) . in order to achieve the agreement between the dft and ff values of e , we modified the equilibrium bond lengths and angles from the opls force - field while retaining the orginal force - constants . this measure gives a better agreement in the geometries and minimal energies obtained from the force - field . a full discussion of the implications of this modification is given in section 3 of the supporting information . as expected from the dft results for alkylfluorenes ( figure 5(a ) ) , addition of methyl side - chains to fluorene , the dihedral potential needs no further modification to accommodate for this . in section 3 , we argued that the steric interactions responsible for large changes in dihedral potential for alkylthiophenes should be incorporated by the force - field . however , as is shown in table 2 , utilizing the dihedral potential fitted from a thiophene with a shorter side - chain leads to drastically overestimated barriers at the planar positions . as such , reparametrization of the dihedral term must be performed to accommodate for this . given the tendency for inconsistency observed in ethylthiophene due to the side - chain degrees of freedom and that the difference in dft dihedral potential between ethyl- and propylthiophene is slight ( 0.5 kj / mol at the planar barriers , figure 5(b ) ) , we use the ethylthiophene potential for thiophene molecules with longer side - chains . each barrier is calculated relative to the closest local minimum ( i.e. the trans minimum for e0 and the cis minimum for e180 ) . the labels tx ( ty ) denote the energies of a 2mer with an x - yl side - chain with energetic profile taken from a y - yl side - chain scan . the dft values shown are those from the dihedral scan of the tx molecule . the force - field parameters obtained from the above procedure are available , in a form suitable for use with the gromacs md package , along with the supporting information . all md simulations were carried out using gromacs 4.6.5 with an integrator step - size of 2 fs and system coordinates sampled every 10 ps . each simulation was performed at ambient temperature and pressure ( 273.15 k , 1.01325 bar ) . in all cases , the following measures have been taken prior to the md run : steepest - descent minimization ; followed by 0.5 ns of both nvt and npt ensemble equilibration with position restraints on the heavy atoms of the molecule followed by 5 ns of unrestrained npt equilibration . we perform our calculations in a fully solvated manner i.e. in a periodic cubic box large enough to avoid any possible interactions of periodic images . in the largest molecules we have looked at ( 32 units in length ) , this condition leads to the majority of computational effort being spent on calculating the solvent dynamics . each simulation was performed with chloroform as a solvent , and solvent parameters were acquired from the molecule database at virtualchemistry.org . long - range electrostatics in all simulations have been treated with reaction - field with a dielectric constant of 4.81 for chloroform . we have found from preliminary simulations that this choice gives results comparable to those obtained using the particle - mesh ewald method with a significant reduction in the simulation run - time . full details of our tests are provided in section 4 of the supporting information . as a test of our parameter sets , we performed simulations of 32mers of fluorene with octyl side - chains ( pf8 ) and thiophene with hexyl side - chains ( p3ht ) over the course of 50 and 100 ns , respectively , in chloroform and calculated persistence lengths , np ( in number of monomer units ) and lp ( in nm ) . we perform this calculation by generating vectors , vi , across the first and last carbon of unit i and generating a correlation function , a(n ) , of the angles between each pair of vectors , n ( eq 3)3 the persistence length is defined by the e - point of a(n ) i.e.4 the correlation curves for fluorene and thiophene with their respective side - chains are shown in figure 11 . by fitting these to the exponential decay function given in eq 4 , the persistence lengths are np = 12.9 and np = 8.5 units , respectively . in order to recast these into nm , we calculate the average unit length , l , from the md simulation to be l = 0.832 nm for pf8 and l = 0.397 nm for p3ht which gives lp = 10.8 nm and lp = 3.4 nm , respectively . as a means of comparison , persistence lengths for the polymer in each case have been reported as lp = 8 1 nm for pf8 and lp = 2.4 0.3 nm for p3ht ; both measured by a combination of gel permeation chromatography and light scattering in thf solution . angle correlation functions , a(n ) , of 32mers of dioctylfluorene ( f8 ) and hexylthiophene ( t6 ) simulated in chloroform . each calculated correlation function is given along with their respective fitted curves ( dashed lines ) ( given by eq 4 ) . from the fitted curves , np , indicated by arrows corresponding to the crossing of the fitted curve and the e line , is found to be 12.9 and 8.5 , respectively . the remarkable agreement found between the md and measured persistence lengths should not be overplayed and is , to an extent , deceptive . our simulations are performed in a different solvent ( chloroform ) from the experiments ( thf ) . we make this choice as simulations with thf are 5 times more expensive computationally than those with chloroform . as we are performing our calculations in a fully solvated manner i.e. in a periodic cubic box large enough to avoid any possible interactions of periodic images , this means we have 2 10 and 2 10 solvent molecules per simulation for fluorene and thiophene , respectively . while we are currently working on means of simulating fully solvated molecules in a more efficient manner , as it currently stands , this method restricts us computationally to smaller solvent molecules such as chloroform . furthermore , we have found it is necessary to use molecules considerably larger than the persistence length in order to obtain a converged result . particularly , performing a similar simulation on a 16mer of fluorene resulted in the value lp = 19.5 units which corresponds to 16.24 nm . this values is 2 times that of the 32mer and of the polymer experiment . similar behavior was observed for thiophenes of near persistence - length lengths although this was to a considerably lesser extent ( e.g. , lp = 9.5 units for a 5mer ) . distributions of end - to - end length have also been computed from our simulations . in figure 12 , these distributions are given for 16mers of dioctylfluorene and 16mers and 32mers of hexylthiophene in chloroform . the distributions for 32mers of dioctylfluorene have not been shown as , while the tangent correlation curves were converged , after 50 ns the end - to - end length distributions were far from converged . in each example , the end - to - end length is given as a fraction , r , of the straight length of the molecule , nl , where n is the number of units and end - to - end length distributions ( solid lines ) for a 16mer of dioctylfluorene ( f8 ) and a 16mer and 32mer hexylthiophene ( t6 ) in chloroform . the end - to - end length is scaled to give each length as a fraction of the fully extended length of each molecule . comparison is given for each distribution to the distribution rpeer obtained using eq 5 ( dashed lines ) with a curve for an f8 32mer given based on the calculated persistence length . in comparing each length of thiophene , it can be seen that the 32mer has a much wider distribution which peaks at a lower length fraction ( 0.7 ) than that of the 16mer which is narrower and peaked at 0.85 . given the shorter persistence length calculated , the distribution for the 16mer of fluorene is less spread and peaked at a higher length fraction 0.9 than the 16mer and 32mer of thiophene . the distribution obtained for the 16mer of fluorene is consistent with measurements performed by muls et al . using end - marked hexylfluorenes of a similar length scale ( 42 monomer units with a polydispersity of 1.8 ) in an inert zeonex matrix , they measured end - to - end length distributions which are centered at a length fraction ( based on the fully extended 42mer ) of 0.89 . both the progression of the distributions and the difference in overall spread between fluorene and largely spread thiophene can be understood qualitatively by considering the increase in conformational entropy with increasing length and is consistent with the persistence lengths calculated previously . for a quantitative insight , we have also calculated the end - to - end length distributions , pee(r ) , using the expression derived by a path - integral approach for semiflexible polymers by wilhelm and frey5where r is the end - to - end length fraction , = np / n is the persistence length fraction , and is the normalization constant such that 01drrpee(r ) = 1 . the values of used are = 1.219 , 0.403 , 0.531 , and 0.266 for the 16mer and 32mer of fluorene and the 16mer and 32mer of thiophene , respectively . for each fluorene , the corresponding persistence length was used due to the large difference for both lengths ( as described above ) . the value calculated for the 32mer for all the distributions calculated , each md distribution agrees well with the corresponding calculated result , and we expect that this behavior would be replicated by a fully converged fluorene 32mer distribution . in order to illustrate how our scheme would be applied in practice , this section provides a working example of the calculations required to parametrize the copolymer 9,9-dioctylfluorene - alt - benzothiadiazole , commonly known as f8bt ( figure 13 ) . the example is taken as an instance where one wishes to perform md simulations on an f8bt molecule of greater than 8 units in length . before carrying out the main body of the scheme suitable parameters must be obtained ( either directly from the opls parameter set or from other sources ) for the basic force terms ( e.g. , bond - stretching , l - j terms ) . one must then generate the lists of atoms and bound pairs within the molecule based on the opls atom naming specifications ( which are intrinsically tied to l - j parameters ) . base monomer of the copolymer 9,9-dioctylfluorene - alt - benzothiadiazole ( f8bt ) . the carbons circled in red are an example of where a substitution of a covalent force term ( in this case , bond - stretching between two benzene carbons ) may need to be made . the next stage is to generate lists of the covalently bound force terms in the molecule . these are often generated by the simulation suite ( such as is the case in gromacs 4.6.5 ) . the bonds , angles , and dihedrals which do not have a direct specification must be input from choices of similar bonds . for example , the carbon carbon bond between the two central atoms of the bt unit ( marked in figure 13 ) could be taken from the parameters for bond - stretching between two carbons of a benzene ring . all internal dihedrals which are not specified are to be replaced with the dihedral potential for 4 aromatic carbons . this choice works in general as this potential serves primarily to ensure the rigidity of the monomeric structure . the first stage of the quantum chemistry calculations is obtaining a dihedral potential by performing a scan of the rotation between the fluorene ( f8 ) and benzothiadiazole ( bt ) subunits of the monomer . as shown in section 3 , the inclusion of side - chains is unnecessary and may lead to complications . with this in mind , the octyl side - chains of the f8 subunit should be replaced with hydrogens and the scan performed on the monomer shown in figure 14 ( hereafter referred to as the f0bt monomer ) . the scan is performed using cam - b3lyp/6 - 31 g * to obtain geometries and cam - b3lyp / cc - pvtz for corrected energies by sp calculation . base monomer of the copolymer fluorene - alt - benzothiadiazole ( f0bt ) which would be used for calculations of dihedral profile and in the subtraction procedure . to obtain charges , one would take an 8mer and 9mer of the f8bt molecule optimized with cam - b3lyp/6 - 31 g * and electron density calculated by sp calculation using cam - b3lyp / cc - pvtz . if there is no notable difference in the total charges of the monomers between 8mer and 9mer , then the charges are converged and will be suitable to build charge sets . the above also applies if one wishes to match the end units ( e.g. , put an extra f8 or bt on either end ) to symmetrize it . as discussed , the convergence of the charges would occur at smaller lengths than in standard f8bt . however , it is likely that using an 8mer and 9mer will be sufficient . this would be performed with the f0bt monomer in the manner described in section 5 . we have determined that it is possible to replicate dihedral potentials of high levels of theory using an economical , two - step method of cam - b3lyp/6 - 31 g * and cam - b3lyp / cc - pvtz for geometry optimization scans and single - point energy calculations , respectively . we find that dihedral potentials are invariant to the length of the molecular conjugated backbone and that any effect due to the inclusion of side - chains seems to stem from long - range interactions alone . furthermore , we find that partial charge distributions converge quickly with varying length of molecule , but one must take into consideration deviations in end monomer distributions for molecules which do not possess end - to - end reflection symmetry . in terms of varying side - chains , it seems that there is no simple way of generalizing the inclusion of partial charges to this variation . with these points in mind , we have developed a parametrization scheme which is applicable to a wide range of conjugated molecules . we have tested the transferability of parameters expected in molecules with high steric contributions to the dihedral energetics from close side - chains ( such as in 3-alkylthiophenes ) and found that delicate reparametrization is necessary , while , in molecules with side - chains far from the intermonomer junction , this careful approach is not required . our preliminary tests indicate that our md simulations are consistent with experimental measurements of persistence lengths and end - to - end lengths with long thiophene oligomers displaying far greater flexibility than their fluorene counterparts .

we describe a general scheme to obtain force - field parameters for classical molecular dynamics simulations of conjugated polymers . we identify a computationally inexpensive methodology for calculation of accurate intermonomer dihedral potentials and partial charges . our findings indicate that the use of a two - step methodology of geometry optimization and single - point energy calculations using dft methods produces potentials which compare favorably to high level theory calculation . we also report the effects of varying the conjugated backbone length and alkyl side - chain lengths on the dihedral profiles and partial charge distributions and determine the existence of converged lengths above which convergence is achieved in the force - field parameter sets . we thus determine which calculations are required for accurate parametrization and the scope of a given parameter set for variations to a given molecule . we perform simulations of long oligomers of dioctylfluorene and hexylthiophene in explicit solvent and find peristence lengths and end - length distributions consistent with experimental values .

Introduction Generating Molecular Dynamics Parameters Determining Dihedral Energy Parameters Partial Charge Calculations Force-Field Implementation Molecular Dynamics Results A Parametrization Example Conclusions

while it is understood that many of the desirable properties , such as the control over solubility allowing solution processing and low bulk modulus providing for flexibility , arise due to the polymeric nature of the constituent molecules and the inherently statistical nature of their mixing , it is often the case that this same principle can lead to the existence of phenomena such as deep - tail trap - states which can inhibit the conductivity . this is primarily due to the delicate physics of both intramolecular conjugation - its sensitivity to local distortions along the backbone of a polymer and the effect this has on the resulting optical absorption and emission dynamics - and the intermolecular excitation transfer dynamics - the interplay of alignment and separation of conjugated segments or chromophores and their spectral overlaps with the frster - type transfer of excitons and polarons . in such simulations , in lieu of an explicit quantum mechanical treatment , the classical dynamics of a molecule are generated using force - fields , which appropriately describe the averaged effect of the molecular electrons on the covalent bonding and van der waal type forces . it is imperative that the force - fields used to describe molecular motion are of sufficiently high accuracy to reproduce observed experimental behavior . the majority of methodological approaches for generating parameters for md simulations of conjugated polymers give strong weight to the importance of the acquisition of appropriate intermonomer dihedral angle profiles and partial charges in order to obtain accurate predictions from the resulting md . furthermore , there is often a lack of systematic parameter development and benchmarking of given methodology , and , as such , there is a large degree of ambiguity in the accuracy of a given method as well as whether or not a prescribed computational route could be replaced by a significantly less computationally expensive one . in light of the above , the work presented here establishes a systematic approach to the force - field parametrization . in addition , our methodology also determines the domain of applicability of a given set of parameters for molecules of varying length and varying length of side chain . we also highlight cases in which the universality of a given parameter set to these variations is broken . a minimal set of force - field parameters contains parameters of five types : three describing the energetics due to covalent bonding between atoms and two describing noncovalent interactions . we choose the opls force - field parameter scheme ( as implemented in gromacs 4.6.5 ) as our starting point due to its provision of parameters for many atoms in a multitude of different molecular frameworks as well as its use in previous works to parametrize conjugated polymers . to parametrize a given molecule , the first step is to build on the appropriate parameters found within opls for a monomer . the missing force - field parameters are the bond - stretching term between the two carbons circled in red ; the angle - bending term between each combination of three blue - circled atoms ( two from one unit and one from the other ) ; and the dihedral angle term for each combination of four blue - circled atoms ( two from one unit and two from the other ) . we expect that bond - stretching and angle - bending within the monomer are reliably parametrized by the opls force - field terms thus the intermonomer dihedral profile is the primary unknown from the covalent terms . in the case of a nonbonding interaction , we can use existing lennard - jones terms without any modifications and focus on partial charge specification and validation . in order to generate sufficiently accurate dihedral potentials at low computational expense , we utilize the method of performing geometry optimizations over the span of a dihedral rotation ( scan ) using computationally inexpensive techniques and refine these results with a further single - point energy ( sp ) calculation using more accurate methods . we find that the dihedral potentials for all lengths of conjugated backbone are accurately described by those of their corresponding 2mer . this indicates converged charge distributions which can be built into a general parameter set . once the appropriate partial charges have been determined , these are directly implemented into the force - field . the dihedral profile , on the other hand , must be implemented by means of a so as to ensure that energetic terms already described by the force - field and partial charges are not double - counted . this is , in spirit , performing an analogous scan using the force - field parameters ( without the required dihedral ) in order to obtain the contribution already accounted for by the force - field and subtracting this from the calculated dft profile . to obtain a reliable dihedral potential , the ethos of the scan - sp approach is to use lower levels of theory such as dft for obtaining geometries and higher levels of theory , for example , local methods ( mp2 , ccsd , ccsd(t ) ) or dft with a larger basis set for sp energy calculations . following our scan - sp approach , we have calculated 2mer dihedral profiles using various basis sets from the 6 - 31 g family with added polarization and diffuse functions for scans and the cc - pvtz basis set for sp calculations . from the results for fluorene ( figure 3(a ) ) , it is observed that the energetics of the geometry optimization alone are quite inaccurate when compared to the cc - pvtz result : those without diffuse functions considerably overestimate ( 2 kj / mol ) both the barriers at the planar conformations and the barrier at 90 ; and those with diffuse functions overestimate the planar barrier but considerably underestimate the 90 barrier to a similar degree . this implies that all discussed choices of the basis set provide very similar geometries and that the final accuracy of the dihedral profile is more dependent on the choice of the basis set for the following sp calculation . now that a computational methodology is established , we examine to what extent the dihedral profile is sensitive to the length of the conjugated backbone . we also demonstrated that the dihedral energetic profile is invariant to its position along a chain longer than a 2mer and that a profile is invariant to the value of the neighboring dihedral angle . we now turn to the influence of alkyl side chains on the dihedral energy profiles . due to this , the calculations both become increasingly computationally expensive and generate results which are strongly dependent on initial conditions , resulting in ambiguity in their translation into force - field parameters . with the above considered , figure 5(a ) gives the resulting dihedral profiles calculated for fluorene with various lengths of side - chain . we observe that increasing side - chain length has very little effect on the energetics of the dihedral . however , in the case of thiophene ( figure 5(b ) ) , the problem becomes considerably more complex due to the close proximity of the side - chain to the dihedral itself . schematic highlighting the difference in the location of the alkyl side - chains in 2mers of fluorene and thiophene . the blue area represents the location of the intermonomer dihedral ; the green represents the alkyl side - chain ; and , in thiophene , the red area highlights the steric conflict involved . as our parametrization is built on the ability to transfer parameters for long - range dispersive forces , the steric contribution to the dihedral profile due to the side - chains is captured by the existing terms in the force - field . as such , we wish to determine the effect of the side - chain on the covalently bound component of the dihedral rotation i.e. the effect it has on orbital conjugation as it is this component which comprises the required parameters in the force - field . in the mirrored 2mer , the side - chain of one unit is moved from the 3 carbon position to the 2 carbon position so as to remove the steric conflict . the nature of the approximation is that the effect of the side - chain on the conjugation - dependent component of the dihedral energetics is the same in both cases . we attribute this deviation , in a similar manner to those seen in figure 4 , to small remaining dispersive forces between the mirrored side - chain and the neighboring unit . these results strongly suggest that any variance in the dihedral energetics on account of the inclusion of side - chains is mediated entirely by known long - range , noncovalent interactions . this suggests that the dihedral profile of a force - field for these molecules is invariant to the addition of side - chains though this statement is analyzed quantitatively in section 5 . not only does this result in more accurate partial charges for utilization within an md force - field , the robustness of the method also allows us to accurately determine variations due to the changes in molecular environment that we are interested in . when generating parameters for md simulations of repeating structures of many units , such as polymers or oligomers , partial charges are often implemented using a three - residue model in which three sets of charges are built : one for each end unit and one for the central unit . in order to build such a model that is fully scalable to a variety of lengths , a first prerequisite is the requirement that the net charge for each individual central residue and the sum of the end residues have a net zero charge . a more exhaustive breakdown of our partial charge analyses ( such as the progression of total monomer charges and individual partial charges with varying backbone and side - chain length ) is given in the supporting information ( section 2 ) . at one end of the hexylthiophene molecule , the hexyl side - chain is on the side of the thiophene nearest the neighboring unit , while , at the other end , the side - chain is on the side further from the neighboring unit . to generate a model which is generalizable to all lengths of side - chain , two requirements must be met : first , there must exist a length of side - chain beyond which the charge distribution of the conjugated backbone is invariant ; and , second , it must be possible to generate a standard methylene ( ch2 ) group and a methyl ( ch3 ) group which can be added in repeatedly to create longer structures . this is because , while there are convergences in the end - most and innermost groups on a given side - chain , those in the middle of a long chain vary considerably as side - chain length is varied . it is seen here that the methylene group taken from the middle of the side - chain still fluctuates greatly in charge , primarily on the carbon atoms , even up to a 10 carbon long side - chain , while the two end groups have stabilized in charge . this is symptomatic of the strong asymmetry between each end of the side - chain - one is connected to a large molecule while the other is terminated only by a hydrogen . as lengths of side chain greater than 12 are not often used in practical settings , it remains that if one wishes to include partial charges for use with side - chains , it is necessary to compute these charges for each length of side - chain required . sample of beginning , middle , and end side - chain charges for various lengths of a side - chain in a monomer of alkylthiophene . it is observed that , in contrast to the end groups , the charge of the carbon on the inner methylene group ( yellow triangles ) does not converge with increased side - chain length . partial charges are implemented in the force - field by means of generating a three - residue model as described in the previous section and inputting these into the force - field parameter set . the required force - field contribution to the dihedral potential is isolated by performing scans over intervals of 10 from 0 to 180 in a manner analogous to that of the scans performed using dft . in the case of ethylthiophene , a large reduction is necessary kc = 10 kj / mol which we attribute to the prevalence of large forces in the side - chain - dihedral area . with the force - field ( ff ) contribution isolated , the required dihedral profile is obtained by subtracting the ff contribution from the dft scan . in the cases with no side - chains ( figure 10(a ) and ( b ) ) , it can be seen that this procedure and the fitting with the ryckaert - bellemans function result in a force - field which quantitatively mimics the dft dihedral potential to a very high degree of accuracy . however , this does not , in all cases , lead to a perfect correspondence in the side - chain conformations between the dft and ff scans . as is shown in figure 10 , the subtraction procedure leads to an overall dihedral potential from the force - field which corresponds closely to that of the dft . to further test this correspondence , we have calculated the difference in energy , em , between the two local - minima ( one in the first quadrant of the dihedral and one in the second ) of each molecule from dft and our force - field and compare them in table 1 . in order to achieve the agreement between the dft and ff values of e , we modified the equilibrium bond lengths and angles from the opls force - field while retaining the orginal force - constants . this measure gives a better agreement in the geometries and minimal energies obtained from the force - field . in section 3 , we argued that the steric interactions responsible for large changes in dihedral potential for alkylthiophenes should be incorporated by the force - field . however , as is shown in table 2 , utilizing the dihedral potential fitted from a thiophene with a shorter side - chain leads to drastically overestimated barriers at the planar positions . given the tendency for inconsistency observed in ethylthiophene due to the side - chain degrees of freedom and that the difference in dft dihedral potential between ethyl- and propylthiophene is slight ( 0.5 kj / mol at the planar barriers , figure 5(b ) ) , we use the ethylthiophene potential for thiophene molecules with longer side - chains . the labels tx ( ty ) denote the energies of a 2mer with an x - yl side - chain with energetic profile taken from a y - yl side - chain scan . the force - field parameters obtained from the above procedure are available , in a form suitable for use with the gromacs md package , along with the supporting information . as a test of our parameter sets , we performed simulations of 32mers of fluorene with octyl side - chains ( pf8 ) and thiophene with hexyl side - chains ( p3ht ) over the course of 50 and 100 ns , respectively , in chloroform and calculated persistence lengths , np ( in number of monomer units ) and lp ( in nm ) . we perform this calculation by generating vectors , vi , across the first and last carbon of unit i and generating a correlation function , a(n ) , of the angles between each pair of vectors , n ( eq 3)3 the persistence length is defined by the e - point of a(n ) i.e.4 the correlation curves for fluorene and thiophene with their respective side - chains are shown in figure 11 . in figure 12 , these distributions are given for 16mers of dioctylfluorene and 16mers and 32mers of hexylthiophene in chloroform . the distributions for 32mers of dioctylfluorene have not been shown as , while the tangent correlation curves were converged , after 50 ns the end - to - end length distributions were far from converged . in each example , the end - to - end length is given as a fraction , r , of the straight length of the molecule , nl , where n is the number of units and end - to - end length distributions ( solid lines ) for a 16mer of dioctylfluorene ( f8 ) and a 16mer and 32mer hexylthiophene ( t6 ) in chloroform . using end - marked hexylfluorenes of a similar length scale ( 42 monomer units with a polydispersity of 1.8 ) in an inert zeonex matrix , they measured end - to - end length distributions which are centered at a length fraction ( based on the fully extended 42mer ) of 0.89 . both the progression of the distributions and the difference in overall spread between fluorene and largely spread thiophene can be understood qualitatively by considering the increase in conformational entropy with increasing length and is consistent with the persistence lengths calculated previously . with this in mind , the octyl side - chains of the f8 subunit should be replaced with hydrogens and the scan performed on the monomer shown in figure 14 ( hereafter referred to as the f0bt monomer ) . we have determined that it is possible to replicate dihedral potentials of high levels of theory using an economical , two - step method of cam - b3lyp/6 - 31 g * and cam - b3lyp / cc - pvtz for geometry optimization scans and single - point energy calculations , respectively . we find that dihedral potentials are invariant to the length of the molecular conjugated backbone and that any effect due to the inclusion of side - chains seems to stem from long - range interactions alone . furthermore , we find that partial charge distributions converge quickly with varying length of molecule , but one must take into consideration deviations in end monomer distributions for molecules which do not possess end - to - end reflection symmetry . in terms of varying side - chains , it seems that there is no simple way of generalizing the inclusion of partial charges to this variation . our preliminary tests indicate that our md simulations are consistent with experimental measurements of persistence lengths and end - to - end lengths with long thiophene oligomers displaying far greater flexibility than their fluorene counterparts .

although iron exists in abundance in the earth 's crust and the environment , iron is found in relatively low concentrations in aqueous systems under aerobic conditions . fe occurs in two main oxidation states : the reduced fe ( fe ii , ferrous ) and the oxidized fe ( fe iii , ferric ) form . iron represents an essential trace element for almost all forms of life ; however , iron has paradoxical properties . it readily accepts and donates electrons , converting between the more soluble ferrous form and the insoluble ferric form , and thus plays an integral role in electron transfer and oxygen transport as well as adenosine triphosphate and deoxyribonucleic acid synthesis . however , iron can also catalyze the formation of reactive oxygen species ( ros ) via redox reactions . the fenton and haber - weiss reactions of h2o2 with fe generate hydroxyl radicals that promote oxidative stress and are responsible for lipid , protein , and dna damage . importantly , dysregulated iron homeostasis is associated with progressive inflammatory and degenerative diseases , as well as cancer . iron withdrawal is part of the natural innate immune response in inflammation . during inflammation and infection a given the role of iron in development of inflammatory diseases , pharmaceutical agents targeting this pathway promise to improve the clinical outcome . the objective of this review is to highlight the mechanisms of iron regulation and iron chelation and to demonstrate the potential impact of this approach in the management of several acute and chronic inflammatory diseases , including cancer . for this purpose , we reviewed the literature regarding experimental and clinical evidence for iron - related anti - inflammatory strategies and discuss implications and limitations of iron removal in inflammation . dietary iron uptake is closely regulated , which is critical to cell physiology and to ensure minimal concentrations of potentially dangerous free intracellular iron . mechanisms for iron homeostasis are complex compared to other metals , which are typically controlled by a simple elimination process . absorption declines to around 1 mg / day in men and 2 mg / day in women when growth declines . heme iron is abundant in meat as hemeprotein and myoglobin , released from hemeprotein by proteolytic enzymes in the stomach and small intestine . nonheme iron crosses the apical brush border membrane of enterocytes after conversion into ferrous iron by duodenal cytochrome b . iron has two fates according to the requirement of the body once within the enterocytes . when iron demand in the body is low , iron remains sequestered in the enterocyte within ferritin as a mechanism of iron storage . when the iron demand of the body is high , iron crosses the basolateral membrane via the iron export protein ferroportin1 ( fpn ) and enters the circulation , ultimately binding to transferrin . fpn is found in the basolateral membrane of the enterocytes and in large quantities on macrophages . ferroxidase activity of ceruloplasmin or hephaestin is required to load iron safely onto transferrin . to mitigate iron losses from the body from shedding of epithelial cells and menstruation , the body must absorb an equivalent amount of iron from the gut , maintaining an overall iron balance . in total , the human body contains approximately 3 to 4 g of iron in the form of both heme and nonheme iron . human hemoglobin accounts for 65% of total body iron , while 25% of body iron is bound to iron storage proteins ferritin and hemosiderin . the remaining 10% are constituents of myoglobin , cytochrome , and other iron - containing enzymes . only about 0.1% of body iron is bound to transferrin and this circulates as a soluble exchangeable pool in the plasma . tissue macrophages phagocytize senescent red blood cells to recover , turnover , and recycle iron . this recycling of iron by macrophages is 20-fold greater than the amount of iron absorbed daily by the intestine . iron absorption by enterocytes and the recycled iron efflux from macrophages are controlled by hepcidin , a key iron regulatory hormone produced by hepatocytes [ 12 , 13 ] . it limits the cellular iron efflux by binding to fpn and facilitates its internalization and ultimate degradation . hepcidin expression is increased in times when iron stores are sufficient and is decreased when the requirement of iron is high . the regulation of iron absorption is critical because humans do not have physiological pathways for its excretion . its release is upregulated in response to inflammatory cytokines , in particular interleukin-6 ( il-6 ) [ 1618 ] . hepcidin is also produced by macrophages and neutrophils during the innate immune response through a toll - like receptor 4-dependent pathway . in contrast , the level of hepcidin decreases during hypoxia , during iron deficiency , and when erythropoietic needs are increased . ros possess paradoxical cellular effects , as they can both induce cell growth and survival or trigger cell death . multiple factors influence the cellular effects of ros , including ros type as well as the level , localization , and duration of the assault [ 2022 ] . low levels of ros act as second messengers in many important signaling pathways , while prolonged exposure to higher levels can exceed cellular antioxidant defenses and damage dna , lipids , and proteins [ 21 , 23 ] . a complex relationship exists between iron metabolism and oxidant status ( figures 1 and 2 ) . free iron plays an important role in ros production as it can participate in the fenton and haber - weiss reactions to yield superoxide anion and the highly reactive hydroxyl radical [ 2022 ] . to prevent iron - catalyzed ros production , iron levels are strictly regulated by multiple factors including hepcidin and fpn , as described above . during inflammation , various pathways in immune cells ros and rns generation can lead to significant tissue damage and is responsible for a variety of inflammatory disease processes [ 2022 ] . proinflammatory cytokines induced by inflammation can stimulate anemia of inflammation by limiting serum iron and increasing cellular iron stores by modulating the expression and activity of various iron regulatory proteins including hepcidin , fpn , ferritin , and the iron importer divalent metal transporter 1 ( dmt1 ) [ 2530 ] . due to their role in iron recycling from erythrocytes , iron accumulation , in addition to increased levels of ros and rns induced by other facets of inflammation , may lead to increased labile iron , which can further perpetuate ros production and damage through participation in the fenton and haber - weiss reactions ( figure 2 ) . in addition , increased labile iron can contribute to the activation of redox - sensitive transcription factors such as nf-b , which is a potent regulator of inflammatory gene products including tnf- . an inhibitory effect on nf-b - mediated generation of tnf- and other inflammatory cytokines has been reported in kupffer cells following the reduction of free iron by chelation . ros and rns are produced both during normal aerobic respiration of cells and at increased rates and concentrations as part of the inflammatory response . of the various reactive species , ros have free iron directly involved in their production through the fenton and haber - weiss reactions . thus , the harnessed and controlled redox cycling of iron , which contributes activity for cell - essential iron - dependent enzymes such as the cytochromes , myeloperoxidase , or superoxide dismutase , also provides a less controlled source of cell - damaging reactive chemicals ; that is , should free iron be available for fenton or haber - weiss chemistry , the latter comprises essentially side reactions . iron can achieve hexadentate coordination with electron - donating ligands and when fully coordinated it is relatively redox stable . studies have shown that fenton - induced hydroxyl radical formation requires at least one free coordination site on the catalyzing iron . moreover , these studies were done using various chelating molecules with varying abilities to coordinate up to the six available coordination sites on iron and these therefore illustrated a key feature of iron chelators . iron - edta and iron - bleomycin chelates are redox - active liberating radicals while the hexadentate fe - desferal chelate is relatively redox - stable . by definition ( iupac , 1997 ) a metal chelate includes at least two coordinate bonds with a metal contributed by a chelating ligand and this , therefore , has important implications for the selection and use of iron chelators for treating inflammation or other disease conditions . it is interesting to note that the transferrins which are the main vertebrate iron chelator and transport protein molecules hold iron protected , within the transferrin structure itself , fully coordinating bound iron in a stable form using carbonate as a coligand . therefore , inappropriate or weak chelating molecules that retain their chelated iron with free , otherwise redox reactive coordination sites could be problematic as to undesirable radical generation . this issue might be further compounded on the basis that such chelators might solubilize and hold additional reactive iron spatially available at or near sensitive sites or , alternately , might deliver reactive iron to sensitive sites for participation in hydroxyl radical formation . an example of this problem is evident with the aminoglycoside antibiotics used to treat bacterial infection but known to possess ototoxicity related to oxidative radical formation when these agents bind iron in a redox active state . desferal ( novartis ) , also known as desferrioxamine or deferoxamine b , is a natural microbial product and an excellent example of a siderophore produced by microbes , in this case by streptomyces spp . . both pathogenic bacteria and fungi can produce iron chelating siderophores as one strategy for obtaining host iron that is essential to their growth and pathogenesis [ 37 , 39 ] . iron withholding and microbial acquisition are key aspects of the host parasite battle during the pathogenesis of infection [ 40 , 41 ] . while desferrioxamine reduced experimental lps - induced inflammation in mice , its use and that of other microbial chelators need to be carefully considered given that these agents could potentially promote infection owing to their microbial origin , for example , as has been shown for desferrioxamine with yersinia enterocolitica or candida albicans . a variety of natural phytochemicals have been described as having anti - inflammatory activity and some of these have been shown to possess iron - chelating activity . for example , curcuminoids bind ferric iron in vitro and alleviate iron toxicity in thalassemic mice [ 45 , 46 ] . in addition , recently reviewed the iron chelating activity of african walnut and wheat grass extracts as to their potential for natural iron removal agents for iron overload associated with thalassemia , which is currently typically treated clinically with desferal . however , the role that iron chelation plays with various phytochemicals in relation to their anti - inflammatory activities remains unclear . as to synthetic agents with anti - inflammatory activity , ibuprofen , a widely used synthetic anti - inflammatory agent , has been shown to chelate iron in a stable form without a free fenton - reactive coordination site and to protect from lipid peroxidation in vitro and phosgene - induced septic lung injury in rabbits . this evidence suggests at least part of ibuprofen 's mode of action is related to iron chelation and suppression of ros activity . research on newer generation synthetic iron chelators has produced a number of candidate molecules primarily in relation for their potential for treating iron overload or as anticancer agents . in this regard , agents that hold iron in a ros - reactive manner have been reviewed , their enhanced ros being proposed to provide killing of cancer cells . such ros - reactive molecules would not likely provide anti - inflammatory activity but , conversely , could induce inflammatory responses . on the other hand , new iron chelators that bind iron in a stable manner would be useful for treating iron overload diseases and thus , presumably , inflammation . kalinowski and richardson have recently reviewed synthetic chelators based on various chelating chemical groupings such as catechol , hydroxamate , and hydroxypyridinone , concluding that structures providing hexadentate hydroxypyridinone functionality ( an example being deferiprone ) have particular promise as bacteriostatic agents in relation to outcompeting siderophores of microbes . however , the hydroxamate desferal and the hydroxypyridinone deferiprone have been shown to be accessible by various micoorganisms thereby restricting their potential use as microbial control agents . holbein and mira reported that dibi , a new chelator , which possesses modified hydroxypyridinone activity , provided fe - specific growth inhibition of candida albicans . dibi is one example of a new approach to providing controlled molecular weight chelating - functional polymers . these agents may provide an additional advantage through providing compartmentalized sinks for iron thus reducing its participation in ros inflammatory reactions or its bioavailability to either pathogenic cancer or microbial cells . deficient regulation of iron homeostasis can contribute to tumor development through a number of different mechanisms ( figure 1 , table 1 ) . the iron - catalyzed production of ros and subsequent damage to dna can result in the loss of tumor suppressors and activation of oncogenes [ 51 , 52 ] . cancer cells require considerably more iron than normal cells due to their increased rate of dna synthesis and utilization of the iron - dependent enzyme ribonucleotide reductase . thus , cancer cells have increased expression of transferrin receptor-1 and a higher rate of iron uptake from transferrin [ 55 , 56 ] , as well as decreased expression of iron exporters . in addition , iron regulates the activity of the transcription factors nf-b and hif-1 , which promote the expression of genes involved in the survival and metastasis of cancer cells [ 59 , 60 ] . interestingly , higher concentrations of free iron in breast cancer cells are associated with a more aggressive tumor phenotype . it follows that cancer cells are more sensitive to iron deprivation than normal cells and may therefore be susceptible to treatment with iron chelators . it has been suggested that the development of atherosclerosis is associated with the amount of iron stored in the body and iron may contribute to the pathogenesis of atherosclerosis by acting as a regulator of vascular oxidative stress and inflammatory immune responses in atherosclerosis . increased levels of iron and oxidized lipids are both found in high - cholesterol diet - fed rabbits associated with atherosclerotic lesions . iron chelation by deferoxamine reduced the expression of oxidative stress markers and delayed the formation of atherosclerotic lesions , indicating that chelation therapy may aid in prevention of atherosclerosis [ 63 , 76 ] . diabetes mellitus is characterized by an impaired glucose metabolism with the main symptom of hyperglycemia , caused either by impaired insulin secretion or impaired insulin action or both . obesity is characterized by an increase in the number and/or the size of the fat cells . by activating adipocytes and stimulating their growth iron reduction by deferoxamine resulted in amelioration of adiposity via the regulation of oxidative stress and inflammation in obese and type 2 diabetes mice . interestingly , despite of deferoxamine administration for two weeks , there was only a mild but not significant reduction in the haemoglobin concentration and the hematocrit between vehicle - treated and chelator - treated mice ( table 1 ) . the uptake of unbound , that is , non - transferrin - bound iron , into pancreatic beta cells causes oxidative stress via the fenton reaction . due to their reduced antioxidant capacity , cell death is induced in the beta cells of isolated pancreatic islets by pharmacological - relevant iron concentrations that may occur during intravenous iron supplementation . renal interstitial fibrosis is characterized by the accumulation of collagen and related molecules in the interstitium , involving cells like tubular epithelial cells , fibroblasts , fibrocytes , myofibroblasts , monocyte / macrophages , and mast cells , and can be caused inter alia by inflammatory processes . in a recent study , the impact of deferoxamine on experimentally induced renal fibrosis was explored , again with an emphasis on inflammation caused by iron induced oxidative stress . after a surgically induced unilateral ureter obstruction ( uuo ) , mice treated with dfo showed significantly less fibrotic progression , less interstitial macrophage infiltration , and thereby a reduced expression of il-1 and mcp-1 and a suppressed uuo - induced accumulation of myofibroblasts , compared with untreated mice . the authors concluded that in mice iron reduction by deferoxamine may prevent renal interstitial fibrosis by regulating tgf - beta / smad signaling , oxidative stress , and inflammatory responses . elevated intraocular pressure , the main risk factor for glaucoma , triggers the initiation and progression of oxidative stress - induced cell damage . the disease is marked by loss of optic nerve axons and retinal ganglion cells , resulting in characteristic optic nerve atrophy and visual field defects . wang et al . observed a correlation of iron intake and the odds of glaucoma in humans . in an in vivo model in rats , chelation treatment ameliorated ocular sequelae caused by increased intraocular pressure . the topically administered metal chelator , edta , combined with a permeability enhancer reduced signs of oxidative stress and inflammation in glaucoma in the rat 's eyes , increased retinal ganglion cell survival , and decreased demyelination of optic nerve compared with untreated eyes . systemic inflammatory response syndrome ( sirs ) is characterized by specific physiological alterations , including temperature , white blood cell count , heart rate , and respiratory rate , caused by a broad spectrum of noninfectious and infectious triggers . hypoferremia in sirs is observed in humans and animals [ 84 , 85 ] . in acute hepatic ischemia induced sirs and consecutive multiple organ dysfunction ( mod ) in pigs , vlahakos et al . demonstrated that dfo attenuated lipid peroxidation , inhibited il-6 production , and substantially diminished sirs and mod . tubulointerstitial damage in the porcine kidney as expression of severity of sirs induced organ failure was reduced by a bolus followed by a continuous infusion of the chelator . these results suggest that iron plays a pivotal role in the pathogenesis of sirs and mod including acute kidney injury by its involvement in various inflammatory pathways and in the generation of reactive oxygen species . using the same model , the researchers also demonstrated that application of dfo significantly reduced brain edema , intracranial pressure , and lung injury [ 71 , 72 ] . in an endotoxemic mouse model , lactoferrin , a nonheme iron - binding glycoprotein , decreased lps - induced oxidative burst and reactive oxygen species in cultured cells and attenuated mitochondrial dysfunction in liver of endotoxemic mice . in a carbon tetrachloride induced acute hepatic injury rat model , application of dfo significantly decreased oxidative stress and limited inflammatory infiltration and hepatocyte necrosis , resulting in reduced mortality rate . ischemia reperfusion injury can be regarded as the most exaggerated form of oxidative stress for cells . onoo and h2o2 produced in the reperfusion phase are known to release iron ions from intracellular iron - sulphur proteins thus increasing intracellular labile iron pool , which in turn promotes by the above - outlined reactions the production of further reactive species . in the postischemic renal injury model in rats , acute iron loading exacerbated postischemic lipid peroxidation and renal injury , while reducing iron level by dfo suppressed lipid peroxidation and improved renal function . in a cardioplegia - ischemia and reperfusion model via cardiopulmonary bypass in sheep , dfo protected lung injury by inhibiting endothelial injury and eliminating postischemic cardiac stunning . colitis is another example for chronic inflammation , leading to anemia of inflammation as a consequence of low serum iron and low iron - binding capacity . using a trinitrobenzene sulfonic acid induced colitis rat model , minaiyan and coworkers compared the effect of deferiprone and deferoxamine with the newer iron chelators , maltol and kojic acid , on inflammatory response . in the highest dosage maltol was comparable with prednisolone as standard anti - inflammatory drug and also deferoxamine and deferiprone as reference iron binding agents . anemia represents a common clinical finding in patients with acute or chronic inflammation . in acute inflammation ( e.g. , trauma and surgery ) the two major mechanisms leading to anemia are blood loss and blunted erythropoiesis due to decreased iron availability [ 93 , 94 ] . in the event of chronic blood loss and/or persisting decreased iron absorption , acute anemia may evolve to anemia of chronic disease ( acd ) with true iron deficiency ( acd+id ) . anemia in chronic inflammation ( without blood loss or decreased baseline iron availability ) represents a specific entity , that is , acd . it is observed , for example , in cancer , rheumatoid arthritis , inflammatory bowel diseases , and congestive heart failure , as well as in sepsis and chronic renal failure . this anemia is the result of activation of the immune system by the underlying process and certain immune and inflammatory cytokines including tumor necrosis factor - alpha , interferon - gamma , il-1 , il-6 , il-8 , and il-10 [ 4 , 95 ] . the discovery of hepcidin yielded significant insight into the link between the immune response in inflammation and systemic iron homoeostasis . circulating hepcidin expression in vivo has also been correlated with acute phase proteins such as c - reactive protein , -1-acid - glycoprotein , ferritin , and amyloid a [ 96 , 97 ] . hepcidin produced by the hepatocytes binds to its receptor fpn , which is an iron export protein that is present on limited cells such as macrophages , hepatocytes , duodenal enterocytes , and placental syncytiotrophoblasts . hepcidin binding to ferroportin leads to ubiquitinization , followed by internalization and degradation of fpn . the end result is that iron can not be released into the plasma and remains trapped inside the macrophages and hepatocytes , resulting in an increase in iron stores reflected in high levels of serum ferritin . therefore , acd is characterized by low serum iron , transferrin , and total iron binding capacity , by normal transferrin saturation , and by increased ferritin , the latter in contrast to iron deficiency anemia . the low transferrin levels are due to downregulation of transferrin synthesis as a result of an increase in ferritin . the increase in hepcidin production in response to inflammation is a protective mechanism in the case of infections in which iron restriction would limit bacterial growth . however , in acd or inflammation without infection , this mechanism can have detrimental consequences when iron remains sequestered in the macrophages and hepatocytes and is not available for erythropoiesis , resulting in anemia . iron overload can be the result of hereditary ( primary ) or acquired ( secondary ) increase in iron storage . most forms of hemochromatosis result from dysregulation of hepcidin or defects of hepcidin or ferroportin themselves . when hepcidin binds to ferroportin it causes internalization of ferroportin and its proteolytic destruction . thus , hepcidin serves to prevent the egress of iron both from intestinal cells and from macrophages . this serves to prevent further absorption of iron from the gastrointestinal tract and its release from macrophages . iron overload associated with hereditary hemochromatosis has been reported to confer susceptibility to infectious pathogens , such as yersinia enterocolitica and vibrio vulnificus . acquired iron overload is frequently observed in thalassemia , myelodysplastic syndromes , congenital dyserythropoietic anemias , sickle cell disease , and other hemoglobinopathies . patients with thalassemia , whose erythroid precursor populations are greatly expanded but fail to mature into functional erythrocytes , have increased intestinal iron absorption despite often severe systemic iron overload . although blood transfusions given for severe anemia ( e.g. , thalassemia major ) contribute to the lethal iron overload in ineffective erythropoiesis , many patients with less severe anemia ( e.g. , thalassemia intermedia ) receive few or no transfusions but still become severely iron - overloaded . they described three potential mechanisms : ( i ) the excess -chains in -thalassemic erythrocytes and erythroblasts being unstable and prone to denaturation and oxidation , ( ii ) peroxidation of tissues that leak malondialdehyde into the blood , and ( iii ) depleted antioxidant capacity lowering defense against oxidants . infections and inflammations are more frequent in thalassemic patients with iron overload induced by frequent blood transfusions . iron accumulation and increased oxidative stress were also described in the pathogenesis of preeclampsia , diabetes , the metabolic syndrome , obesity , hypertension , cardiovascular diseases , heart failure , atherosclerosis , stroke , alzheimer 's , parkinson 's and other major neurodegenerative diseases , friedreich 's ataxia , amyotrophic lateral sclerosis , rheumatoid arthritis , systemic lupus erythematosus , asthma , inflammatory bowel diseases , age - related macula degeneration , psoriasis , gout , chronic obstructive pulmonary disorder , cancer , malaria , and other diseases ( for overviews see kell 2009 and weinberg 2010 ) . iron deficiency ( i d ) can be caused by inadequate oral iron uptake ( dietary ) , inadequate iron absorption ( e.g. , celiac disease ) , excessive blood loss , or increased iron demand ( e.g. growth or pregnancy ) . the clinical picture of i d patients is usually characterized by mild to severe anemias and further consequences , including epithelial changes ( stomatitis and glossitis ) , and neurocognitive effects , that is , impaired motor and mental functioning . however , in the same way that primary or secondary iron storage diseases can cause oxidative stress , artificial iron overload due to therapeutic administration can cause harm for the patients . the pemba trial , which led to adverse events in children receiving iron in a malaria - endemic region , , it has been demonstrated that parenteral or oral iron increase mycobacterial growth and that morbidity and mortality increase in patients receiving iron supplementation . the iron hypothesis of the benefits of some iron depletion due to menstruation was devised to account for the lowering of heart - disease risk in young women that disappears in those who are postmenopausal [ 131 , 132 ] . depending on the localization and severity of the inflammatory condition , different approaches and routes of administration for iron chelators are feasible . the route of administration as well as needs for extra- and/or intracellular action determines the required molecular structure and weight of the iron chelating substances . for wound infections , topical / local administration of large molecular size substances might be feasible , whereas for systemic ( sc / iv / ip ) administration ( e.g. , sepsis ) smaller molecules might be necessary . due to the bacteriostatic / bactericidal effects of iron removal from media , the use of iron chelators as preservatives in standard medications ( e.g. , eye drops ) is also an option . in the same way , application of iron chelators as adjunct to peritoneal lavage fluids appears to be an option for both prophylactic and therapeutic purposes . suggesting that inflammatory disorders might be treatable by the induction of a second disorder , that is , iron depletion , might be seen as a controversial proposal due to the prevalent assumption that iron in storage is inherently safe . it has been previously stated that the benefit of iron depletion can only be rigorously demonstrated in relation to the state of iron in excess of needs ( i.e. , the condition of having iron in storage ) . it has not been widely appreciated that the safety of stored iron can only be shown with clarity in studies of the same design . absence of proof that iron depletion is beneficial implies an absence of proof of the safety of stored iron . because of the deeply rooted assumption that stored iron is safe , appropriate trials to rule out the potential hazards of iron stores have not been undertaken . but even if there exists a potential ( short - term ) harm of iron depletion ( e.g. , anemia ) when administered in inflammatory diseases , this can be compared with appears acceptable , if it is possible to get control over the main , potentially life - threatening condition . iron is involved in almost every clinical condition of acute or chronic inflammation . since iron can contribute to detrimental ros and rns generating processes , it seems to be a reasonable approach to modulate iron - related pathways in inflammation . in humans ( with normal baseline iron levels ) , a counterregulatory reduction in iron availability is observed during inflammatory diseases ( anemia of inflammation ) . under pathological conditions with reduced or increased baseline iron levels different consequences regarding protection or susceptibility to inflammation have to be considered . therapeutic interventions with iron or iron chelators have an impact on the oxidant status and iron chelation might be utilized as a potential antioxidative strategy in inflammatory diseases . new iron chelators designed to sequester iron in a nonredox active state may prove to provide increased efficacy in this regard . additional requirements for new iron chelators apply in infection - induced , local , and systemic inflammation . conventional clinical chelators do not effectively deny iron to pathogens ; in fact pathogens can utilize these conventional chelators as iron sources . this underscores the need for superior chelators since agents like desferal or deferiprone as antimicrobials agents have provided varying results depending on the microbe being tested .

since iron can contribute to detrimental radical generating processes through the fenton and haber - weiss reactions , it seems to be a reasonable approach to modulate iron - related pathways in inflammation . in the human organism a counterregulatory reduction in iron availability is observed during inflammatory diseases . under pathological conditions with reduced or increased baseline iron levels different consequences regarding protection or susceptibility to inflammation have to be considered . given the role of iron in development of inflammatory diseases , pharmaceutical agents targeting this pathway promise to improve the clinical outcome . the objective of this review is to highlight the mechanisms of iron regulation and iron chelation , and to demonstrate the potential impact of this strategy in the management of several acute and chronic inflammatory diseases , including cancer .

1. Introduction 2. Basic Mechanisms 3. Iron Chelators 4. Experimental Studies 5. Clinical Evidence 6. Conclusions

although iron exists in abundance in the earth 's crust and the environment , iron is found in relatively low concentrations in aqueous systems under aerobic conditions . it readily accepts and donates electrons , converting between the more soluble ferrous form and the insoluble ferric form , and thus plays an integral role in electron transfer and oxygen transport as well as adenosine triphosphate and deoxyribonucleic acid synthesis . however , iron can also catalyze the formation of reactive oxygen species ( ros ) via redox reactions . the fenton and haber - weiss reactions of h2o2 with fe generate hydroxyl radicals that promote oxidative stress and are responsible for lipid , protein , and dna damage . importantly , dysregulated iron homeostasis is associated with progressive inflammatory and degenerative diseases , as well as cancer . iron withdrawal is part of the natural innate immune response in inflammation . during inflammation and infection a given the role of iron in development of inflammatory diseases , pharmaceutical agents targeting this pathway promise to improve the clinical outcome . the objective of this review is to highlight the mechanisms of iron regulation and iron chelation and to demonstrate the potential impact of this approach in the management of several acute and chronic inflammatory diseases , including cancer . for this purpose , we reviewed the literature regarding experimental and clinical evidence for iron - related anti - inflammatory strategies and discuss implications and limitations of iron removal in inflammation . dietary iron uptake is closely regulated , which is critical to cell physiology and to ensure minimal concentrations of potentially dangerous free intracellular iron . heme iron is abundant in meat as hemeprotein and myoglobin , released from hemeprotein by proteolytic enzymes in the stomach and small intestine . when iron demand in the body is low , iron remains sequestered in the enterocyte within ferritin as a mechanism of iron storage . fpn is found in the basolateral membrane of the enterocytes and in large quantities on macrophages . to mitigate iron losses from the body from shedding of epithelial cells and menstruation , the body must absorb an equivalent amount of iron from the gut , maintaining an overall iron balance . in total , the human body contains approximately 3 to 4 g of iron in the form of both heme and nonheme iron . the remaining 10% are constituents of myoglobin , cytochrome , and other iron - containing enzymes . only about 0.1% of body iron is bound to transferrin and this circulates as a soluble exchangeable pool in the plasma . this recycling of iron by macrophages is 20-fold greater than the amount of iron absorbed daily by the intestine . multiple factors influence the cellular effects of ros , including ros type as well as the level , localization , and duration of the assault [ 2022 ] . low levels of ros act as second messengers in many important signaling pathways , while prolonged exposure to higher levels can exceed cellular antioxidant defenses and damage dna , lipids , and proteins [ 21 , 23 ] . free iron plays an important role in ros production as it can participate in the fenton and haber - weiss reactions to yield superoxide anion and the highly reactive hydroxyl radical [ 2022 ] . to prevent iron - catalyzed ros production , iron levels are strictly regulated by multiple factors including hepcidin and fpn , as described above . during inflammation , various pathways in immune cells ros and rns generation can lead to significant tissue damage and is responsible for a variety of inflammatory disease processes [ 2022 ] . proinflammatory cytokines induced by inflammation can stimulate anemia of inflammation by limiting serum iron and increasing cellular iron stores by modulating the expression and activity of various iron regulatory proteins including hepcidin , fpn , ferritin , and the iron importer divalent metal transporter 1 ( dmt1 ) [ 2530 ] . due to their role in iron recycling from erythrocytes , iron accumulation , in addition to increased levels of ros and rns induced by other facets of inflammation , may lead to increased labile iron , which can further perpetuate ros production and damage through participation in the fenton and haber - weiss reactions ( figure 2 ) . in addition , increased labile iron can contribute to the activation of redox - sensitive transcription factors such as nf-b , which is a potent regulator of inflammatory gene products including tnf- . of the various reactive species , ros have free iron directly involved in their production through the fenton and haber - weiss reactions . thus , the harnessed and controlled redox cycling of iron , which contributes activity for cell - essential iron - dependent enzymes such as the cytochromes , myeloperoxidase , or superoxide dismutase , also provides a less controlled source of cell - damaging reactive chemicals ; that is , should free iron be available for fenton or haber - weiss chemistry , the latter comprises essentially side reactions . iron - edta and iron - bleomycin chelates are redox - active liberating radicals while the hexadentate fe - desferal chelate is relatively redox - stable . by definition ( iupac , 1997 ) a metal chelate includes at least two coordinate bonds with a metal contributed by a chelating ligand and this , therefore , has important implications for the selection and use of iron chelators for treating inflammation or other disease conditions . it is interesting to note that the transferrins which are the main vertebrate iron chelator and transport protein molecules hold iron protected , within the transferrin structure itself , fully coordinating bound iron in a stable form using carbonate as a coligand . an example of this problem is evident with the aminoglycoside antibiotics used to treat bacterial infection but known to possess ototoxicity related to oxidative radical formation when these agents bind iron in a redox active state . while desferrioxamine reduced experimental lps - induced inflammation in mice , its use and that of other microbial chelators need to be carefully considered given that these agents could potentially promote infection owing to their microbial origin , for example , as has been shown for desferrioxamine with yersinia enterocolitica or candida albicans . a variety of natural phytochemicals have been described as having anti - inflammatory activity and some of these have been shown to possess iron - chelating activity . for example , curcuminoids bind ferric iron in vitro and alleviate iron toxicity in thalassemic mice [ 45 , 46 ] . however , the role that iron chelation plays with various phytochemicals in relation to their anti - inflammatory activities remains unclear . as to synthetic agents with anti - inflammatory activity , ibuprofen , a widely used synthetic anti - inflammatory agent , has been shown to chelate iron in a stable form without a free fenton - reactive coordination site and to protect from lipid peroxidation in vitro and phosgene - induced septic lung injury in rabbits . this evidence suggests at least part of ibuprofen 's mode of action is related to iron chelation and suppression of ros activity . in this regard , agents that hold iron in a ros - reactive manner have been reviewed , their enhanced ros being proposed to provide killing of cancer cells . on the other hand , new iron chelators that bind iron in a stable manner would be useful for treating iron overload diseases and thus , presumably , inflammation . kalinowski and richardson have recently reviewed synthetic chelators based on various chelating chemical groupings such as catechol , hydroxamate , and hydroxypyridinone , concluding that structures providing hexadentate hydroxypyridinone functionality ( an example being deferiprone ) have particular promise as bacteriostatic agents in relation to outcompeting siderophores of microbes . however , the hydroxamate desferal and the hydroxypyridinone deferiprone have been shown to be accessible by various micoorganisms thereby restricting their potential use as microbial control agents . deficient regulation of iron homeostasis can contribute to tumor development through a number of different mechanisms ( figure 1 , table 1 ) . the iron - catalyzed production of ros and subsequent damage to dna can result in the loss of tumor suppressors and activation of oncogenes [ 51 , 52 ] . cancer cells require considerably more iron than normal cells due to their increased rate of dna synthesis and utilization of the iron - dependent enzyme ribonucleotide reductase . thus , cancer cells have increased expression of transferrin receptor-1 and a higher rate of iron uptake from transferrin [ 55 , 56 ] , as well as decreased expression of iron exporters . in addition , iron regulates the activity of the transcription factors nf-b and hif-1 , which promote the expression of genes involved in the survival and metastasis of cancer cells [ 59 , 60 ] . interestingly , higher concentrations of free iron in breast cancer cells are associated with a more aggressive tumor phenotype . it has been suggested that the development of atherosclerosis is associated with the amount of iron stored in the body and iron may contribute to the pathogenesis of atherosclerosis by acting as a regulator of vascular oxidative stress and inflammatory immune responses in atherosclerosis . increased levels of iron and oxidized lipids are both found in high - cholesterol diet - fed rabbits associated with atherosclerotic lesions . iron chelation by deferoxamine reduced the expression of oxidative stress markers and delayed the formation of atherosclerotic lesions , indicating that chelation therapy may aid in prevention of atherosclerosis [ 63 , 76 ] . obesity is characterized by an increase in the number and/or the size of the fat cells . by activating adipocytes and stimulating their growth iron reduction by deferoxamine resulted in amelioration of adiposity via the regulation of oxidative stress and inflammation in obese and type 2 diabetes mice . interestingly , despite of deferoxamine administration for two weeks , there was only a mild but not significant reduction in the haemoglobin concentration and the hematocrit between vehicle - treated and chelator - treated mice ( table 1 ) . the uptake of unbound , that is , non - transferrin - bound iron , into pancreatic beta cells causes oxidative stress via the fenton reaction . due to their reduced antioxidant capacity , cell death is induced in the beta cells of isolated pancreatic islets by pharmacological - relevant iron concentrations that may occur during intravenous iron supplementation . renal interstitial fibrosis is characterized by the accumulation of collagen and related molecules in the interstitium , involving cells like tubular epithelial cells , fibroblasts , fibrocytes , myofibroblasts , monocyte / macrophages , and mast cells , and can be caused inter alia by inflammatory processes . in a recent study , the impact of deferoxamine on experimentally induced renal fibrosis was explored , again with an emphasis on inflammation caused by iron induced oxidative stress . the authors concluded that in mice iron reduction by deferoxamine may prevent renal interstitial fibrosis by regulating tgf - beta / smad signaling , oxidative stress , and inflammatory responses . observed a correlation of iron intake and the odds of glaucoma in humans . the topically administered metal chelator , edta , combined with a permeability enhancer reduced signs of oxidative stress and inflammation in glaucoma in the rat 's eyes , increased retinal ganglion cell survival , and decreased demyelination of optic nerve compared with untreated eyes . systemic inflammatory response syndrome ( sirs ) is characterized by specific physiological alterations , including temperature , white blood cell count , heart rate , and respiratory rate , caused by a broad spectrum of noninfectious and infectious triggers . hypoferremia in sirs is observed in humans and animals [ 84 , 85 ] . tubulointerstitial damage in the porcine kidney as expression of severity of sirs induced organ failure was reduced by a bolus followed by a continuous infusion of the chelator . these results suggest that iron plays a pivotal role in the pathogenesis of sirs and mod including acute kidney injury by its involvement in various inflammatory pathways and in the generation of reactive oxygen species . using the same model , the researchers also demonstrated that application of dfo significantly reduced brain edema , intracranial pressure , and lung injury [ 71 , 72 ] . in an endotoxemic mouse model , lactoferrin , a nonheme iron - binding glycoprotein , decreased lps - induced oxidative burst and reactive oxygen species in cultured cells and attenuated mitochondrial dysfunction in liver of endotoxemic mice . onoo and h2o2 produced in the reperfusion phase are known to release iron ions from intracellular iron - sulphur proteins thus increasing intracellular labile iron pool , which in turn promotes by the above - outlined reactions the production of further reactive species . colitis is another example for chronic inflammation , leading to anemia of inflammation as a consequence of low serum iron and low iron - binding capacity . in the highest dosage maltol was comparable with prednisolone as standard anti - inflammatory drug and also deferoxamine and deferiprone as reference iron binding agents . , trauma and surgery ) the two major mechanisms leading to anemia are blood loss and blunted erythropoiesis due to decreased iron availability [ 93 , 94 ] . anemia in chronic inflammation ( without blood loss or decreased baseline iron availability ) represents a specific entity , that is , acd . it is observed , for example , in cancer , rheumatoid arthritis , inflammatory bowel diseases , and congestive heart failure , as well as in sepsis and chronic renal failure . the discovery of hepcidin yielded significant insight into the link between the immune response in inflammation and systemic iron homoeostasis . circulating hepcidin expression in vivo has also been correlated with acute phase proteins such as c - reactive protein , -1-acid - glycoprotein , ferritin , and amyloid a [ 96 , 97 ] . hepcidin produced by the hepatocytes binds to its receptor fpn , which is an iron export protein that is present on limited cells such as macrophages , hepatocytes , duodenal enterocytes , and placental syncytiotrophoblasts . the end result is that iron can not be released into the plasma and remains trapped inside the macrophages and hepatocytes , resulting in an increase in iron stores reflected in high levels of serum ferritin . therefore , acd is characterized by low serum iron , transferrin , and total iron binding capacity , by normal transferrin saturation , and by increased ferritin , the latter in contrast to iron deficiency anemia . the increase in hepcidin production in response to inflammation is a protective mechanism in the case of infections in which iron restriction would limit bacterial growth . however , in acd or inflammation without infection , this mechanism can have detrimental consequences when iron remains sequestered in the macrophages and hepatocytes and is not available for erythropoiesis , resulting in anemia . iron overload can be the result of hereditary ( primary ) or acquired ( secondary ) increase in iron storage . thus , hepcidin serves to prevent the egress of iron both from intestinal cells and from macrophages . this serves to prevent further absorption of iron from the gastrointestinal tract and its release from macrophages . iron overload associated with hereditary hemochromatosis has been reported to confer susceptibility to infectious pathogens , such as yersinia enterocolitica and vibrio vulnificus . acquired iron overload is frequently observed in thalassemia , myelodysplastic syndromes , congenital dyserythropoietic anemias , sickle cell disease , and other hemoglobinopathies . , thalassemia major ) contribute to the lethal iron overload in ineffective erythropoiesis , many patients with less severe anemia ( e.g. , thalassemia intermedia ) receive few or no transfusions but still become severely iron - overloaded . they described three potential mechanisms : ( i ) the excess -chains in -thalassemic erythrocytes and erythroblasts being unstable and prone to denaturation and oxidation , ( ii ) peroxidation of tissues that leak malondialdehyde into the blood , and ( iii ) depleted antioxidant capacity lowering defense against oxidants . iron accumulation and increased oxidative stress were also described in the pathogenesis of preeclampsia , diabetes , the metabolic syndrome , obesity , hypertension , cardiovascular diseases , heart failure , atherosclerosis , stroke , alzheimer 's , parkinson 's and other major neurodegenerative diseases , friedreich 's ataxia , amyotrophic lateral sclerosis , rheumatoid arthritis , systemic lupus erythematosus , asthma , inflammatory bowel diseases , age - related macula degeneration , psoriasis , gout , chronic obstructive pulmonary disorder , cancer , malaria , and other diseases ( for overviews see kell 2009 and weinberg 2010 ) . , celiac disease ) , excessive blood loss , or increased iron demand ( e.g. the clinical picture of i d patients is usually characterized by mild to severe anemias and further consequences , including epithelial changes ( stomatitis and glossitis ) , and neurocognitive effects , that is , impaired motor and mental functioning . however , in the same way that primary or secondary iron storage diseases can cause oxidative stress , artificial iron overload due to therapeutic administration can cause harm for the patients . the pemba trial , which led to adverse events in children receiving iron in a malaria - endemic region , , it has been demonstrated that parenteral or oral iron increase mycobacterial growth and that morbidity and mortality increase in patients receiving iron supplementation . due to the bacteriostatic / bactericidal effects of iron removal from media , the use of iron chelators as preservatives in standard medications ( e.g. in the same way , application of iron chelators as adjunct to peritoneal lavage fluids appears to be an option for both prophylactic and therapeutic purposes . suggesting that inflammatory disorders might be treatable by the induction of a second disorder , that is , iron depletion , might be seen as a controversial proposal due to the prevalent assumption that iron in storage is inherently safe . it has been previously stated that the benefit of iron depletion can only be rigorously demonstrated in relation to the state of iron in excess of needs ( i.e. , the condition of having iron in storage ) . because of the deeply rooted assumption that stored iron is safe , appropriate trials to rule out the potential hazards of iron stores have not been undertaken . , anemia ) when administered in inflammatory diseases , this can be compared with appears acceptable , if it is possible to get control over the main , potentially life - threatening condition . since iron can contribute to detrimental ros and rns generating processes , it seems to be a reasonable approach to modulate iron - related pathways in inflammation . in humans ( with normal baseline iron levels ) , a counterregulatory reduction in iron availability is observed during inflammatory diseases ( anemia of inflammation ) . under pathological conditions with reduced or increased baseline iron levels different consequences regarding protection or susceptibility to inflammation have to be considered . therapeutic interventions with iron or iron chelators have an impact on the oxidant status and iron chelation might be utilized as a potential antioxidative strategy in inflammatory diseases . new iron chelators designed to sequester iron in a nonredox active state may prove to provide increased efficacy in this regard . additional requirements for new iron chelators apply in infection - induced , local , and systemic inflammation .

growing evidence in the literature supports the role of increased dietary intake of saturated fats in the initiation of inflammation [ 1 , 2 ] . although elevated plasma levels of nonesterified fas have been extensively correlated to insulin resistance , the role of specific fas , such as palmitic acid ( pa ) , is not well understood [ 3 , 4 ] . visceral adipose tissue is a major site of obesity - induced inflammation , and dyslipidemia is a major factor in the recruitment of activated immune cells such as macrophages , t cells , nk cells , dendritic cells , and b cells to visceral adipose tissue . infiltrating adipose immune cells are a major source of proinflammatory cytokines in obesity - induced inflammation and type 2 diabetes [ 57 ] . in particular , the proinflammatory cytokine il-1 can directly cause insulin resistance in insulin - sensitive cells [ 5 , 811 ] . moreover , pa has been shown to activate toll - like receptor 4 on immune cells and induce secretion of il-1 . recently , b cells have been recognized as a major contributor to obesity - induced inflammation [ 5 , 1315 ] . b cells are recruited to adipose tissue in response to a high fat diet [ 16 , 17 ] . the importance of igg antibodies secreted by b cells has been established in a mouse model of type 2 diabetes . for example , depletion of b cells results in protection against diabetes in mice fed with a high fat diet . in addition , the transfer of igg antibodies from obesity induced - diabetic mice to nondiabetic mice rapidly induces insulin resistance and glucose intolerance . these findings suggest that b cell secretion of antibodies may be critical regulators of insulin resistance . parallel to mice studies , humans with type 2 diabetes have disease - associated changes in b cell function , but the role of these changes in disease pathogenesis is not well established . insulin resistance in obese individuals is linked to antibodies directed against intracellular protein antigens such as golgi snap receptor complex 1 and bruton 's tyrosine kinase . there is the possibility that antibodies to lipids are generated in response to a high fat diet because the authors of that study only screen serum for protein antigens ( b cells promote insulin resistance through modulation of t cells and production of pathogenic igg antibodies ) . moreover , igm antibodies against fas have been reported in multiple sclerosis as well as in human immunodeficiency virus ( hiv ) patients [ 2022 ] . however , there is a gap in the literature of studies demonstrating the presence of igg antibodies against fas such as palmitic acid . the purpose of the present study was to investigate whether humans produce class switched igg antibodies that recognize saturated fas such as pa . to answer this question , we retrospectively analyzed serum from 2 different cohorts of obese individuals , including patients with and without type 2 diabetes and patients who participated in the diabetes intervention program , en balance . this study consisted of analysis of serum samples from the bioserve biorepository in addition to serum samples from a 3-month diabetes education intervention ( en balance ) designed to promote improved type 2 diabetes management in hispanic adults [ 2427 ] . we measured il-1 and antibodies which recognize palmitic acid in these samples and correlated the values obtained from the en balance samples with the original primary outcomes of that study . a total of 73 hispanic males and females with type 2 diabetes met the en balance participation criteria as previously described [ 26 , 27 ] . the loma linda university institutional review board ( irb ) approved the en balance study protocol and all participants gave written informed consent to participate . signed consent forms for the study are stored in locked filing cabinets and can not be linked to participant data according to loma linda university irb protocol . two blood samples ( 1214 hr fasting ) were drawn from the participants at both baseline and 3 months and analyzed for glucose , a1c , and insulin . anthropometric measures ( height , weight , waist circumference , hip circumference , and waist / hip ratio ) were assessed at baseline and 3 months as previously described [ 25 , 28 ] . body composition was assessed at baseline and at 3 months using a tanita scale ( detecto , web city , missouri ) , bioelectric impendence technology , and a fan beam dual x - ray absorptiometry ( dxa ) hologic discovery a software version 12.6 ( waltham , ma ) as previously described [ 26 , 27 ] . serum samples from 46 en balance participants were available for anti - lcsfa antibody and il-1 testing . the baseline characteristics of these participants are presented in table 1 with the exception of missing participant data . twenty - one of these participants had paired serum samples from baseline and 3 months available for longitudinal analysis . twelve participants had serum samples from only baseline and 13 participants had serum samples from only 3 months . serum samples ( four groups with 20 serum samples each : hispanic control , hispanics with type 2 diabetes , caucasian control , and caucasians with type 2 diabetes ) purchased from the bioserve biorepository ( beltsville , md ) were also tested for anti - lcsfa antibody and il-1. the samples were gender , age , and bmi matched against the 21 en balance participants with paired samples ( table 2 and figure 1 ) . exclusion criteria consisted of history of renal disease , self - declared diabetic complications , tobacco use , and other medical histories including cancer history . palmitoylation of bsa was performed by modifying a protocol developed by the geffard laboratory [ 2022 ] . for this reaction , 10 mg of pa was dissolved in 10 ml of anhydrous methanol ( sigma - aldrich ) containing 100 l of triethylamine ( sigma - aldrich ) . to activate the carboxylic acid group , 200 ml of anhydrous dimethylformamide ( dmf ) solution containing ethylchloroformate ( etococl ) ( sigma - aldrich ) diluted 1/16 was added . next , 200 mg fa - free bsa ( gemini bio - products ) was dissolved in 10 ml of 1 m phosphate buffer ( ph 6.8 ) , containing 1 mm cacl2 and 100 l of triethylamine ( sigma - aldrich ) . the bsa solution was added to the pa solution and the mixture was stirred for 1 hr at room temperature . the bsa - pa conjugates were purified by dialysis against 150 ml of 1 mm cacl2 phosphate buffer ( ph 6.8 ) , containing dimethylformamide and methanol , ( vol / vol / vol ) overnight at room temperature ( rt ) . the conjugate was then dialyzed against 1 m phosphate buffer ( ph 6.8 ) , containing 1 mm cacl2 for 24 hours at rt . the mixture was then dialyzed against pbs ( ph 7.2 ) until precipitated phosphate became soluble . the bsa - pa was filtered to remove any remaining precipitate and the bsa - pa concentration was determined by measuring the optical density ( od ) at 280 nm . an elisa was developed for identifying igg antibodies in human serum that recognize pa using the ready - set - go elisa igg kit ( ebioscience ) according to manufacturer 's protocol with the following exceptions . plates were coated with 100 l of 50 g / ml pa - bsa or bsa . purified igg at 2 g / ml in pbs was preincubated for 16 hrs at 4c with bsa - pa at concentrations varying between 10 and 10 m. after centrifugation at 10,000 g for 30 min , 100 l of the supernatants was analyzed by elisa as described above . antibody avidity was defined as the concentration of bsa - pa required for 50% inhibition ( ic50 ) of antibody binding to immobilized bsa - pa . total igg from 7 diabetic patient participant sera was isolated and purified on pierce protein g chromatography cartridges ( thermo scientific ) according to the manufacturer 's protocol . igg that recognized bsa - pa was purified using the sulfolink immobilization kit for proteins ( thermo scientific ) according to manufacturer 's instructions . bsa - pa was immobilized to resin via covalent thioester bonds , and this resin was used to pack the chromatography column . the anti - lcsfa antibodies ( antibodies isolated by affinity to bsa - pa ) were then purified from their respective igg fraction by affinity chromatography according to the protocol outlined for the sulfolink immobilization kit ( thermo scientific ) . a lipid dot blot method was established to determine the reactivity of a panel of nonesterified fas ( from sigma - aldrich ) and esterified palmitic acid ( glycerol tripalmitate from sigma - aldrich and n - palmitoyl phosphatidyl ethanolamine from enzo life sciences ) to purified anti - lcsfa igg . m fas dissolved in 100% chloroform + 0.1% hcl was spotted onto nitrocellulose . after allowing the samples to completely dry , the lipid blots were probed overnight at 4c with purified antibodies ( diluted 1 : 1000 ) that recognize bsa - pa . after washing the blot with pbs , anti - human igg alkaline phosphatase conjugate ( sigma ) was used as the secondary ( 1 : 10,000 ) detection ab . monocytes ( cd14 ) were isolated from human peripheral blood ( lifestream blood bank ) and cultured at 37c and 5% co2 in 96-well plates at a density of 200,000 per well in rpmi-1640 supplemented with 10% fbs , 1,000 u penicillin and streptomycin , 50 ng / ml gm - csf , and 10 ng / ml il-4 for 6 days to differentiate them into dendritic cells ( dcs ) . the dcs were treated with 150 m pa in a 1 : 1 ratio with bsa for 24 hrs in the presence of igg ab containing anti - lcsfa igg . a mixture of total igg isolated from 5 hispanics with type 2 diabetes with high anti - lcsfa igg levels ( measured by elisa ) or a mixture of total igg isolated from 2 samples which tested negative for anti - lcsfa igg ( measured by elisa ) was used at a concentration of 1.4 mg / ml . preabsorption of pa was performed at 37c for 2 hrs . fifty l of culture supernatant was analyzed by cytometric bead array . using the free fatty assay colorimetric kit ( cell biolabs , inc . ) , the concentration of free fatty acid in the serum samples diluted 1 : 2 was determined in duplicate following the manufacturer 's recommendations . en balance serum samples and cell culture supernatants were analyzed with a human il-1 flex set as described by the manufacturer ( bd biosciences ) on a macsquant flow cytometer ( miltenyi biotech ) . western blots were performed on ~10 g bsa or 60 g bsa - pa . due to the diffuse banding pattern of bsa - pa , increased amounts of bsa - pa ( 60 g ) were loaded for the western blot ( figure 2(c ) ) in order to detect differences between samples . the proteins were separated on a 12% acrylamide gel at 120 v and then transferred to pvdf membranes and blocked with 5% nonfat milk solution in dulbecco 's pbs ( caisson labs , logan , ut ) for 1 hr at room temperature . after blocking , the membrane was incubated in primary antibody ( complete patient serum ) diluted 1 : 100 in 5% nonfat milk solution overnight at 4c . the membrane was washed three times with pbs + 0.02% tween 20 and incubated with anti - human igg conjugated to alkaline phosphatase ( sigma ) diluted 1 : 5,000 for 1 hr at room temperature . the membrane was washed three times with pbs + 0.02% tween 20 and then one time with pbs and the protein bands were visualized on photographic film ( kodak ) using novex ap chemiluminescent substrate ( invitrogen ) . statistical analyses were calculated using spss for windows version 22 ( spss , inc . , spearman 's correlation coefficient was used to determine associations between body composition , glucose , and insulin levels with il-1 and anti - lcsfa antibodies . the wilcoxon signed - rank test was used to identify baseline to 3-month paired differences . mann - whitney test was used for nonparametric unpaired data analysis of changes in anti - lcsfa antibody and ffa levels . lipids are generally considered to be poor immunogens , and immunoassays targeting lipids like pa are rare . we detected igm ( data not shown ) and igg antibodies which recognize bsa - pa in serum by using a specialized elisa ( figure 2 ) . because adsorbent 96-well plates bind protein and not fas , we conjugated the fa of interest , pa , to bsa ( bsa - pa ) in order to stably coat the plates with pa ( figures 2(a ) and 2(c ) ) . the acylation reaction of bsa with pa occurs via an sn2 mechanism which results in o - palmitoylation of serine , tyrosine , and threonine in bsa ( figures 2(a ) , 3(a ) , and 3(b ) ) . due to the orientation of the palmitoyl groups on bsa , only the carbon chain is an accessible epitope for ab binding ( figure 3(b ) ) . we adopted this published approach with slight modifications and demonstrated that class switched igg antibodies which recognize pa can be identified in human serum ( figure 2(b ) ) . additionally , we tested whether serum samples with differential reactivity for bsa - pa according to elisa could also be distinguished by western blot ( figure 2(d ) ) . the serum with higher od450 did exhibit stronger reactivity with bsa - pa than sera with a lower od450 . to further characterize the antibodies which recognize bsa - pa , we isolated the total igg fraction and from this igg fraction we also isolated igg antibodies which recognize bsa - pa from 5 en balance participants with the highest antibody levels to determine ab specificity and avidity . using a novel lipid dot blot method and a panel of fatty acids ( table 3 ) , we found that these antibodies can recognize nonesterified palmitic acid ( 16 : 0 ) . in addition , we found that the antibodies cross - react with stearic acid ( 18 : 0 ) , lignoceric acid ( 24 : 0 ) , and elaidic acid ( 19 : 1 ) ( figure 4(a ) ) . because the bsa - pa reactive antibodies cross - react with long chain saturated fatty acids ( lcsfa ) , we termed these antibodies anti - lcsfa igg . we also determined that the bsa - pa reactive igg recognizes palmitic acid esterified to glycerol but not to phosphate ( figure 4(b ) ) . both total igg fractions and next , we modified this dot blot approach to determine whether the antibodies could bind to nonesterified pa in the presence of physiological concentrations of bsa . figure 4(c ) indicates that a pa dot blot probed with total igg fraction containing bsa - pa reactive igg still binds to pa in the presence of 750 m bsa . in addition , we determined the avidity of the igg antibodies for bsa - pa . following the protocol published by boullerne et al . , we used a competition elisa to determine that the avidity of anti - lcsfa igg to bsa - pa was approximately 2.07 10 ( figure 4(d ) ) . after validation of the elisa technique to identify anti - lcsfa antibodies , we set out to determine whether these antibodies play a role in type 2 diabetes . we tested for the presence of pa antibodies in the sera of 46 hispanics with type 2 diabetes and assessed the impact of the en balance program on antibody levels . out of a total of 67 serum samples , paired samples ( baseline versus 3-month intervention ) were available for only 21 participants . we found that 100% ( 36/36 ) of the hispanics with type 2 diabetes tested positive for anti - lcsfa igg antibodies before undergoing the diabetes education program ( figure 5(a ) ) . interestingly , after 3 months of participation in the en balance program , the frequency of participants that tested positive for anti - lcsfa antibodies significantly decreased to 49% ( 20/41 ) ( figure 5(a ) ) . more importantly , the relative levels of anti - lcsfa antibodies in individual participants ( n = 21 ) from baseline ( mean sem = 1.05 0.121 ) to 3 months ( mean sem = 0.25 0.030 ) were significantly reduced ( figure 5(b ) ) . this reduction in anti - lcsfa antibodies coincides with improved diabetes control as described in previous publications ( table 4 ) [ 23 , 2528 ] . to determine whether these results are cohort , disease , or ethnicity specific , we tested serum samples from the bioserve biorepository . we found that approximately 30% of caucasian control samples ( 7/21 ) and 33% of samples from caucasians with type 2 diabetes ( 6/20 ) tested positive for anti - lcsfa igg by elisa ( figure 5(c ) ) . overall , samples from hispanic cohorts showed an increased frequency of patients who tested positive for anti - lcsfa igg . sera from hispanic controls were 55% positive ( 11/20 ) while sera from hispanics with type 2 diabetes were 50% positive ( 10/20 ) . interestingly , anti - lcsfa antibodies levels were higher in the bioserve hispanic control group than in the bioserve hispanic diabetic cohort . the frequencies of serum samples positive for anti - lcsfa igg in the hispanic cohorts from the bioserve biorepository ( 55% and 50% ) were similar to that of serum samples from the en balance ( 49% ) cohort after 3 months of intervention . lastly , to compare anti - lcsfa antibody levels across all groups , we performed multiple of the median ( mom ) analysis . this type of analysis normalizes data with 1 corresponding to normal levels and also measures how far an individual test result deviates from the median . mom is commonly used to report the results of medical screening tests that tend to be highly variable . first , we corrected the anti - lcsfa igg optical density values at od450 by subtracting out the od450 values obtained by reacting each serum sample with bsa alone . we then found the median of the all the corrected od450 values from both the bioserve and the en balance samples . each sample 's od450 value was divided by this median to obtain the mom value . we found that only the baseline serum samples from the en balance study significantly deviated from in addition to measuring pa antibodies , we determined the concentration of il-1 present in the serum of en balance participants . we found that serum il-1 levels did not change from baseline to 3 months ( table 4 ) . next , we performed multivariate analysis of anti - lcsfa antibodies , il-1 , and the health variables collected from each participant at baseline and at 3 months . surprisingly , we found that anti - lcsfa antibodies negatively correlated with body fat ( table 5 ) . in addition , the levels of il-1 correlated to hba1c values ( table 5 ) . we measured the concentration of total nonesterified fatty acid ( both bound and free ) in all serum samples . confirming the literature , total fa in serum was significantly higher in the cohorts with type 2 diabetes as compared to the controls without diabetes ( figure 6(a ) ) . when combining all cohorts , the levels of fa did not correlate with the levels of anti - lcsfa igg detected in the serum samples ( figure 6(b ) ) . because no significant correlations of anti - lcsfa antibodies with serum fa were found overall , we subcategorized the samples and analyzed the data according to the published american diabetes association target ranges for fasting blood glucose ( fbg ) . we found that the levels of ffa correlate to anti - lcsfa igg levels ( n = 24 , spearman 's rho 0.4862 , p = 0.16 ) for individuals whose fbg was in the target range of 70130 mg / dl . in other words , the levels of ffa correlate to anti - lcsfa igg levels for individuals whose diabetes is managed . finally , to further understand the functions anti - lcsfa antibodies may play in humans , we determined the effect of anti - lcsfa antibodies on pa - induced secretion of il-1 from dendritic cells in the presence of bsa ( figure 6(c ) ) . we found that preabsorption of pa with igg antibodies from patients with high levels of anti - lcsfa igg significantly reduced dc secretion of il-1 ( figure 6(c ) ) . however , a significant reduction in dc secretion of il1 was not observed in patients who tested negative for anti - lcsfa by elisa . this result suggests that anti - lcsfa igg may sequester nonesterified fas in the blood . in support of this hypothesis , we found that anti - lcsfa antibodies negatively correlate with serum il-1 in the en balance cohort at baseline ( n = 33 , spearman 's r = 0.34 , p = 0.026 ) and at 3 months ( n = 33 , spearman 's r = 0.26 , p = 0.05 ) . in this paper we show that igg antibodies against pa and other saturated fatty acids are detectable in human serum ( figures 2 and 5 ) . the activity of igg antibodies occurs principally during a secondary antibody response driven by t cells . therefore , the emergence of anti - lcsfa igg antibodies coincides with maturation of an antibody response , which occurs upon repeated exposure to an antigen . we have also demonstrated that conjugation of a fatty acid to the carrier protein bsa is unnecessary for anti - lcsfa igg recognition of saturated fatty acids ( figure 4(a ) ) . the anti - lcsfa antibodies we detected can recognize nonesterified palmitic acid as well as palmitic acid esterified to glycerol ( figure 4(b ) ) , indicating that these antibodies could potentially function both in the blood and in the tissue where saturated fatty acids are stored as triglycerides . characterization of anti - lcsfa igg antibodies revealed that they are not specific for pa but can also bind to other long chain saturated fas ( figure 4 and table 3 ) . anti - lcsfa igg did not recognize butyric acid ( 6 : 0 ) , which indicates that these antibodies may be unable to recognize short chain saturated fas . interestingly , we found that anti - lcsfa did not recognize oleic acid ( 18 : 1 ) but did recognize elaidic acid ( 19 : 1 ) . both oleic and elaidic acid are monounsaturated long chain fas ; however , only elaidic acid is recognized . we speculate that this result is due to the double bond being in the trans- rather than cis - configuration ( table 3 , figure 3(c ) ) , leading to a configuration structurally mimicking a saturated fa ( figure 3(a ) ) . further , we did not detect any igg antibodies which bind to polyunsaturated fas , supporting the notion that polyunsaturated fas are anti - inflammatory . overall , the results presented in figure 3 implicate that the epitope which anti - lcsfa antibodies recognize is dependent on the carbon chain length of the fatty acid as well as its configuration . thus , it is possible that a diet high in saturated fat may be linked to anti - lcsfa antibody generation . of particular significance is that our data clearly show that anti - lcsfa antibodies were reduced in serum samples obtained from hispanics with type 2 diabetes after 3 months of culturally sensitive diabetes education ( figure 5 ) . in this same cohort of diabetic patient participants , blood glucose , hba1c , body fat , and dietary fat were also found to be significantly reduced , indicating that managing diabetes through diet and exercise results in a reduction of pa antibody titers ( table 4 and [ 23 , 2528 ] ) . therefore , we can speculate that a mechanism indirectly related to serum fa levels or dietary intake of saturated fat may be responsible for the generation of anti - lcsfa antibodies . our data also showed that anti - lcsfa igg antibodies are found in both the diabetic and nondiabetic condition ( figure 5 ) . in the caucasian cohort this finding indicates that the generation of anti - lcsfa antibodies may be a natural response to fas rather than a pathological response . the detection of bsa - pa reactive antibodies by western blot in serum samples which tested negative by elisa further support this notion ( figure 2(b ) ) . as a natural antibody , anti - lcsfa could potentially play a role in the clearance of saturated fatty acids from the blood stream . this hypothesis is supported by our data that show that the antibodies have the ability to sequester pa and protect dcs from a proinflammatory response ( figure 6(c ) ) . interestingly , hispanic participants presented with the anti - lcsfa antibody about 20% more frequently than caucasian subjects ( figure 5(c ) ) . to our surprise , the serum from bioserve hispanic nondiabetic controls had significantly higher levels of anti - lcsfa igg than the serum from bioserve hispanic diabetes samples ( figure 5(c ) ) . this observed difference could be due to variables for which we could not control in the bioserve samples such as fasting blood glucose , time of sample draw , dietary intake , and ethnic homogeneity . the en balance serum samples were from mexican americans as opposed to the hispanic bioserve samples , which represent 17 countries of origin in addition to different levels of socioeconomic status . although the bioserve hispanic controls had higher levels of anti - lcsfa igg than the bioserve hispanic diabetes group , mom analysis indicated that both of these groups reside within the prospective normal the only group that had serum levels of anti - lcsfa igg above control or normal levels were from the en balance participants at baseline ( figure 5(d ) ) . not only did these participants have type 2 diabetes , but at baseline , their diabetes was unmanaged ( table 4 ) . this may suggest that high anti - lcsfa antibody titers are related to how well type 2 diabetes is managed . further characterization of the relationship between diabetes management and anti - lcsfa igg antibody levels could potentially lead to using this antibody as an additional biological indicator in the management of type 2 diabetes . table 5 demonstrates that anti - lcsfa antibodies negatively correlate with body fat and that il-1 positively correlates with hba1c . these correlations seem counterintuitive because increased adiposity is associated with low - grade chronic inflammation . however , obesity and increased bmi have been associated with impaired antibody responses [ 3234 ] . our study shows that increased fat is associated with reduced production of anti - lcsfa antibodies , a result which corroborates the literature findings [ 3234 ] . the correlation of serum il-1 with hba1c supports the current convention that il-1 is strongly associated with insulin resistance and type 2 diabetes [ 5 , 35 ] . we found that igg from hispanics with type 2 diabetes neutralized the secretion of il-1 from dcs ( figure 6(c ) ) . previous studies have determined that igg antibodies generated in type 2 diabetes are pathogenic [ 17 , 18 ] . an alternative possibility that requires further exploration is that anti - lcsfa igg antibodies are generated as a protective mechanism against increased plasma nonesterified saturated fas and subsequent inflammation including insulin resistance ( figure 6(d ) ) . in managed diabetes , total serum nonesterified fas correlate with anti - lcsfa igg levels . however , nonesterified fas do not correlate in unmanaged diabetes , indicating dysregulation of what may be a normal process of antibody production . further research is necessary to determine what mechanisms link saturated fatty acids to the production of anti - lcsfa igg . based on the available data , our current hypothesis is that increased nonesterified pa resulting from excess calories ( or overeating ) and obesity stimulates the production of anti - lcsfa antibodies through an indirect mechanism such as palmitoylating serum proteins ( figure 6(d ) ) . because we observed class switched igg antibodies ( figures 2 , 4 , and 5 ) , we speculate that generation of anti - lcsfa antibodies may be antigen and t cell dependent . these antibodies however , the natural function of anti - lcsfa igg in the body remains unknown . if the role of anti - lcsfa igg in the healthy state and in unmanaged type 2 diabetes can be further validated , a new avenue in understanding and treatment of these conditions is possible . alternatively , they could be used as indicators of the level of diabetes management . understanding how poor type 2 diabetes management impacts anti - lcsfa igg production and lipid immunology could be a major influence in the future of type 2 diabetes research . the main objective of the present study was to determine whether there are detectable levels of anti - lcsfa antibodies in normal or diabetic patients and our findings support the existence of such antibodies . future studies need to explore in depth the significance of this finding using larger cohorts of normal , prediabetic and diabetic patients . we propose that our findings raise the need to investigate the role of lipid antibodies in healthy conditions and in type 2 diabetes to further our understanding of lipid immunology .

high levels of serum long chain saturated fatty acids ( lcsfas ) have been associated with inflammation in type 2 diabetes . dietary sfas can promote inflammation , the secretion of igg antibodies , and secretion of the proinflammatory cytokine il-1. this study characterizes anti - lcsfa igg antibodies from patients with type 2 diabetes . serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti - lcsfa igg , the cytokine il-1 , and nonesterified fatty acids . anti - lcsfa igg was isolated from patient samples and used for in vitro characterization of avidity and specificity . a cohort participating in en balance , a diabetes health education program that improved diabetes management , tested positive for anti - lcsfa igg . following the 3-month program , the cohort showed a significant reduction in anti - lcsfa igg levels . anti - lcsfa antibodies isolated from these patients demonstrated high avidity , were specific for long chain sfas , and correlated with serum fatty acids in patients with managed type 2 diabetes . interestingly , anti - lcsfa igg neutralized pa - induced il-1 secretion by dendritic cells . our data shows that nonesterified sfas are recognized by igg antibodies present in human blood . the identification of anti - lcsfa igg antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients .

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

although elevated plasma levels of nonesterified fas have been extensively correlated to insulin resistance , the role of specific fas , such as palmitic acid ( pa ) , is not well understood [ 3 , 4 ] . visceral adipose tissue is a major site of obesity - induced inflammation , and dyslipidemia is a major factor in the recruitment of activated immune cells such as macrophages , t cells , nk cells , dendritic cells , and b cells to visceral adipose tissue . infiltrating adipose immune cells are a major source of proinflammatory cytokines in obesity - induced inflammation and type 2 diabetes [ 57 ] . in particular , the proinflammatory cytokine il-1 can directly cause insulin resistance in insulin - sensitive cells [ 5 , 811 ] . recently , b cells have been recognized as a major contributor to obesity - induced inflammation [ 5 , 1315 ] . the importance of igg antibodies secreted by b cells has been established in a mouse model of type 2 diabetes . in addition , the transfer of igg antibodies from obesity induced - diabetic mice to nondiabetic mice rapidly induces insulin resistance and glucose intolerance . parallel to mice studies , humans with type 2 diabetes have disease - associated changes in b cell function , but the role of these changes in disease pathogenesis is not well established . moreover , igm antibodies against fas have been reported in multiple sclerosis as well as in human immunodeficiency virus ( hiv ) patients [ 2022 ] . however , there is a gap in the literature of studies demonstrating the presence of igg antibodies against fas such as palmitic acid . the purpose of the present study was to investigate whether humans produce class switched igg antibodies that recognize saturated fas such as pa . to answer this question , we retrospectively analyzed serum from 2 different cohorts of obese individuals , including patients with and without type 2 diabetes and patients who participated in the diabetes intervention program , en balance . this study consisted of analysis of serum samples from the bioserve biorepository in addition to serum samples from a 3-month diabetes education intervention ( en balance ) designed to promote improved type 2 diabetes management in hispanic adults [ 2427 ] . we measured il-1 and antibodies which recognize palmitic acid in these samples and correlated the values obtained from the en balance samples with the original primary outcomes of that study . a total of 73 hispanic males and females with type 2 diabetes met the en balance participation criteria as previously described [ 26 , 27 ] . two blood samples ( 1214 hr fasting ) were drawn from the participants at both baseline and 3 months and analyzed for glucose , a1c , and insulin . serum samples from 46 en balance participants were available for anti - lcsfa antibody and il-1 testing . twenty - one of these participants had paired serum samples from baseline and 3 months available for longitudinal analysis . twelve participants had serum samples from only baseline and 13 participants had serum samples from only 3 months . serum samples ( four groups with 20 serum samples each : hispanic control , hispanics with type 2 diabetes , caucasian control , and caucasians with type 2 diabetes ) purchased from the bioserve biorepository ( beltsville , md ) were also tested for anti - lcsfa antibody and il-1. the samples were gender , age , and bmi matched against the 21 en balance participants with paired samples ( table 2 and figure 1 ) . an elisa was developed for identifying igg antibodies in human serum that recognize pa using the ready - set - go elisa igg kit ( ebioscience ) according to manufacturer 's protocol with the following exceptions . the anti - lcsfa antibodies ( antibodies isolated by affinity to bsa - pa ) were then purified from their respective igg fraction by affinity chromatography according to the protocol outlined for the sulfolink immobilization kit ( thermo scientific ) . a lipid dot blot method was established to determine the reactivity of a panel of nonesterified fas ( from sigma - aldrich ) and esterified palmitic acid ( glycerol tripalmitate from sigma - aldrich and n - palmitoyl phosphatidyl ethanolamine from enzo life sciences ) to purified anti - lcsfa igg . after washing the blot with pbs , anti - human igg alkaline phosphatase conjugate ( sigma ) was used as the secondary ( 1 : 10,000 ) detection ab . monocytes ( cd14 ) were isolated from human peripheral blood ( lifestream blood bank ) and cultured at 37c and 5% co2 in 96-well plates at a density of 200,000 per well in rpmi-1640 supplemented with 10% fbs , 1,000 u penicillin and streptomycin , 50 ng / ml gm - csf , and 10 ng / ml il-4 for 6 days to differentiate them into dendritic cells ( dcs ) . the dcs were treated with 150 m pa in a 1 : 1 ratio with bsa for 24 hrs in the presence of igg ab containing anti - lcsfa igg . a mixture of total igg isolated from 5 hispanics with type 2 diabetes with high anti - lcsfa igg levels ( measured by elisa ) or a mixture of total igg isolated from 2 samples which tested negative for anti - lcsfa igg ( measured by elisa ) was used at a concentration of 1.4 mg / ml . , the concentration of free fatty acid in the serum samples diluted 1 : 2 was determined in duplicate following the manufacturer 's recommendations . en balance serum samples and cell culture supernatants were analyzed with a human il-1 flex set as described by the manufacturer ( bd biosciences ) on a macsquant flow cytometer ( miltenyi biotech ) . , spearman 's correlation coefficient was used to determine associations between body composition , glucose , and insulin levels with il-1 and anti - lcsfa antibodies . mann - whitney test was used for nonparametric unpaired data analysis of changes in anti - lcsfa antibody and ffa levels . we adopted this published approach with slight modifications and demonstrated that class switched igg antibodies which recognize pa can be identified in human serum ( figure 2(b ) ) . to further characterize the antibodies which recognize bsa - pa , we isolated the total igg fraction and from this igg fraction we also isolated igg antibodies which recognize bsa - pa from 5 en balance participants with the highest antibody levels to determine ab specificity and avidity . using a novel lipid dot blot method and a panel of fatty acids ( table 3 ) , we found that these antibodies can recognize nonesterified palmitic acid ( 16 : 0 ) . because the bsa - pa reactive antibodies cross - react with long chain saturated fatty acids ( lcsfa ) , we termed these antibodies anti - lcsfa igg . both total igg fractions and next , we modified this dot blot approach to determine whether the antibodies could bind to nonesterified pa in the presence of physiological concentrations of bsa . figure 4(c ) indicates that a pa dot blot probed with total igg fraction containing bsa - pa reactive igg still binds to pa in the presence of 750 m bsa . in addition , we determined the avidity of the igg antibodies for bsa - pa . , we used a competition elisa to determine that the avidity of anti - lcsfa igg to bsa - pa was approximately 2.07 10 ( figure 4(d ) ) . after validation of the elisa technique to identify anti - lcsfa antibodies , we set out to determine whether these antibodies play a role in type 2 diabetes . we tested for the presence of pa antibodies in the sera of 46 hispanics with type 2 diabetes and assessed the impact of the en balance program on antibody levels . we found that 100% ( 36/36 ) of the hispanics with type 2 diabetes tested positive for anti - lcsfa igg antibodies before undergoing the diabetes education program ( figure 5(a ) ) . interestingly , after 3 months of participation in the en balance program , the frequency of participants that tested positive for anti - lcsfa antibodies significantly decreased to 49% ( 20/41 ) ( figure 5(a ) ) . more importantly , the relative levels of anti - lcsfa antibodies in individual participants ( n = 21 ) from baseline ( mean sem = 1.05 0.121 ) to 3 months ( mean sem = 0.25 0.030 ) were significantly reduced ( figure 5(b ) ) . this reduction in anti - lcsfa antibodies coincides with improved diabetes control as described in previous publications ( table 4 ) [ 23 , 2528 ] . to determine whether these results are cohort , disease , or ethnicity specific , we tested serum samples from the bioserve biorepository . we found that approximately 30% of caucasian control samples ( 7/21 ) and 33% of samples from caucasians with type 2 diabetes ( 6/20 ) tested positive for anti - lcsfa igg by elisa ( figure 5(c ) ) . overall , samples from hispanic cohorts showed an increased frequency of patients who tested positive for anti - lcsfa igg . sera from hispanic controls were 55% positive ( 11/20 ) while sera from hispanics with type 2 diabetes were 50% positive ( 10/20 ) . interestingly , anti - lcsfa antibodies levels were higher in the bioserve hispanic control group than in the bioserve hispanic diabetic cohort . the frequencies of serum samples positive for anti - lcsfa igg in the hispanic cohorts from the bioserve biorepository ( 55% and 50% ) were similar to that of serum samples from the en balance ( 49% ) cohort after 3 months of intervention . lastly , to compare anti - lcsfa antibody levels across all groups , we performed multiple of the median ( mom ) analysis . first , we corrected the anti - lcsfa igg optical density values at od450 by subtracting out the od450 values obtained by reacting each serum sample with bsa alone . we then found the median of the all the corrected od450 values from both the bioserve and the en balance samples . we found that only the baseline serum samples from the en balance study significantly deviated from in addition to measuring pa antibodies , we determined the concentration of il-1 present in the serum of en balance participants . next , we performed multivariate analysis of anti - lcsfa antibodies , il-1 , and the health variables collected from each participant at baseline and at 3 months . surprisingly , we found that anti - lcsfa antibodies negatively correlated with body fat ( table 5 ) . in addition , the levels of il-1 correlated to hba1c values ( table 5 ) . we measured the concentration of total nonesterified fatty acid ( both bound and free ) in all serum samples . confirming the literature , total fa in serum was significantly higher in the cohorts with type 2 diabetes as compared to the controls without diabetes ( figure 6(a ) ) . when combining all cohorts , the levels of fa did not correlate with the levels of anti - lcsfa igg detected in the serum samples ( figure 6(b ) ) . because no significant correlations of anti - lcsfa antibodies with serum fa were found overall , we subcategorized the samples and analyzed the data according to the published american diabetes association target ranges for fasting blood glucose ( fbg ) . we found that the levels of ffa correlate to anti - lcsfa igg levels ( n = 24 , spearman 's rho 0.4862 , p = 0.16 ) for individuals whose fbg was in the target range of 70130 mg / dl . in other words , the levels of ffa correlate to anti - lcsfa igg levels for individuals whose diabetes is managed . finally , to further understand the functions anti - lcsfa antibodies may play in humans , we determined the effect of anti - lcsfa antibodies on pa - induced secretion of il-1 from dendritic cells in the presence of bsa ( figure 6(c ) ) . we found that preabsorption of pa with igg antibodies from patients with high levels of anti - lcsfa igg significantly reduced dc secretion of il-1 ( figure 6(c ) ) . however , a significant reduction in dc secretion of il1 was not observed in patients who tested negative for anti - lcsfa by elisa . this result suggests that anti - lcsfa igg may sequester nonesterified fas in the blood . in support of this hypothesis , we found that anti - lcsfa antibodies negatively correlate with serum il-1 in the en balance cohort at baseline ( n = 33 , spearman 's r = 0.34 , p = 0.026 ) and at 3 months ( n = 33 , spearman 's r = 0.26 , p = 0.05 ) . in this paper we show that igg antibodies against pa and other saturated fatty acids are detectable in human serum ( figures 2 and 5 ) . the activity of igg antibodies occurs principally during a secondary antibody response driven by t cells . therefore , the emergence of anti - lcsfa igg antibodies coincides with maturation of an antibody response , which occurs upon repeated exposure to an antigen . we have also demonstrated that conjugation of a fatty acid to the carrier protein bsa is unnecessary for anti - lcsfa igg recognition of saturated fatty acids ( figure 4(a ) ) . the anti - lcsfa antibodies we detected can recognize nonesterified palmitic acid as well as palmitic acid esterified to glycerol ( figure 4(b ) ) , indicating that these antibodies could potentially function both in the blood and in the tissue where saturated fatty acids are stored as triglycerides . characterization of anti - lcsfa igg antibodies revealed that they are not specific for pa but can also bind to other long chain saturated fas ( figure 4 and table 3 ) . anti - lcsfa igg did not recognize butyric acid ( 6 : 0 ) , which indicates that these antibodies may be unable to recognize short chain saturated fas . interestingly , we found that anti - lcsfa did not recognize oleic acid ( 18 : 1 ) but did recognize elaidic acid ( 19 : 1 ) . further , we did not detect any igg antibodies which bind to polyunsaturated fas , supporting the notion that polyunsaturated fas are anti - inflammatory . overall , the results presented in figure 3 implicate that the epitope which anti - lcsfa antibodies recognize is dependent on the carbon chain length of the fatty acid as well as its configuration . thus , it is possible that a diet high in saturated fat may be linked to anti - lcsfa antibody generation . of particular significance is that our data clearly show that anti - lcsfa antibodies were reduced in serum samples obtained from hispanics with type 2 diabetes after 3 months of culturally sensitive diabetes education ( figure 5 ) . therefore , we can speculate that a mechanism indirectly related to serum fa levels or dietary intake of saturated fat may be responsible for the generation of anti - lcsfa antibodies . our data also showed that anti - lcsfa igg antibodies are found in both the diabetic and nondiabetic condition ( figure 5 ) . in the caucasian cohort this finding indicates that the generation of anti - lcsfa antibodies may be a natural response to fas rather than a pathological response . as a natural antibody , anti - lcsfa could potentially play a role in the clearance of saturated fatty acids from the blood stream . interestingly , hispanic participants presented with the anti - lcsfa antibody about 20% more frequently than caucasian subjects ( figure 5(c ) ) . to our surprise , the serum from bioserve hispanic nondiabetic controls had significantly higher levels of anti - lcsfa igg than the serum from bioserve hispanic diabetes samples ( figure 5(c ) ) . the en balance serum samples were from mexican americans as opposed to the hispanic bioserve samples , which represent 17 countries of origin in addition to different levels of socioeconomic status . although the bioserve hispanic controls had higher levels of anti - lcsfa igg than the bioserve hispanic diabetes group , mom analysis indicated that both of these groups reside within the prospective normal the only group that had serum levels of anti - lcsfa igg above control or normal levels were from the en balance participants at baseline ( figure 5(d ) ) . not only did these participants have type 2 diabetes , but at baseline , their diabetes was unmanaged ( table 4 ) . this may suggest that high anti - lcsfa antibody titers are related to how well type 2 diabetes is managed . further characterization of the relationship between diabetes management and anti - lcsfa igg antibody levels could potentially lead to using this antibody as an additional biological indicator in the management of type 2 diabetes . table 5 demonstrates that anti - lcsfa antibodies negatively correlate with body fat and that il-1 positively correlates with hba1c . however , obesity and increased bmi have been associated with impaired antibody responses [ 3234 ] . our study shows that increased fat is associated with reduced production of anti - lcsfa antibodies , a result which corroborates the literature findings [ 3234 ] . the correlation of serum il-1 with hba1c supports the current convention that il-1 is strongly associated with insulin resistance and type 2 diabetes [ 5 , 35 ] . we found that igg from hispanics with type 2 diabetes neutralized the secretion of il-1 from dcs ( figure 6(c ) ) . previous studies have determined that igg antibodies generated in type 2 diabetes are pathogenic [ 17 , 18 ] . an alternative possibility that requires further exploration is that anti - lcsfa igg antibodies are generated as a protective mechanism against increased plasma nonesterified saturated fas and subsequent inflammation including insulin resistance ( figure 6(d ) ) . in managed diabetes , total serum nonesterified fas correlate with anti - lcsfa igg levels . further research is necessary to determine what mechanisms link saturated fatty acids to the production of anti - lcsfa igg . based on the available data , our current hypothesis is that increased nonesterified pa resulting from excess calories ( or overeating ) and obesity stimulates the production of anti - lcsfa antibodies through an indirect mechanism such as palmitoylating serum proteins ( figure 6(d ) ) . because we observed class switched igg antibodies ( figures 2 , 4 , and 5 ) , we speculate that generation of anti - lcsfa antibodies may be antigen and t cell dependent . these antibodies however , the natural function of anti - lcsfa igg in the body remains unknown . if the role of anti - lcsfa igg in the healthy state and in unmanaged type 2 diabetes can be further validated , a new avenue in understanding and treatment of these conditions is possible . alternatively , they could be used as indicators of the level of diabetes management . understanding how poor type 2 diabetes management impacts anti - lcsfa igg production and lipid immunology could be a major influence in the future of type 2 diabetes research . the main objective of the present study was to determine whether there are detectable levels of anti - lcsfa antibodies in normal or diabetic patients and our findings support the existence of such antibodies . we propose that our findings raise the need to investigate the role of lipid antibodies in healthy conditions and in type 2 diabetes to further our understanding of lipid immunology .

doctor - patient communication is considered an important marker of health - care quality,1 and the social - psychological aspects of the patient - physician interaction are increasingly recognized as complementary to the more technical aspects of medical care.2,3 high quality doctor - patient communication involves multiple domains , including ( 1 ) building an effective relationship , ( 2 ) gathering information , ( 3 ) understanding the patient s perspective , ( 4 ) giving information and ( 5 ) decision making.4,5 patients rate communication with their physicians as a valuable part of the medical encounter,6,7 and improved doctor - patient communication has been associated with higher patient satisfaction,6,8 self - management of chronic diseases,912 more appropriate prescribing of medications13 and improved health outcomes.1417 doctor - patient communication is particularly important in the management of chronic diseases , which may require frequent encounters with the medical system and complex treatment decisions . patients with chronic disease often have multiple medical comorbidities and severe illness , presenting clinical challenges for physicians that may result in less attention to the social - psychological aspects of the patient - physician interaction.18,19 patients with chronic disease also suffer disproportionately from depression,20,21 which may complicate doctor - patient communication through the challenge of managing depression along with other medical conditions or because depressed patients experience or recall communication with their physicians differently.2224 however , little is known about the relative association of medical comorbidities , disease severity and depressive symptoms with doctor - patient communication . we conducted this study to evaluate the association of medical comorbidities , disease severity and depressive symptoms with patient reports of doctor - patient communication in a cohort of adult patients with coronary heart disease ( chd ) . we chose to examine predictors of doctor - patient communication in this cohort because chd is a chronic illness associated with high rates of medical comorbidities and depression . the heart and soul study is an ongoing prospective cohort study of adults with coronary heart disease ( chd ) . study recruitment procedures have been published previously.25 briefly , we used administrative databases to identify outpatients with documented chd at two veterans affairs medical centers in northern california ( san francisco and palo alto ) , one university medical center ( university of california at san francisco ) and nine local community health clinics . we defined documented chd as at least one of the following : a history of myocardial infarction , angiographic evidence of at least 50% stenosis in one or more coronary arteries , previous evidence of exercise - induced ischemia by treadmill or nuclear testing , a history of coronary artery revascularization or a diagnosis of chd by an internist or a cardiologist . subjects were excluded if they had a history of myocardial infarction in the prior 6 months , could not walk one block or were planning to move from the local area within 3 years . the majority of participants spoke english ; a small number of non - english - speaking patients participated through the assistance of an english - speaking family member . after 315 participants had already been enrolled , additional funding enabled us to add instruments measuring doctor - patient communication as described below . of the 709 remaining participants , 703 answered one or more questions about doctor - patient communication and were included in this cross - sectional study . all subjects completed a day - long initial visit that involved an extensive structured questionnaire to measure demographic variables , medical comorbidities and psychosocial functioning , a resting echocardiogram and an exercise treadmill test with stress echocardiography . the protocol was approved by the appropriate institutional review boards , and all participants provided written informed consent . we assessed doctor - patient communication using the two - item explanations of condition subscale and the four - item responsiveness to patient preferences subscale from the interpersonal processes of care ( ipc ) instrument , an established self - report questionnaire designed to measure specific components of doctor - patient communication in diverse populations.2 details of the reliability and validity of the ipc instrument have been published previously.2 the questionnaire was self - administered with a research assistant available in the same room to help patients understand and interpret questions as necessary . the explanations of condition subscale consisted of two questions : how often did your doctor(s ) give you enough information about your health problems ? and how often did your doctor(s ) make sure you understood your health problems ? the responsiveness to patient preferences subscale consisted of four questions : how often did your doctor(s ) try to involve you or include you in decisions about your treatment ? how often did your doctor(s ) ask how you felt about different treatments ? how often did your doctor(s ) make decisions without taking your preferences or opinions into account ? and how often did you feel pressured by your doctor(s ) to have a treatment you were nt sure you wanted ? for each question , patients were asked to report their experiences with their physician over the past 6 months on a five - point likert scale ranging from never to always . we generated a summary score for each subscale by adding up the total scores within the subscale and dividing by the number of questions . for participants who did not complete all questions in the subscale ( n = 6 for explanations of condition and n = 79 for responsiveness to patient preferences ) , we divided by the number of answered questions . higher scores indicate better experiences of doctor - patient communication ( the likert scale was reverse scored for negative characteristics ) . we evaluated the ipc subscales both as continuous variables and as dichotomous variables , with a score of < 4 on each subscale ( corresponding to never / rarely / sometimes for positive characteristics and always / often / sometimes for negative characteristics ) considered poor doctor - patient communication.26 we assessed depressive symptoms using the nine - item patient health questionnaire ( phq).27,28 we evaluated depressive symptoms both as a continuous variable ( range of scores 0 to 27 ) and as an ordinal variable , using previously established categories of none - minimal ( phq score of 03 ) , mild ( phq score of 49 ) or moderate - severe ( phq score 10 ) depressive symptoms.28 medical comorbidities ( history of hypertension , diabetes and myocardial infarction ) were assessed by patient questionnaire . we assessed disease severity using three objective measures of cardiac function : a resting two - dimensional echocardiogram for measurement of left ventricular ejection fraction ( lvef ) , an exercise treadmill test for measurement of exercise capacity and a stress echocardiogram for assessment of inducible ischemia . we performed a symptom - limited , graded exercise treadmill test according to a standard bruce protocol , a widely adopted and validated measure of exercise capacity and ischemic heart disease.29 peak exercise capacity was defined as the total number of metabolic equivalent tasks ( mets ) achieved . poor exercise capacity was defined a priori as < 5 mets.30 inducible ischemia was defined as the presence of exercise - induced electrocardiographic changes or new echocardiographic wall motion abnormalities at peak exercise . education was measured by asking participants what is the highest level of education that you have completed ? participants who indicated grade 12 ( or equivalent ) or higher were considered to have a high school education . income was measured by asking participants which of the following categories best describes your total combined household income for the past 12 months ? primary care site was determined from administrative data . our goal was to examine the associations of medical comorbidities , disease severity and depressive symptoms with patient reports of doctor - patient communication . in univariate analyses , we compared differences in characteristics of participants ( including demographic variables , medical comorbidities , disease severity and depressive symptoms ) in patients with low ( poor communication ) vs. high ( good communication ) ipc subscale scores . chi - squared tests were used for dichotomous variables and t tests for continuous variables . we then used logistic regression to evaluate the independent association of depressive symptoms with poor reported doctor - patient communication , adjusted for demographic characteristics ( age , sex , race , education , income ) , medical comorbidities ( history of myocardial infarction , hypertension and diabetes ) and disease severity ( resting left ventricular ejection fraction , exercise capacity , inducible ischemia ) . the heart and soul study is an ongoing prospective cohort study of adults with coronary heart disease ( chd ) . study recruitment procedures have been published previously.25 briefly , we used administrative databases to identify outpatients with documented chd at two veterans affairs medical centers in northern california ( san francisco and palo alto ) , one university medical center ( university of california at san francisco ) and nine local community health clinics . we defined documented chd as at least one of the following : a history of myocardial infarction , angiographic evidence of at least 50% stenosis in one or more coronary arteries , previous evidence of exercise - induced ischemia by treadmill or nuclear testing , a history of coronary artery revascularization or a diagnosis of chd by an internist or a cardiologist . subjects were excluded if they had a history of myocardial infarction in the prior 6 months , could not walk one block or were planning to move from the local area within 3 years . the majority of participants spoke english ; a small number of non - english - speaking patients participated through the assistance of an english - speaking family member . after 315 participants had already been enrolled , additional funding enabled us to add instruments measuring doctor - patient communication as described below . of the 709 remaining participants , 703 answered one or more questions about doctor - patient communication and were included in this cross - sectional study . all subjects completed a day - long initial visit that involved an extensive structured questionnaire to measure demographic variables , medical comorbidities and psychosocial functioning , a resting echocardiogram and an exercise treadmill test with stress echocardiography . the protocol was approved by the appropriate institutional review boards , and all participants provided written informed consent . we assessed doctor - patient communication using the two - item explanations of condition subscale and the four - item responsiveness to patient preferences subscale from the interpersonal processes of care ( ipc ) instrument , an established self - report questionnaire designed to measure specific components of doctor - patient communication in diverse populations.2 details of the reliability and validity of the ipc instrument have been published previously.2 the questionnaire was self - administered with a research assistant available in the same room to help patients understand and interpret questions as necessary . the explanations of condition subscale consisted of two questions : how often did your doctor(s ) give you enough information about your health problems ? and how often did your doctor(s ) make sure you understood your health problems ? the responsiveness to patient preferences subscale consisted of four questions : how often did your doctor(s ) try to involve you or include you in decisions about your treatment ? how often did your doctor(s ) ask how you felt about different treatments ? how often did your doctor(s ) make decisions without taking your preferences or opinions into account ? and how often did you feel pressured by your doctor(s ) to have a treatment you were nt sure you wanted ? for each question , patients were asked to report their experiences with their physician over the past 6 months on a five - point likert scale ranging from never to always . we generated a summary score for each subscale by adding up the total scores within the subscale and dividing by the number of questions . for participants who did not complete all questions in the subscale ( n = 6 for explanations of condition and n = 79 for responsiveness to patient preferences ) , we divided by the number of answered questions . higher scores indicate better experiences of doctor - patient communication ( the likert scale was reverse scored for negative characteristics ) . we evaluated the ipc subscales both as continuous variables and as dichotomous variables , with a score of < 4 on each subscale ( corresponding to never / rarely / sometimes for positive characteristics and always / often / sometimes for negative characteristics ) considered poor doctor - patient communication.26 we assessed depressive symptoms using the nine - item patient health questionnaire ( phq).27,28 we evaluated depressive symptoms both as a continuous variable ( range of scores 0 to 27 ) and as an ordinal variable , using previously established categories of none - minimal ( phq score of 03 ) , mild ( phq score of 49 ) or moderate - severe ( phq score 10 ) depressive symptoms.28 medical comorbidities ( history of hypertension , diabetes and myocardial infarction ) were assessed by patient questionnaire . we assessed disease severity using three objective measures of cardiac function : a resting two - dimensional echocardiogram for measurement of left ventricular ejection fraction ( lvef ) , an exercise treadmill test for measurement of exercise capacity and a stress echocardiogram for assessment of inducible ischemia . we performed a symptom - limited , graded exercise treadmill test according to a standard bruce protocol , a widely adopted and validated measure of exercise capacity and ischemic heart disease.29 peak exercise capacity was defined as the total number of metabolic equivalent tasks ( mets ) achieved . poor exercise capacity was defined a priori as < 5 mets.30 inducible ischemia was defined as the presence of exercise - induced electrocardiographic changes or new echocardiographic wall motion abnormalities at peak exercise . we assessed doctor - patient communication using the two - item explanations of condition subscale and the four - item responsiveness to patient preferences subscale from the interpersonal processes of care ( ipc ) instrument , an established self - report questionnaire designed to measure specific components of doctor - patient communication in diverse populations.2 details of the reliability and validity of the ipc instrument have been published previously.2 the questionnaire was self - administered with a research assistant available in the same room to help patients understand and interpret questions as necessary . the explanations of condition subscale consisted of two questions : how often did your doctor(s ) give you enough information about your health problems ? and how often did your doctor(s ) make sure you understood your health problems ? the responsiveness to patient preferences subscale consisted of four questions : how often did your doctor(s ) try to involve you or include you in decisions about your treatment ? how often did your doctor(s ) ask how you felt about different treatments ? how often did your doctor(s ) make decisions without taking your preferences or opinions into account ? and how often did you feel pressured by your doctor(s ) to have a treatment you were nt sure you wanted ? for each question , patients were asked to report their experiences with their physician over the past 6 months on a five - point likert scale ranging from never to always . we generated a summary score for each subscale by adding up the total scores within the subscale and dividing by the number of questions . for participants who did not complete all questions in the subscale ( n = 6 for explanations of condition and n = 79 for responsiveness to patient preferences ) , we divided by the number of answered questions . higher scores indicate better experiences of doctor - patient communication ( the likert scale was reverse scored for negative characteristics ) . we evaluated the ipc subscales both as continuous variables and as dichotomous variables , with a score of < 4 on each subscale ( corresponding to never / rarely / sometimes for positive characteristics and always / often / sometimes for negative characteristics ) considered poor doctor - patient communication.26 we assessed depressive symptoms using the nine - item patient health questionnaire ( phq).27,28 we evaluated depressive symptoms both as a continuous variable ( range of scores 0 to 27 ) and as an ordinal variable , using previously established categories of none - minimal ( phq score of 03 ) , mild ( phq score of 49 ) or moderate - severe ( phq score 10 ) depressive symptoms.28 medical comorbidities ( history of hypertension , diabetes and myocardial infarction ) were assessed by patient questionnaire . we assessed disease severity using three objective measures of cardiac function : a resting two - dimensional echocardiogram for measurement of left ventricular ejection fraction ( lvef ) , an exercise treadmill test for measurement of exercise capacity and a stress echocardiogram for assessment of inducible ischemia . we performed a symptom - limited , graded exercise treadmill test according to a standard bruce protocol , a widely adopted and validated measure of exercise capacity and ischemic heart disease.29 peak exercise capacity was defined as the total number of metabolic equivalent tasks ( mets ) achieved . poor exercise capacity was defined a priori as < 5 mets.30 inducible ischemia was defined as the presence of exercise - induced electrocardiographic changes or new echocardiographic wall motion abnormalities at peak exercise . education was measured by asking participants what is the highest level of education that you have completed ? participants who indicated grade 12 ( or equivalent ) or higher were considered to have a high school education . income was measured by asking participants which of the following categories best describes your total combined household income for the past 12 months ? our goal was to examine the associations of medical comorbidities , disease severity and depressive symptoms with patient reports of doctor - patient communication . in univariate analyses , we compared differences in characteristics of participants ( including demographic variables , medical comorbidities , disease severity and depressive symptoms ) in patients with low ( poor communication ) vs. high ( good communication ) ipc subscale scores . chi - squared tests were used for dichotomous variables and t tests for continuous variables . we then used logistic regression to evaluate the independent association of depressive symptoms with poor reported doctor - patient communication , adjusted for demographic characteristics ( age , sex , race , education , income ) , medical comorbidities ( history of myocardial infarction , hypertension and diabetes ) and disease severity ( resting left ventricular ejection fraction , exercise capacity , inducible ischemia ) . of the 703 participants , 526 ( 75% ) were male , and the mean age was 65 ( range 3697 ) . overall , 407 ( 58% ) participants were white , 126 ( 18% ) were black , 87 ( 13% ) were asian or pacific islander , and 63 ( 9% ) were latino . of the 703 participants , 122 ( 17% ) were recruited from the san francisco and palo alto va medical centers , 340 ( 48% ) from the university of california , san francisco , and 236 ( 34% ) from public health clinics in the community health network of san francisco . overall , 27% ( 186/701 ) reported poor doctor - patient communication on the explanations of condition subscale ( score < 4 ) and 41% ( 286/702 ) reported poor doctor - patient communication on the responsiveness to patient preferences subscale ( score < 4 ) . participant responses were skewed toward reporting more positive communication experiences ( fig . 1 ) . figure 1distribution of summary scores on the explanations of condition and responsiveness to patient preferences subscales*. * participants rated doctor - patient communication on a five - point likert scale ranging from never to always . a summary score was generated for each subscale by adding up the total scores within the scale and dividing by the number of answered questions . distribution of summary scores on the explanations of condition and responsiveness to patient preferences subscales*. * participants rated doctor - patient communication on a five - point likert scale ranging from never to always . a summary score was generated for each subscale by adding up the total scores within the scale and dividing by the number of answered questions . participants who reported poor explanations of condition were more likely to be female , more likely to be asian , and less likely to be black than those who reported good explanations of condition ( table 1 ) . those who reported poor responsiveness to patient preferences were more likely to be white and less likely to be black than those who reported good responsiveness to patient preferences . participants who reported poor communication on either subscale were more likely to have depressive symptoms . there were no other significant differences in the characteristics of patients reporting poor vs. good doctor - patient communication on either subscale . ratings of communication did not differ by primary care site and were not associated with medical comorbidities or objective measures of disease severity . table 1characteristics of participants by reports of doctor - patient communication * explanations of conditionresponsiveness to patient preferencespoorgoodp valuepoorgoodp value(n = 186)(n = 515)(n = 286)(n = 416)demographics age , years , mean sd65 1165 110.7965 1165 110.74 male128 ( 69%)396 ( 77%)0.03211 ( 74%)314 ( 76%)0.61 race0.020.01 white110 ( 59%)297 ( 58%)180 ( 63%)227 ( 55% ) black21 ( 11%)105 ( 20%)34 ( 12%)92 ( 22% ) asian31 ( 17%)55 ( 11%)38 ( 13%)48 ( 12% ) latino17 ( 9%)46 ( 9%)25 ( 9%)38 ( 9% ) other7 ( 4%)11 ( 2%)9 ( 3%)10 ( 2% ) high school education162 ( 88%)446 ( 87%)0.78254 ( 89%)354 ( 85%)0.1 annual income 20 k102 ( 55%)267 ( 52%)0.56139 ( 49%)231 ( 56%)0.07 primary care site0.10.55 san francisco va21 ( 11%)91 ( 18%)43 ( 15%)69 ( 17% ) palo alto va1 ( 1%)9 ( 2%)4 ( 1%)6 ( 1% ) ucsf99 ( 54%)240 ( 47%)147 ( 52%)192 ( 46% ) public health clinic64 ( 35%)171 ( 33%)89 ( 31%)147 ( 36%)medical comorbidities hypertension129 ( 70%)357 ( 69%)0.92194 ( 68%)293 ( 70%)0.5 diabetes40 ( 22%)142 ( 28%)0.170 ( 24%)113 ( 27%)0.41 myocardial infarction97 ( 52%)264 ( 52%)0.86148 ( 52%)213 ( 52%)0.98disease severity resting lvef , mean sd61.2 9.661.1 9.80.8461.6 9.460.9 10.00.35 exercise capacity , mets7.5 3.37.6 3.50.767.6 3.47.6 3.50.84 inducible ischemia33 ( 20%)93 ( 20%)0.9849 ( 19%)77 ( 21%)0.67depressive symptoms phq-9 , mean sd7.1 5.84.8 5.1<0.00016.1 5.45.0 5.30.006abbreviations : va , veterans affairs medical center ; ucsf , university of california , san francisco ; lvef , left ventricular ejection fraction ; mets , metabolic equivalent tasks ; phq-9 , nine - item patient health questionaire*data are given as number ( percentage ) except where indicated otherwisesubscale score < 4 = poor characteristics of participants by reports of doctor - patient communication * abbreviations : va , veterans affairs medical center ; ucsf , university of california , san francisco ; lvef , left ventricular ejection fraction ; mets , metabolic equivalent tasks ; phq-9 , nine - item patient health questionaire * data are given as number ( percentage ) except where indicated otherwise subscale score < 4 = poor of the 703 participants , 47% had none - minimal depressive symptoms ( phq 03 ) , 32% had mild depressive symptoms ( phq 49 ) and 21% had moderate - severe depressive symptoms ( phq 10 ) . in describing communication with their doctor , 18% of participants with none - minimal depressive symptoms , 31% of participants with mild depressive symptoms and 39% of participants with moderate - severe depressive symptoms reported poor explanations of condition ( p < 0.001 , fig . 2 ) . similarly , 34% of participants with none - minimal depressive symptoms , 48% of participants with mild depressive symptoms and 45% of participants with moderate - severe depressive symptoms reported poor responsiveness to patient preferences ( p = 0.003 , fig . 2 ) . figure 2percentage of participants reporting poor communication on the explanations of condition and responsiveness to patient preferences subscales , * stratified by depressive symptom score . * subscale score < 4 = poor doctor - patient communication . depressive systems assessed using the 9-item patient health questionaire ( phq range of scores 0 - 27 ) . phq score 0 - 3 = none - minimal ; phq score 4 - 9 = mild ; phq score 10 = moderate - severe . percentage of participants reporting poor communication on the explanations of condition and responsiveness to patient preferences subscales , * stratified by depressive symptom score . * subscale score < 4 = poor doctor - patient communication . depressive systems assessed using the 9-item patient health questionaire ( phq range of scores 0 - 27 ) . phq score 0 - 3 = none - minimal ; phq score 4 - 9 = mild ; phq score 10 = moderate - severe . in logistic regression models adjusted for age , with doctor - patient communication coded as a dichotomous outcome variable , each standard deviation ( 5.4 point ) increase in depressive symptom score was associated with a 50% greater odds of poor reported explanations of condition ( or 1.5 , 95% ci , 1.31.8 ; p < 0.0001 ) ( table 2 ) and a 30% greater odds of poor reported responsiveness to patient preferences ( or 1.3 , 95% ci , 1.11.5 ; p = 0.004 ) ( table 3 ) . in multivariable models , each sd increase in depressive symptoms remained strongly associated with poor reported explanations of conditions ( or 1.5 , 95% ci 1.21.8 , p < 0.0001 ) and poor reported responsiveness to patient preferences ( or 1.3 , 95% ci 1.11.5 , p = 0.01 ) , adjusting for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , left ventricular ejection fraction 50% , poor exercise capacity ( mets < 5 ) , and inducible ischemia . we found the same association of depressive symptoms with poor doctor - patient communication in models adjusted only for age , race and sex ( or 1.5 , 95% ci 1.31.8 ; p < 0.0001 for explanations of condition ; or 1.3 , 95% ci 1.11.5 ; p = 0.006 for responsiveness to patient preferences , data not shown ) . in contrast , three objective measures of disease severity ( systolic function , exercise capacity and inducible ischemia ) and three common medical comorbidities ( history of hypertension , diabetes and myocardial infarction ) were not associated with reports of doctor - patient communication on either subscale . in a sensitivity analysis including only participants who answered all questions on either subscale in the same multivariable models ( n = 695 for the explanations of condition subscale and n = 623 for the responsiveness to patient preferences subscale ) , results were unchanged ( data not shown ) . table 2predictors of poor reports of doctor - patient communication on the explanations of condition subscale * age - adjustedmv adjusted or ( 95% ci)p valueor ( 95% ci)p valuedepressive symptoms phq-9 , per sd increase1.5 ( 1.31.8)<0.00011.5 ( 1.21.8)<0.0001medical comorbidities history of hypertension1.0 ( 0.71.5)0.911.1 ( 0.71.7)0.63 history of diabetes0.7 ( 0.51.1)0.10.7 ( 0.41.1)0.09 history of myocardial infarction1.0 ( 0.71.4)0.871.1 ( 0.71.6)0.7disease severity systolic dysfunction ( lvef 50%)1.2 ( 0.71.9)0.491.4 ( 0.82.6)0.23 poor exercise capacity ( mets < 5)0.9 ( 0.61.4)0.520.7 ( 0.41.1)0.15 inducible ischemia1.0 ( 0.61.6)0.971.0 ( 0.61.6)0.87abbreviations : phq-9 , nine - item patient health questionnaire ; lvef , left ventricular ejection fraction ; mets , metabolic equivalents*poor reports of doctor - patient communication defined as explanations of condition subscale score < 4odds ratio for a model adjusted for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , lvef 50% , mets < 5 , inducible ischemia , phq-9table 3predictors of poor reports of doctor - patient communication on the responsiveness to patient preferences subscale * age - adjustedmv adjusted or ( 95% ci)p valueor ( 95% ci)p valuedepressive symptoms phq-9 , per sd increase1.3 ( 1.11.5)0.0041.3 ( 1.11.5)0.01medical comorbidities history of hypertension0.9 ( 0.61.2)0.51.0 ( 0.71.4)0.96 history of diabetes0.9 ( 0.61.2)0.431.0 ( 0.71.5)0.95 history of myocardial infarction1.0 ( 0.71.4)1.001.1 ( 0.81.5)0.75disease severity systolic dysfunction ( lvef 50%)1.0 ( 0.61.6)0.961.0 ( 0.61.6)0.88 poor exercise capacity ( mets < 5)1.0 ( 0.61.4)0.661.0 ( 0.61.5)0.89 inducible ischemia0.9 ( 0.61.3)0.60.8 ( 0.51.2)0.33abbreviations : phq-9 , nine - item patient health questionnaire ; lvef , left ventricular ejection fraction ; mets , metabolic equivalents*poor reports of doctor - patient communication defined as responsiveness to patient preferences subscale score < 4odds ratio for a model adjusted for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , lvef 50% , mets < 5 , inducible ischemia , phq-9 predictors of poor reports of doctor - patient communication on the explanations of condition subscale * abbreviations : phq-9 , nine - item patient health questionnaire ; lvef , left ventricular ejection fraction ; mets , metabolic equivalents * poor reports of doctor - patient communication defined as explanations of condition subscale score < 4 odds ratio for a model adjusted for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , lvef 50% , mets < 5 , inducible ischemia , phq-9 predictors of poor reports of doctor - patient communication on the responsiveness to patient preferences subscale * abbreviations : phq-9 , nine - item patient health questionnaire ; lvef , left ventricular ejection fraction ; mets , metabolic equivalents * poor reports of doctor - patient communication defined as responsiveness to patient preferences subscale score < 4 odds ratio for a model adjusted for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , lvef 50% , mets < 5 , inducible ischemia , phq-9 because the association between depressive symptoms and responsiveness to patient preferences did not appear to be linear ( fig . 2 ) , we also evaluated the association of depressive symptom score by category with reports of poor responsiveness to patient preferences ( data not shown ) . in the multivariable model adjusted for the above - listed variables , mild depressive symptoms ( phq 49 ) were associated with a 60% increased odds of poor responsiveness to patient preferences ( or 1.6 , 95% ci 1.12.4 , p = 0.01 ) , and moderate - severe depressive symptoms ( phq 10 ) were associated with a 70% increased odds of poor responsiveness to patient preferences ( or 1.7 , 95% ci 1.12.8 , p = 0.02 ) compared with none - minimal depressive symptoms ( phq 03 ) . among patients with chronic chd , we found that depressive symptoms were strongly associated with patient reports of poor doctor - patient communication , as measured on two subscales from the established interpersonal processes of care questionnaire . depressive symptoms were independently associated with poor reported explanations of condition and poor reported responsiveness to patient preferences , after adjustment for demographic factors , medical comorbidities and disease severity . in contrast , common medical comorbidities and objective measures of cardiac disease severity were not associated with reported doctor - patient communication . these findings raise questions about whether self - reports of doctor - patient communication are in part a reflection of the psychological state of the patient . patients with underlying mental disorders may be more likely to have unmet patient expectations,31 less likely to have their symptoms understood by their physicians,32 less involved in shared decision making33 and less satisfied with their care.34 to our knowledge , only one prior study has evaluated the influence of depressive symptom severity on patient reports of doctor - patient communication across different domains.24 this study , conducted in a cohort of adults with diabetes , found that depressive symptoms were associated with worse reports of doctor - patient communication on four out of seven subscales from the same interpersonal processes of care instrument . however , it was unclear how the association of depressive symptoms with poor reports of doctor - patient communication compared with medical comorbidities and objective measures of disease severity . our study extends these important findings by reporting an association of depressive symptoms with reports of poor explanations of condition and responsiveness to patient preferences in a cohort of adults with chronic chd and by demonstrating that the association is not explained by medical comorbidities or worse cardiac function in depressed patients . consistent with previous data , we found a high prevalence of depressive symptoms in patients with chronic coronary disease.21,35,36 depression is a significant risk factor for adverse outcomes in this population . patients with chd and concomitant depression are at increased risk for recurrence of cardiac events and for adverse cardiovascular outcomes after coronary artery bypass grafting surgery,21 independent of baseline cardiac disease severity.37 depression is associated with poor health - related quality of life in patients with chronic chd and more strongly predicts health status outcomes than objective measures of disease severity.25 our findings suggest that patients with depression are at increased risk for experiencing poor communication with their physicians . poor experiences of doctor - patient communication similar to poor perceived health status may also lead to depressive symptoms . the lack of association between medical comorbidities or disease severity and reports of poor communication implies that medical complexity may not compromise patients experiences of communication with their doctors to the same extent as depressive symptoms . in addition to interventions focused on clinician communication skills , efforts to improve doctor - patient communication should include increased screening and treatment of patients with depressive symptoms . referral to mental health services , when available , may assist primary care physicians in such evaluation and treatment . physicians may also wish to explore different strategies for communicating information and sharing decisions with patients who are depressed . poor experiences of communication in this population may signal a need for alternative communication models . swenson hypothesized that differences in content , process or recall among patients with depression may result in poor communication.24 content refers to the challenge of dealing with multiple issues during a medical visit , which could lead to less time - sharing information . our findings do not support the content explanation because communication was not associated with medical comorbidities or cardiac function . the fact that sicker patients did not report worse communication suggests that visit content did not interfere with information exchange or shared decision making . depressed patients may have difficulty discussing their health problems with physicians,22 have a more negative affect , be less well - liked by their doctors38 and/or make physicians feel more frustrated or less engaged,18 thereby placing a strain on interpersonal relationships , which may impede physician - patient communication . visit recall means that depressed patients may have inaccurate or skewed recollections of what happened during a visit . depressed patients may report poor communication in part because they view everything in a negative light . alternatively , difficulty with concentration , a symptom of major depression,39 may impair patient recall of communication during the medical visit . while the process of communication may be objectively worse in patients with depressive symptoms , it is also possible that patients with depression perceive the same interactions differently . depressive symptoms are associated with poor reports of doctor - patient communication , and the perceived quality of doctor - patient communication predicts patient satisfaction.40 thus , if patients with depressive symptoms report lower satisfaction with their physicians , is this because depressed patients perceive things differently or because their physicians are actually providing poorer quality care ? the possibility that patients with depressive symptoms view clinical encounters through different lenses raises questions about the validity of patient self - report questionnaires used as measures of health - care quality . first , we evaluated two aspects of doctor - patient communication , explanations of condition and responsiveness to patient preferences , using two subscales from the interpersonal processes of care instrument . our study did not address other important components of doctor - patient communication including empathy , respectfulness , listening or empowerment.2,3,41 while explanations of condition and responsiveness to patient preferences are critical aspects of care , it is possible that the inclusion of additional measures of doctor - patient communication would have provided different results . second , we studied patients with coronary heart disease , so our results may not generalize to patients with other illnesses . however , chd is a common chronic illness requiring frequent physician visits and associated with multiple medical comorbidities and high rates of depression . third , it is possible that depression itself may have presented a barrier to participation in this study . however , we found a high prevalence of depressive symptoms consistent with previous data.36 fourth , our participants spoke english , and the majority were male and white . although the interpersonal processes of care instrument was designed to compare doctor - patient communication across diverse racial / ethnic groups , our study sample did not allow us to make such comparisons . fifth , we did not collect information about individual physicians . while previous work has demonstrated an association between physician characteristics and patient reports of doctor - patient communication,42 we were unable to evaluate physician predictors of communication or account for clustering at the physician level . sixth , the interpersonal processes of care instrument is a patient self - report questionnaire ; we did not observe doctor - patient communication directly . while patient perceptions of communication are important in and of themselves , direct observation might have allowed us to examine whether perceived differences in communication were more strongly associated with visit process or recall . finally , this was a cross - sectional study ; thus , we were unable to evaluate causality . in summary , patients with depressive symptoms are more likely to report poor doctor - patient communication than those without depressive symptoms . whether differences in perceived communication are due to differences in the communication process or to differences in recall among patients with depressive symptoms deserves further study . patient reports are important in and of themselves and signal a need for increased awareness of the influence of depressive symptoms on experiences of doctor - patient communication . however , further correlation with physician reports and/or direct observation may be necessary before such reports can be used as objective measures of health - care quality .

backgrounddoctor - patient communication is an important marker of health - care quality . little is known about the extent to which medical comorbidities , disease severity and depressive symptoms influence perceptions of doctor - patient communication in patients with chronic disease.methodsin a cross - sectional study of 703 outpatients with chronic coronary disease , we evaluated the extent to which patient reports of doctor - patient communication were influenced by medical comorbidities , disease severity and depressive symptoms . we assessed patient reports of doctor - patient communication using the explanations of condition and responsiveness to patient preferences subscales from the interpersonal processes of care instrument . poor doctor - patient communication was defined as a score of < 4 ( range 1 to 5 ) on either subscale . all patients completed the nine - item patient health questionnaire ( phq ) for measurement of depressive symptoms and underwent an extensive evaluation of medical comorbidities and cardiac function.resultsin univariate analyses , the following patient characteristics were associated with poor reported doctor - patient communication on one or both subscales : female sex , white or asian race and depressive symptoms . after adjusting for demographic factors , medical comorbidities and disease severity , each standard deviation ( 5.4-point ) increase in depressive symptom score was associated with a 50% greater odds of poor reported explanations of condition ( or 1.5 , 95% ci , 1.21.8 ; p < 0.001 ) and a 30% greater odds of poor reported responsiveness to patient preferences ( or 1.3 , 95% ci , 1.11.5 ; p = 0.01 ) . in contrast , objective measures of disease severity ( left ventricular ejection fraction , exercise capacity , inducible ischemia ) and medical comorbidities ( hypertension , diabetes , myocardial infarction ) were not associated with reports of doctor - patient communication.conclusionsin outpatients with chronic coronary heart disease , depressive symptoms are associated with perceived deficits in doctor - patient communication , while medical comorbidities and disease severity are not . these findings suggest that patient reports of doctor - patient communication may partly reflect the psychological state of the patient .

BACKGROUND METHODS Subjects and Setting Measurements Doctor-Patient Communication Depressive Symptoms Medical Comorbidities and Disease Severity Other Patient Characteristics Statistical Analysis RESULTS DISCUSSION Conclusion

doctor - patient communication is considered an important marker of health - care quality,1 and the social - psychological aspects of the patient - physician interaction are increasingly recognized as complementary to the more technical aspects of medical care.2,3 high quality doctor - patient communication involves multiple domains , including ( 1 ) building an effective relationship , ( 2 ) gathering information , ( 3 ) understanding the patient s perspective , ( 4 ) giving information and ( 5 ) decision making.4,5 patients rate communication with their physicians as a valuable part of the medical encounter,6,7 and improved doctor - patient communication has been associated with higher patient satisfaction,6,8 self - management of chronic diseases,912 more appropriate prescribing of medications13 and improved health outcomes.1417 doctor - patient communication is particularly important in the management of chronic diseases , which may require frequent encounters with the medical system and complex treatment decisions . patients with chronic disease often have multiple medical comorbidities and severe illness , presenting clinical challenges for physicians that may result in less attention to the social - psychological aspects of the patient - physician interaction.18,19 patients with chronic disease also suffer disproportionately from depression,20,21 which may complicate doctor - patient communication through the challenge of managing depression along with other medical conditions or because depressed patients experience or recall communication with their physicians differently.2224 however , little is known about the relative association of medical comorbidities , disease severity and depressive symptoms with doctor - patient communication . we conducted this study to evaluate the association of medical comorbidities , disease severity and depressive symptoms with patient reports of doctor - patient communication in a cohort of adult patients with coronary heart disease ( chd ) . we assessed doctor - patient communication using the two - item explanations of condition subscale and the four - item responsiveness to patient preferences subscale from the interpersonal processes of care ( ipc ) instrument , an established self - report questionnaire designed to measure specific components of doctor - patient communication in diverse populations.2 details of the reliability and validity of the ipc instrument have been published previously.2 the questionnaire was self - administered with a research assistant available in the same room to help patients understand and interpret questions as necessary . we evaluated the ipc subscales both as continuous variables and as dichotomous variables , with a score of < 4 on each subscale ( corresponding to never / rarely / sometimes for positive characteristics and always / often / sometimes for negative characteristics ) considered poor doctor - patient communication.26 we assessed depressive symptoms using the nine - item patient health questionnaire ( phq).27,28 we evaluated depressive symptoms both as a continuous variable ( range of scores 0 to 27 ) and as an ordinal variable , using previously established categories of none - minimal ( phq score of 03 ) , mild ( phq score of 49 ) or moderate - severe ( phq score 10 ) depressive symptoms.28 medical comorbidities ( history of hypertension , diabetes and myocardial infarction ) were assessed by patient questionnaire . our goal was to examine the associations of medical comorbidities , disease severity and depressive symptoms with patient reports of doctor - patient communication . we then used logistic regression to evaluate the independent association of depressive symptoms with poor reported doctor - patient communication , adjusted for demographic characteristics ( age , sex , race , education , income ) , medical comorbidities ( history of myocardial infarction , hypertension and diabetes ) and disease severity ( resting left ventricular ejection fraction , exercise capacity , inducible ischemia ) . we assessed doctor - patient communication using the two - item explanations of condition subscale and the four - item responsiveness to patient preferences subscale from the interpersonal processes of care ( ipc ) instrument , an established self - report questionnaire designed to measure specific components of doctor - patient communication in diverse populations.2 details of the reliability and validity of the ipc instrument have been published previously.2 the questionnaire was self - administered with a research assistant available in the same room to help patients understand and interpret questions as necessary . we evaluated the ipc subscales both as continuous variables and as dichotomous variables , with a score of < 4 on each subscale ( corresponding to never / rarely / sometimes for positive characteristics and always / often / sometimes for negative characteristics ) considered poor doctor - patient communication.26 we assessed depressive symptoms using the nine - item patient health questionnaire ( phq).27,28 we evaluated depressive symptoms both as a continuous variable ( range of scores 0 to 27 ) and as an ordinal variable , using previously established categories of none - minimal ( phq score of 03 ) , mild ( phq score of 49 ) or moderate - severe ( phq score 10 ) depressive symptoms.28 medical comorbidities ( history of hypertension , diabetes and myocardial infarction ) were assessed by patient questionnaire . we assessed doctor - patient communication using the two - item explanations of condition subscale and the four - item responsiveness to patient preferences subscale from the interpersonal processes of care ( ipc ) instrument , an established self - report questionnaire designed to measure specific components of doctor - patient communication in diverse populations.2 details of the reliability and validity of the ipc instrument have been published previously.2 the questionnaire was self - administered with a research assistant available in the same room to help patients understand and interpret questions as necessary . we evaluated the ipc subscales both as continuous variables and as dichotomous variables , with a score of < 4 on each subscale ( corresponding to never / rarely / sometimes for positive characteristics and always / often / sometimes for negative characteristics ) considered poor doctor - patient communication.26 we assessed depressive symptoms using the nine - item patient health questionnaire ( phq).27,28 we evaluated depressive symptoms both as a continuous variable ( range of scores 0 to 27 ) and as an ordinal variable , using previously established categories of none - minimal ( phq score of 03 ) , mild ( phq score of 49 ) or moderate - severe ( phq score 10 ) depressive symptoms.28 medical comorbidities ( history of hypertension , diabetes and myocardial infarction ) were assessed by patient questionnaire . our goal was to examine the associations of medical comorbidities , disease severity and depressive symptoms with patient reports of doctor - patient communication . we then used logistic regression to evaluate the independent association of depressive symptoms with poor reported doctor - patient communication , adjusted for demographic characteristics ( age , sex , race , education , income ) , medical comorbidities ( history of myocardial infarction , hypertension and diabetes ) and disease severity ( resting left ventricular ejection fraction , exercise capacity , inducible ischemia ) . table 1characteristics of participants by reports of doctor - patient communication * explanations of conditionresponsiveness to patient preferencespoorgoodp valuepoorgoodp value(n = 186)(n = 515)(n = 286)(n = 416)demographics age , years , mean sd65 1165 110.7965 1165 110.74 male128 ( 69%)396 ( 77%)0.03211 ( 74%)314 ( 76%)0.61 race0.020.01 white110 ( 59%)297 ( 58%)180 ( 63%)227 ( 55% ) black21 ( 11%)105 ( 20%)34 ( 12%)92 ( 22% ) asian31 ( 17%)55 ( 11%)38 ( 13%)48 ( 12% ) latino17 ( 9%)46 ( 9%)25 ( 9%)38 ( 9% ) other7 ( 4%)11 ( 2%)9 ( 3%)10 ( 2% ) high school education162 ( 88%)446 ( 87%)0.78254 ( 89%)354 ( 85%)0.1 annual income 20 k102 ( 55%)267 ( 52%)0.56139 ( 49%)231 ( 56%)0.07 primary care site0.10.55 san francisco va21 ( 11%)91 ( 18%)43 ( 15%)69 ( 17% ) palo alto va1 ( 1%)9 ( 2%)4 ( 1%)6 ( 1% ) ucsf99 ( 54%)240 ( 47%)147 ( 52%)192 ( 46% ) public health clinic64 ( 35%)171 ( 33%)89 ( 31%)147 ( 36%)medical comorbidities hypertension129 ( 70%)357 ( 69%)0.92194 ( 68%)293 ( 70%)0.5 diabetes40 ( 22%)142 ( 28%)0.170 ( 24%)113 ( 27%)0.41 myocardial infarction97 ( 52%)264 ( 52%)0.86148 ( 52%)213 ( 52%)0.98disease severity resting lvef , mean sd61.2 9.661.1 9.80.8461.6 9.460.9 10.00.35 exercise capacity , mets7.5 3.37.6 3.50.767.6 3.47.6 3.50.84 inducible ischemia33 ( 20%)93 ( 20%)0.9849 ( 19%)77 ( 21%)0.67depressive symptoms phq-9 , mean sd7.1 5.84.8 5.1<0.00016.1 5.45.0 5.30.006abbreviations : va , veterans affairs medical center ; ucsf , university of california , san francisco ; lvef , left ventricular ejection fraction ; mets , metabolic equivalent tasks ; phq-9 , nine - item patient health questionaire*data are given as number ( percentage ) except where indicated otherwisesubscale score < 4 = poor characteristics of participants by reports of doctor - patient communication * abbreviations : va , veterans affairs medical center ; ucsf , university of california , san francisco ; lvef , left ventricular ejection fraction ; mets , metabolic equivalent tasks ; phq-9 , nine - item patient health questionaire * data are given as number ( percentage ) except where indicated otherwise subscale score < 4 = poor of the 703 participants , 47% had none - minimal depressive symptoms ( phq 03 ) , 32% had mild depressive symptoms ( phq 49 ) and 21% had moderate - severe depressive symptoms ( phq 10 ) . in logistic regression models adjusted for age , with doctor - patient communication coded as a dichotomous outcome variable , each standard deviation ( 5.4 point ) increase in depressive symptom score was associated with a 50% greater odds of poor reported explanations of condition ( or 1.5 , 95% ci , 1.31.8 ; p < 0.0001 ) ( table 2 ) and a 30% greater odds of poor reported responsiveness to patient preferences ( or 1.3 , 95% ci , 1.11.5 ; p = 0.004 ) ( table 3 ) . in multivariable models , each sd increase in depressive symptoms remained strongly associated with poor reported explanations of conditions ( or 1.5 , 95% ci 1.21.8 , p < 0.0001 ) and poor reported responsiveness to patient preferences ( or 1.3 , 95% ci 1.11.5 , p = 0.01 ) , adjusting for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , left ventricular ejection fraction 50% , poor exercise capacity ( mets < 5 ) , and inducible ischemia . we found the same association of depressive symptoms with poor doctor - patient communication in models adjusted only for age , race and sex ( or 1.5 , 95% ci 1.31.8 ; p < 0.0001 for explanations of condition ; or 1.3 , 95% ci 1.11.5 ; p = 0.006 for responsiveness to patient preferences , data not shown ) . in contrast , three objective measures of disease severity ( systolic function , exercise capacity and inducible ischemia ) and three common medical comorbidities ( history of hypertension , diabetes and myocardial infarction ) were not associated with reports of doctor - patient communication on either subscale . table 2predictors of poor reports of doctor - patient communication on the explanations of condition subscale * age - adjustedmv adjusted or ( 95% ci)p valueor ( 95% ci)p valuedepressive symptoms phq-9 , per sd increase1.5 ( 1.31.8)<0.00011.5 ( 1.21.8)<0.0001medical comorbidities history of hypertension1.0 ( 0.71.5)0.911.1 ( 0.71.7)0.63 history of diabetes0.7 ( 0.51.1)0.10.7 ( 0.41.1)0.09 history of myocardial infarction1.0 ( 0.71.4)0.871.1 ( 0.71.6)0.7disease severity systolic dysfunction ( lvef 50%)1.2 ( 0.71.9)0.491.4 ( 0.82.6)0.23 poor exercise capacity ( mets < 5)0.9 ( 0.61.4)0.520.7 ( 0.41.1)0.15 inducible ischemia1.0 ( 0.61.6)0.971.0 ( 0.61.6)0.87abbreviations : phq-9 , nine - item patient health questionnaire ; lvef , left ventricular ejection fraction ; mets , metabolic equivalents*poor reports of doctor - patient communication defined as explanations of condition subscale score < 4odds ratio for a model adjusted for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , lvef 50% , mets < 5 , inducible ischemia , phq-9table 3predictors of poor reports of doctor - patient communication on the responsiveness to patient preferences subscale * age - adjustedmv adjusted or ( 95% ci)p valueor ( 95% ci)p valuedepressive symptoms phq-9 , per sd increase1.3 ( 1.11.5)0.0041.3 ( 1.11.5)0.01medical comorbidities history of hypertension0.9 ( 0.61.2)0.51.0 ( 0.71.4)0.96 history of diabetes0.9 ( 0.61.2)0.431.0 ( 0.71.5)0.95 history of myocardial infarction1.0 ( 0.71.4)1.001.1 ( 0.81.5)0.75disease severity systolic dysfunction ( lvef 50%)1.0 ( 0.61.6)0.961.0 ( 0.61.6)0.88 poor exercise capacity ( mets < 5)1.0 ( 0.61.4)0.661.0 ( 0.61.5)0.89 inducible ischemia0.9 ( 0.61.3)0.60.8 ( 0.51.2)0.33abbreviations : phq-9 , nine - item patient health questionnaire ; lvef , left ventricular ejection fraction ; mets , metabolic equivalents*poor reports of doctor - patient communication defined as responsiveness to patient preferences subscale score < 4odds ratio for a model adjusted for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , lvef 50% , mets < 5 , inducible ischemia , phq-9 predictors of poor reports of doctor - patient communication on the explanations of condition subscale * abbreviations : phq-9 , nine - item patient health questionnaire ; lvef , left ventricular ejection fraction ; mets , metabolic equivalents * poor reports of doctor - patient communication defined as explanations of condition subscale score < 4 odds ratio for a model adjusted for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , lvef 50% , mets < 5 , inducible ischemia , phq-9 predictors of poor reports of doctor - patient communication on the responsiveness to patient preferences subscale * abbreviations : phq-9 , nine - item patient health questionnaire ; lvef , left ventricular ejection fraction ; mets , metabolic equivalents * poor reports of doctor - patient communication defined as responsiveness to patient preferences subscale score < 4 odds ratio for a model adjusted for age , sex , race , education , income , history of hypertension , diabetes , myocardial infarction , lvef 50% , mets < 5 , inducible ischemia , phq-9 because the association between depressive symptoms and responsiveness to patient preferences did not appear to be linear ( fig . in the multivariable model adjusted for the above - listed variables , mild depressive symptoms ( phq 49 ) were associated with a 60% increased odds of poor responsiveness to patient preferences ( or 1.6 , 95% ci 1.12.4 , p = 0.01 ) , and moderate - severe depressive symptoms ( phq 10 ) were associated with a 70% increased odds of poor responsiveness to patient preferences ( or 1.7 , 95% ci 1.12.8 , p = 0.02 ) compared with none - minimal depressive symptoms ( phq 03 ) . among patients with chronic chd , we found that depressive symptoms were strongly associated with patient reports of poor doctor - patient communication , as measured on two subscales from the established interpersonal processes of care questionnaire . depressive symptoms were independently associated with poor reported explanations of condition and poor reported responsiveness to patient preferences , after adjustment for demographic factors , medical comorbidities and disease severity . patients with underlying mental disorders may be more likely to have unmet patient expectations,31 less likely to have their symptoms understood by their physicians,32 less involved in shared decision making33 and less satisfied with their care.34 to our knowledge , only one prior study has evaluated the influence of depressive symptom severity on patient reports of doctor - patient communication across different domains.24 this study , conducted in a cohort of adults with diabetes , found that depressive symptoms were associated with worse reports of doctor - patient communication on four out of seven subscales from the same interpersonal processes of care instrument . however , it was unclear how the association of depressive symptoms with poor reports of doctor - patient communication compared with medical comorbidities and objective measures of disease severity . our study extends these important findings by reporting an association of depressive symptoms with reports of poor explanations of condition and responsiveness to patient preferences in a cohort of adults with chronic chd and by demonstrating that the association is not explained by medical comorbidities or worse cardiac function in depressed patients . first , we evaluated two aspects of doctor - patient communication , explanations of condition and responsiveness to patient preferences , using two subscales from the interpersonal processes of care instrument .

social anxiety disorder ( sad ) , often referred to as social phobia , is characterized by fear and avoidance of social situations . epidemiological surveys have shown that sad is the fourth most common psychiatric disorder,1 with a lifetime prevalence of 12%.2 sad begins during adolescence and often persists.3 patients with sad often suffer from comorbid depression4,5 and other anxiety disorders.6 according to such characteristics of the disorder , sad causes significant social dysfunction , and patients with sad frequently develop functional impairment at work and in their private lives , which decreases their quality of life.7,8 therefore , providing appropriate treatment for sad is important . previous studies have provided evidence that pharmacotherapy,9 including benzodiazepines , selective serotonin - reuptake inhibitors , and monoamine oxidase inhibitors , are effective during sad treatment as well as during cognitive behavioral therapy ( cbt).10 a number of randomized controlled trials11,12 and strong evidence for a positive effect of cbt on sad have been published . the effect size of cbt has been estimated at 0.71 ( 95% confidence interval [ ci ] 0.560.85 ) by a recent meta - analysis,13 and it showed lower relapse rates than treatments based on pharmacotherapy.14 some researchers have demonstrated the effectiveness of cbt in a group format . because patients with sad are often anxious and avoid small - group work , they can be exposed to fearful situations by attending sessions.10 furthermore , group cbt has greater cost - effectiveness compared with individual cbt.15 from 2003 onward , we conducted group cbt for outpatients with sad at the department of psychiatry , nagoya city university hospital , based on previous studies . our preliminary posttreatment data ( from july 2003 to january 2007 , n = 57 ) show that group cbt is acceptable.16 we have also published the long - term ( 1-year ) effects on quality of life17 ( n = 57 ) and symptomatology18 ( n = 62 ) in patients with sad . these studies1618 also had limitations because of small sample size , and many dropout cases made it difficult to identify predictors . furthermore , we included both the generalized and nongeneralized subtypes of sad in these studies . although both subtypes can be improved by cbt , the generalized subtype has more severe social anxiety symptoms and social function disability than those of the nongeneralized subtype , and patients are more impaired prior to and after treatment.19 our previous studies may have contaminated efficacy by including both subtypes . to overcome these limitations , in the present study we accumulated twice the number of participants ( n = 113 ) as in our previous studies,1618 and we focused on the generalized subtype to present more conclusive data . moreover , we adopted a mixed - model analysis , which is considered the most effective way to identify treatment outcome predictors . many studies have attempted to identify predictors of treatment outcomes , but only a few specific predictors have been found.20 baseline predictors may enable us to provide cbt more effectively and to prevent dropout from treatment . furthermore , although cbt was originally developed in western countries , some previous studies have discussed the cultural boundaries of sad symptoms or sad treatment.21,22 a condition called taijin kyofusho syndrome occurs in japan and some other east asian countries , as stated in the appendix of the diagnostic and statistical manual of mental disorders ( dsm - iv ) . from this perspective thus , we conducted this study with the aim of identifying the long - term efficacy and predictors of group cbt for patients with generalized sad in japan . from july 2003 to august 2010 , 113 outpatients with sad were enrolled in the group - based cbt program at the department of psychiatry , nagoya city university hospital , japan . all patients fulfilled the criteria for generalized sad as the primary disorder according to the structured clinical interview for the dsm - iv . furthermore , all patients met the following criteria : ( 1 ) no history of psychosis or bipolar disorder , or current substance - abuse disorder , ( 2 ) no previous cbt treatments , with agreement not to be involved in any other structured psychosocial therapies during treatment , and ( 3 ) absence of cluster b personality disorder . we included patients with current axis i disorders if symptoms were controlled sufficiently to allow joining a group session . for example , we included major depressive disorder or other current anxiety disorders or patients with axis ii personality disorders except criterion ( 3 ) . this study was approved by the ethics committee of the nagoya city university graduate school of medical sciences . this study was conducted as a single - arm , naturalistic , follow - up study in a routine japanese clinical setting . we followed the cbt manual for sad written by andrews et al,23 and we modified and improved the program according to clark and wells model.24 treatment was conducted in groups of three patients led by one principal therapist and one cotherapist , and were scheduled for 120 minutes once per week . the average number of sessions was 14 ( range 1220 ) , depending on the needs of each group . the program included ( 1 ) psychoeducation about sad ( session 1 ) , ( 2 ) introduction about the individual cognitive behavioral model of sad ( session 2 ) , ( 3 ) experiments to drop safety behavior and self - focused attention ( from session 8 to last session ) , ( 4 ) attention training to shift focus away from themselves to the task or the external social situation ( sessions 4 and 5 ) , ( 5 ) video feedback of role - playing in anxious situations to modify their self - image ( sessions 6 and 7 ) , ( 6 ) in vivo exposure using behavioral experiments to test the patient s catastrophic predictions ( from session 8 to last session ) , and ( 7 ) cognitive restructuring ( session 3 , from session 8 to last session ) . we assigned homework to the patients after every session . among 113 patients , 98 patients ( 86.7% ) completed cbt , and almost all of the patients ( n = 109 ) finished all the exercise kinds , even when they were absent from a few sessions . eight therapists ( five psychiatrists and three doctoral - level clinical psychologists ) , with more than 3 years of clinical practice with anxiety disorders , conducted the treatment program . we allowed patients to use antidepressants and benzodiazepines during cbt , because our study was based in a clinical setting and there is some evidence for combined pharmacologic / cbt therapy.11,25 patients did not participate in any other structured psychotherapy while attending group cbt . the principal therapist conducted the mood- and anxiety - disorder sections of the structured clinical interview for the dsm - iv at baseline , for the sad diagnosis , and any mood and anxiety comorbidities . patients demographic data were gathered at baseline , including such sociodemographic factors as sex , age , educational status , marital status , and employment status . information about age of onset and duration of sad , sad subtype , psychiatric comorbidities , and medication use was also obtained . the patients were assessed with self - report questionnaires at baseline , post - treatment , and by mail at the 1-year follow - up . our primary outcome was the total social phobia scale / social interaction anxiety scale ( sps / sias)26 score at the 1-year follow - up . the sps and sias are 20-item self - report questionnaires with ratings on a 4-point scale from 0 ( not at all characteristic or true of me ) to 4 ( extremely characteristic or true of me ) , and total scores of 080 . the sps measures the fear of being observed , whereas the sias provides a measure of fear of social interaction . sufficient internal consistency , reliability , and discrimination , as well as predictive and concurrent validity have been demonstrated for both original and japanese versions.27 cronbach s alphas of our sample for sps / sias were 0.88/0.600.88 . the fear questionnaire social phobia subscale ( fq - sp)28 is a 5-item self - reported instrument for measuring the fear - motivated avoidance of being observed , performing , being criticized , and talking to authorities . items are rated on a 9-point likert - type scale , from 0 ( would not avoid it ) to 8 ( always avoid it ) . retest reliability and factor validity have been demonstrated.29 we compared treatment completers with patients who dropped out using unpaired t - tests for continuous variables or chi - square tests for categorical variables . treatment completers were defined as participants who had attended at least 80% of all treatment sessions and completed posttreatment and 1-year follow - up questionnaires . the pretreatment and 1-year follow - up scores on sps / sias were compared using paired t - tests to quantify outcomes from the cbt program . furthermore , to examine the outcomes of the cbt program across various aspects of the disorder , pre- and posttreatment scores were compared for sps , sias , and fq - sp using paired t - tests , and pretreatment and 1-year follow - up were compared for fq - sp using paired t - tests . to show the magnitude of the treatment effect , we calculated the effect size ( m pretest all statistical analyses for these treatment outcomes were conducted twice : once based on the intention - to - treat ( itt ) principle and once among the completers only . the itt analyses were conducted using the last - observation - carried forward ( locf ) model , for which we used the mid - treatment data ( after the eighth session ) or the 3-month follow - up data , whichever were the last observational data available . we conducted mixed - model analyses to detect the baseline predictors of treatment outcome with the 1-year follow - up sps / sias score as a dependent variable and the baseline demographic and clinical variables ( sex , age , marital status , educational status , employment status , onset , duration of sad , current mood disorder , current anxiety disorder , antidepressant use at baseline , benzodiazepine use at baseline , number of treatment sessions , severity ) as variables . age and onset of sad age were categorized by medline search criteria ( age was divided into three categories : 1318 years , 1945 years , and 46 years ; onset of sad was divided into three categories : 12 years of age , 1318 years of age , and 1945 years of age ) . the number of treatment sessions was divided into two categories according to our definition of minimal session number ( n = 12 ) . we divided the variable of duration of sad into two categories of 1 year or > 1 year , because we wanted to explore the effectiveness of early treatment intervention . baseline severity of sad was defined by the baseline sps total score , based on heimberg et al,30 with more than 34 being defined as severe . all the statistical tests were two - tailed , and an alpha value of less than 0.05 was considered statistically significant . all the data were examined using spss 19.0 ( ibm , armonk , ny , usa ) for windows . from july 2003 to august 2010 , 113 outpatients with sad were enrolled in the group - based cbt program at the department of psychiatry , nagoya city university hospital , japan . all patients fulfilled the criteria for generalized sad as the primary disorder according to the structured clinical interview for the dsm - iv . furthermore , all patients met the following criteria : ( 1 ) no history of psychosis or bipolar disorder , or current substance - abuse disorder , ( 2 ) no previous cbt treatments , with agreement not to be involved in any other structured psychosocial therapies during treatment , and ( 3 ) absence of cluster b personality disorder . we included patients with current axis i disorders if symptoms were controlled sufficiently to allow joining a group session . for example , we included major depressive disorder or other current anxiety disorders or patients with axis ii personality disorders except criterion ( 3 ) . this study was approved by the ethics committee of the nagoya city university graduate school of medical sciences . this study was conducted as a single - arm , naturalistic , follow - up study in a routine japanese clinical setting . we followed the cbt manual for sad written by andrews et al,23 and we modified and improved the program according to clark and wells model.24 treatment was conducted in groups of three patients led by one principal therapist and one cotherapist , and were scheduled for 120 minutes once per week . the average number of sessions was 14 ( range 1220 ) , depending on the needs of each group . the program included ( 1 ) psychoeducation about sad ( session 1 ) , ( 2 ) introduction about the individual cognitive behavioral model of sad ( session 2 ) , ( 3 ) experiments to drop safety behavior and self - focused attention ( from session 8 to last session ) , ( 4 ) attention training to shift focus away from themselves to the task or the external social situation ( sessions 4 and 5 ) , ( 5 ) video feedback of role - playing in anxious situations to modify their self - image ( sessions 6 and 7 ) , ( 6 ) in vivo exposure using behavioral experiments to test the patient s catastrophic predictions ( from session 8 to last session ) , and ( 7 ) cognitive restructuring ( session 3 , from session 8 to last session ) . we assigned homework to the patients after every session . among 113 patients , 98 patients ( 86.7% ) completed cbt , and almost all of the patients ( n = 109 ) finished all the exercise kinds , even when they were absent from a few sessions . eight therapists ( five psychiatrists and three doctoral - level clinical psychologists ) , with more than 3 years of clinical practice with anxiety disorders , conducted the treatment program . we allowed patients to use antidepressants and benzodiazepines during cbt , because our study was based in a clinical setting and there is some evidence for combined pharmacologic / cbt therapy.11,25 patients did not participate in any other structured psychotherapy while attending group cbt . the principal therapist conducted the mood- and anxiety - disorder sections of the structured clinical interview for the dsm - iv at baseline , for the sad diagnosis , and any mood and anxiety comorbidities . patients demographic data were gathered at baseline , including such sociodemographic factors as sex , age , educational status , marital status , and employment status . information about age of onset and duration of sad , sad subtype , psychiatric comorbidities , and medication use was also obtained . the patients were assessed with self - report questionnaires at baseline , post - treatment , and by mail at the 1-year follow - up . our primary outcome was the total social phobia scale / social interaction anxiety scale ( sps / sias)26 score at the 1-year follow - up . the sps and sias are 20-item self - report questionnaires with ratings on a 4-point scale from 0 ( not at all characteristic or true of me ) to 4 ( extremely characteristic or true of me ) , and total scores of 080 . the sps measures the fear of being observed , whereas the sias provides a measure of fear of social interaction . sufficient internal consistency , reliability , and discrimination , as well as predictive and concurrent validity have been demonstrated for both original and japanese versions.27 cronbach s alphas of our sample for sps / sias were 0.88/0.600.88 . the fear questionnaire social phobia subscale ( fq - sp)28 is a 5-item self - reported instrument for measuring the fear - motivated avoidance of being observed , performing , being criticized , and talking to authorities . items are rated on a 9-point likert - type scale , from 0 ( would not avoid it ) to 8 ( always avoid it ) . the sps and sias are 20-item self - report questionnaires with ratings on a 4-point scale from 0 ( not at all characteristic or true of me ) to 4 ( extremely characteristic or true of me ) , and total scores of 080 . the sps measures the fear of being observed , whereas the sias provides a measure of fear of social interaction . sufficient internal consistency , reliability , and discrimination , as well as predictive and concurrent validity have been demonstrated for both original and japanese versions.27 cronbach s alphas of our sample for sps / sias were 0.88/0.600.88 . the fear questionnaire social phobia subscale ( fq - sp)28 is a 5-item self - reported instrument for measuring the fear - motivated avoidance of being observed , performing , being criticized , and talking to authorities . items are rated on a 9-point likert - type scale , from 0 ( would not avoid it ) to 8 ( always avoid it ) . a high score indicates severe symptoms . we compared treatment completers with patients who dropped out using unpaired t - tests for continuous variables or chi - square tests for categorical variables . treatment completers were defined as participants who had attended at least 80% of all treatment sessions and completed posttreatment and 1-year follow - up questionnaires . the pretreatment and 1-year follow - up scores on sps / sias furthermore , to examine the outcomes of the cbt program across various aspects of the disorder , pre- and posttreatment scores were compared for sps , sias , and fq - sp using paired t - tests , and pretreatment and 1-year follow - up were compared for fq - sp using paired t - tests . to show the magnitude of the treatment effect , we calculated the effect size ( m pretest all statistical analyses for these treatment outcomes were conducted twice : once based on the intention - to - treat ( itt ) principle and once among the completers only . the itt analyses were conducted using the last - observation - carried forward ( locf ) model , for which we used the mid - treatment data ( after the eighth session ) or the 3-month follow - up data , whichever were the last observational data available . we conducted mixed - model analyses to detect the baseline predictors of treatment outcome with the 1-year follow - up sps / sias score as a dependent variable and the baseline demographic and clinical variables ( sex , age , marital status , educational status , employment status , onset , duration of sad , current mood disorder , current anxiety disorder , antidepressant use at baseline , benzodiazepine use at baseline , number of treatment sessions , severity ) as variables . age and onset of sad age were categorized by medline search criteria ( age was divided into three categories : 1318 years , 1945 years , and 46 years ; onset of sad was divided into three categories : 12 years of age , 1318 years of age , and 1945 years of age ) . the number of treatment sessions was divided into two categories according to our definition of minimal session number ( n = 12 ) . we divided the variable of duration of sad into two categories of 1 year or > 1 year , because we wanted to explore the effectiveness of early treatment intervention . baseline severity of sad was defined by the baseline sps total score , based on heimberg et al,30 with more than 34 being defined as severe . all the statistical tests were two - tailed , and an alpha value of less than 0.05 was considered statistically significant . all the data were examined using spss 19.0 ( ibm , armonk , ny , usa ) for windows . one hundred and thirteen outpatients with sad ( 57 males and 56 females ; age range 1463 years ; mean sd 31.8 10.4 years ) were enrolled in our study . table 1 summarizes the demographic and clinical characteristics of the patients and compares treatment completers with dropouts . all participants met the principal diagnostic criteria for the dsm - iv sad generalized subtype . as a result of chi - square tests for categorical variables , onset of sad and sps total score at baseline showed p < 0.05 , but no other major differences were observed between completers and dropouts . the number of sessions taken by patients on average was 14 ( range 1220 ) . of the 113 patients who were enrolled , 98 completed treatment and 70 finished the 1-year follow - up . although pre- and posttreatment sps were not normally distributed , we conducted analyses as we planned , because the other measures were normally distributed . table 2 shows the mean symptom scores and sds of all measures for all participants ( itt population using the locf model ) and pre- and posttreatment completers and pre- and 1-year follow - ups . an examination of the change in symptom measures ( sps , sias , and fq - sp ) between pre- and posttreatment and between pre- and 1-year follow - ups revealed significant improvements not only for the completers but also for the itt samples ( all p < 0.05 ) . next , the effect sizes for each symptom measure were calculated , and the results are presented in tables 4 and 5 . the effect sizes for the total sps / sias scores at the 1-year follow - up , which was our primary outcome , were 0.68 ( 95% ci 0.410.95)/0.76 ( 95% ci , 0.491.03 ) in the itt sample and 0.77 ( 0.421.10)/0.84 ( 0.491.18 ) in completers . based on the itt sample analyses , effect sizes for assessment at posttreatment were sps 0.64 ( 0.370.90 ) , sias 0.76 ( 0.491.03 ) , and fq - sp 0.66 ( 0.390.93 ) , and at 1-year follow - up fq - sp was 0.76 ( 0.481.02 ) . effect sizes for treatment completers at posttreatment were sps 0.81 ( 95% ci , 0.461.15 ) , sias 0.76 ( 0.491.10 ) , and fq - sp 0.81 ( 0.471.15 ) , and the effect size of the fq - sp at 1-year follow up was 0.96 ( 0.611.31 ) , indicating a greater change than that in the itt sample . a significant difference was found for sias in the older age - group at baseline ( p = 0.019 ) , a lower severity on sps ( p = 0.000 ) , and late onset of sad for both sps ( p = 0.001 ) and sias ( p = 0.000 ) as predictors of good treatment outcome . one hundred and thirteen outpatients with sad ( 57 males and 56 females ; age range 1463 years ; mean sd 31.8 10.4 years ) were enrolled in our study . table 1 summarizes the demographic and clinical characteristics of the patients and compares treatment completers with dropouts . all participants met the principal diagnostic criteria for the dsm - iv sad generalized subtype . as a result of chi - square tests for categorical variables , onset of sad and sps total score at baseline showed p < 0.05 , but no other major differences were observed between completers and dropouts . the number of sessions taken by patients on average was 14 ( range 1220 ) . of the 113 patients who were enrolled , 98 completed treatment and 70 finished the 1-year follow - up . although pre- and posttreatment sps were not normally distributed , we conducted analyses as we planned , because the other measures were normally distributed . table 2 shows the mean symptom scores and sds of all measures for all participants ( itt population using the locf model ) and pre- and posttreatment completers and pre- and 1-year follow - ups . an examination of the change in symptom measures ( sps , sias , and fq - sp ) between pre- and posttreatment and between pre- and 1-year follow - ups revealed significant improvements not only for the completers but also for the itt samples ( all p < 0.05 ) . next , the effect sizes for each symptom measure were calculated , and the results are presented in tables 4 and 5 . the effect sizes for the total sps / sias scores at the 1-year follow - up , which was our primary outcome , were 0.68 ( 95% ci 0.410.95)/0.76 ( 95% ci , 0.491.03 ) in the itt sample and 0.77 ( 0.421.10)/0.84 ( 0.491.18 ) in completers . based on the itt sample analyses , effect sizes for assessment at posttreatment were sps 0.64 ( 0.370.90 ) , sias 0.76 ( 0.491.03 ) , and fq - sp 0.66 ( 0.390.93 ) , and at 1-year follow - up fq - sp was 0.76 ( 0.481.02 ) . effect sizes for treatment completers at posttreatment were sps 0.81 ( 95% ci , 0.461.15 ) , sias 0.76 ( 0.491.10 ) , and fq - sp 0.81 ( 0.471.15 ) , and the effect size of the fq - sp at 1-year follow up was 0.96 ( 0.611.31 ) , indicating a greater change than that in the itt sample . a significant difference was found for sias in the older age - group at baseline ( p = 0.019 ) , a lower severity on sps ( p = 0.000 ) , and late onset of sad for both sps ( p = 0.001 ) and sias ( p = 0.000 ) as predictors of good treatment outcome . the results indicate the long - term efficacy of a cbt program for japanese patients with sad generalized subtype . although we focused on patients with the generalized subtype , who have more severe symptoms than those with the nongeneralized subtype , the effect sizes were as large as those in a meta - analysis conducted in western countries13 and our previous study at posttreatment . according to the effect - size calculation , our treatment program had significant effects at posttreatment that were maintained until the 1-year follow - up . this outcome is the same as that of a previous study , which demonstrated the maintenance efficacy of cbt14 and indicates the possibility that patients are able to use treatment elements by themselves after group treatment . few cbt therapists are available for sad treatment in japan , and national health insurance does not include cbt for anxiety disorders . thus , accumulating evidence for a positive effect of cbt in japan is a matter of urgency , and we hope our study contributes to this purpose . group cbt is more cost - effective than individual cbt in this regard , and we would like to diffuse this effective treatment for sad in japan . a number of studies have examined the role of particular variables in predicting the response to treatment ; however , results have been inconsistent and inconclusive.20 the severity of comorbid depression,31,32 symptomatic severity,31 avoidant personality disorder,33 and expectancy32 have been suggested as possible follow - up predictors for group cbt . although some demographic variables ( female , married , higher education ) were possible follow - up predictors in a study34 that conducted individual cbt , and the aforementioned demographic variables were not statistically significant in our group cbt study , we believe that suitable characteristics of patients are different between group cbt and individual therapy . we found that older age , late onset of sad , and less severe symptoms on sps were possible baseline treatment predictors for a good outcome . we may have to pay more attention to patients who are contrary to those features by reflecting on those results . future studies should focus not only on pretreatment variables but also on the treatment process , such as homework compliance and the client therapist relationship , as suggested by scholing and emmelkamp.31 these factors may help improve the clinical practice of cbt for sad . first , this study was conducted in a routine japanese clinical setting as a single - arm , naturalistic , follow - up study . thus , a random control trial is needed to estimate the conservative efficacy of treatment . second , antidepressant and benzodiazepine medications were allowed during treatment , but information about the amount of drug consumption during the course was not collected . we are unable to consider dose effects of medications on cbt ; however , use of medication at baseline was not a significant predictor of treatment outcomes in the present study . third , some may argue that there were no patients with avoidant personality disorder in our study . we used the structured clinical interview of the dsm - iv mood / anxiety module , but we did not use other modules considering patient load . we only excluded patients who were clinically diagnosed with personality b disorders in accordance with group therapy . the diagnosis of avoidance personality disorder is difficult , as is distinguishing between severe generalized sad and avoidant personality disorder , thus we did not diagnose avoidant personality disorder rigidly in the aforementioned way . however , the magnitude of the treatment effectiveness was quantified by effect size as well as the percentage reduction . besides , data for pre- and posttreatment sps were not normally distributed , although those for the other measures were normally distributed . we conducted post hoc mann whitney analysis between completers and dropouts , and the result was not different . some may point out that we did not use the liebowitz social anxiety scale as the primary outcome , which is a widely used measure . because this study was conducted as routine japanese clinical work , follow - up assessments done by post- and self - reporting versions of this35 have not been validated in japan to date . moreover , a recent study36 showed the effectiveness of attention training , which costs less than typical cbt . although our program included attention training , we might be able to improve our program by emphasizing this component , according to the new findings . despite these limitations , this study provided evidence of long - term efficacy of group cbt for japanese patients with generalized sad . although there is still room for improvement , our results favor the use of cbt for generalized sad in japan . the results indicate the long - term efficacy of a cbt program for japanese patients with sad generalized subtype . although we focused on patients with the generalized subtype , who have more severe symptoms than those with the nongeneralized subtype , the effect sizes were as large as those in a meta - analysis conducted in western countries13 and our previous study at posttreatment . according to the effect - size calculation , our treatment program had significant effects at posttreatment that were maintained until the 1-year follow - up . this outcome is the same as that of a previous study , which demonstrated the maintenance efficacy of cbt14 and indicates the possibility that patients are able to use treatment elements by themselves after group treatment . few cbt therapists are available for sad treatment in japan , and national health insurance does not include cbt for anxiety disorders . thus , accumulating evidence for a positive effect of cbt in japan is a matter of urgency , and we hope our study contributes to this purpose . group cbt is more cost - effective than individual cbt in this regard , and we would like to diffuse this effective treatment for sad in japan . a number of studies have examined the role of particular variables in predicting the response to treatment ; however , results have been inconsistent and inconclusive.20 the severity of comorbid depression,31,32 symptomatic severity,31 avoidant personality disorder,33 and expectancy32 have been suggested as possible follow - up predictors for group cbt . although some demographic variables ( female , married , higher education ) were possible follow - up predictors in a study34 that conducted individual cbt , and the aforementioned demographic variables were not statistically significant in our group cbt study , we believe that suitable characteristics of patients are different between group cbt and individual therapy . we found that older age , late onset of sad , and less severe symptoms on sps were possible baseline treatment predictors for a good outcome . we may have to pay more attention to patients who are contrary to those features by reflecting on those results . future studies should focus not only on pretreatment variables but also on the treatment process , such as homework compliance and the client therapist relationship , as suggested by scholing and emmelkamp.31 these factors may help improve the clinical practice of cbt for sad . first , this study was conducted in a routine japanese clinical setting as a single - arm , naturalistic , follow - up study . thus , a random control trial is needed to estimate the conservative efficacy of treatment . second , antidepressant and benzodiazepine medications were allowed during treatment , but information about the amount of drug consumption during the course was not collected . we are unable to consider dose effects of medications on cbt ; however , use of medication at baseline was not a significant predictor of treatment outcomes in the present study . third , some may argue that there were no patients with avoidant personality disorder in our study . we used the structured clinical interview of the dsm - iv mood / anxiety module , but we did not use other modules considering patient load . we only excluded patients who were clinically diagnosed with personality b disorders in accordance with group therapy . the diagnosis of avoidance personality disorder is difficult , as is distinguishing between severe generalized sad and avoidant personality disorder , thus we did not diagnose avoidant personality disorder rigidly in the aforementioned way . multiple t - tests may have increased the risk for type i errors . however , the magnitude of the treatment effectiveness was quantified by effect size as well as the percentage reduction . besides , data for pre- and posttreatment sps were not normally distributed , although those for the other measures were normally distributed . however , we conducted post hoc mann whitney analysis between completers and dropouts , and the result was not different . some may point out that we did not use the liebowitz social anxiety scale as the primary outcome , which is a widely used measure . because this study was conducted as routine japanese clinical work , follow - up assessments done by post- and self - reporting versions of this35 have not been validated in japan to date . moreover , a recent study36 showed the effectiveness of attention training , which costs less than typical cbt . although our program included attention training , we might be able to improve our program by emphasizing this component , according to the new findings . despite these limitations , this study provided evidence of long - term efficacy of group cbt for japanese patients with generalized sad . although there is still room for improvement , our results favor the use of cbt for generalized sad in japan . group cbt resulted in improvements in japanese patients with generalized sad , and these improvements were maintained for up to 1 year after group cbt . we showed that older age at baseline , late onset , and lower severity of sad were predictors of good outcome at 1-year follow - up for group cbt .

backgroundsocial anxiety disorder ( sad ) is one of the most common psychiatric disorders worldwide . cognitive behavioral therapy ( cbt ) is an effective treatment option for patients with sad . in the present study , we examined the efficacy of group cbt for patients with generalized sad in japan at 1-year follow - up and investigated predictors with regard to outcomes.methodsthis study was conducted as a single - arm , naturalistic , follow - up study in a routine japanese clinical setting . a total of 113 outpatients with generalized sad participated in group cbt from july 2003 to august 2010 and were assessed at follow - ups for up to 1 year . primary outcome was the total score on the social phobia scale / social interaction anxiety scale ( sps / sias ) at 1 year . possible baseline predictors were investigated using mixed - model analyses.resultsamong the 113 patients , 70 completed the assessment at the 1-year follow - up . the sps / sias scores showed significant improvement throughout the follow - ups for up to 1 year . the effect sizes of sps / sias at the 1-year follow - up were 0.68 ( 95% confidence interval 0.410.95)/0.76 ( 0.491.03 ) in the intention - to - treat group and 0.77 ( 0.421.10)/0.84 ( 0.491.18 ) in completers . older age at baseline , late onset , and lower severity of sad were significantly associated with good outcomes as a result of mixed - model analyses.conclusionscbt for patients with generalized sad in japan is effective for up to 1 year after treatment . the effect sizes were as large as those in previous studies conducted in western countries . older age at baseline , late onset , and lower severity of sad were predictors for a good outcome from group cbt .

Introduction Methods Subjects Treatments Assessment SPS/SIAS Fear Questionnaire social phobia subscale Statistical analyses Results Demographic and diagnostic characteristics of patients and comparison of treatment completers and dropouts Changes in symptoms and function through treatment Predictors of treatment outcomes at 1-year follow-up Discussion Main findings Limitations Conclusions

social anxiety disorder ( sad ) , often referred to as social phobia , is characterized by fear and avoidance of social situations . epidemiological surveys have shown that sad is the fourth most common psychiatric disorder,1 with a lifetime prevalence of 12%.2 sad begins during adolescence and often persists.3 patients with sad often suffer from comorbid depression4,5 and other anxiety disorders.6 according to such characteristics of the disorder , sad causes significant social dysfunction , and patients with sad frequently develop functional impairment at work and in their private lives , which decreases their quality of life.7,8 therefore , providing appropriate treatment for sad is important . previous studies have provided evidence that pharmacotherapy,9 including benzodiazepines , selective serotonin - reuptake inhibitors , and monoamine oxidase inhibitors , are effective during sad treatment as well as during cognitive behavioral therapy ( cbt).10 a number of randomized controlled trials11,12 and strong evidence for a positive effect of cbt on sad have been published . the effect size of cbt has been estimated at 0.71 ( 95% confidence interval [ ci ] 0.560.85 ) by a recent meta - analysis,13 and it showed lower relapse rates than treatments based on pharmacotherapy.14 some researchers have demonstrated the effectiveness of cbt in a group format . because patients with sad are often anxious and avoid small - group work , they can be exposed to fearful situations by attending sessions.10 furthermore , group cbt has greater cost - effectiveness compared with individual cbt.15 from 2003 onward , we conducted group cbt for outpatients with sad at the department of psychiatry , nagoya city university hospital , based on previous studies . our preliminary posttreatment data ( from july 2003 to january 2007 , n = 57 ) show that group cbt is acceptable.16 we have also published the long - term ( 1-year ) effects on quality of life17 ( n = 57 ) and symptomatology18 ( n = 62 ) in patients with sad . although both subtypes can be improved by cbt , the generalized subtype has more severe social anxiety symptoms and social function disability than those of the nongeneralized subtype , and patients are more impaired prior to and after treatment.19 our previous studies may have contaminated efficacy by including both subtypes . moreover , we adopted a mixed - model analysis , which is considered the most effective way to identify treatment outcome predictors . furthermore , although cbt was originally developed in western countries , some previous studies have discussed the cultural boundaries of sad symptoms or sad treatment.21,22 a condition called taijin kyofusho syndrome occurs in japan and some other east asian countries , as stated in the appendix of the diagnostic and statistical manual of mental disorders ( dsm - iv ) . from this perspective thus , we conducted this study with the aim of identifying the long - term efficacy and predictors of group cbt for patients with generalized sad in japan . from july 2003 to august 2010 , 113 outpatients with sad were enrolled in the group - based cbt program at the department of psychiatry , nagoya city university hospital , japan . this study was conducted as a single - arm , naturalistic , follow - up study in a routine japanese clinical setting . we followed the cbt manual for sad written by andrews et al,23 and we modified and improved the program according to clark and wells model.24 treatment was conducted in groups of three patients led by one principal therapist and one cotherapist , and were scheduled for 120 minutes once per week . the program included ( 1 ) psychoeducation about sad ( session 1 ) , ( 2 ) introduction about the individual cognitive behavioral model of sad ( session 2 ) , ( 3 ) experiments to drop safety behavior and self - focused attention ( from session 8 to last session ) , ( 4 ) attention training to shift focus away from themselves to the task or the external social situation ( sessions 4 and 5 ) , ( 5 ) video feedback of role - playing in anxious situations to modify their self - image ( sessions 6 and 7 ) , ( 6 ) in vivo exposure using behavioral experiments to test the patient s catastrophic predictions ( from session 8 to last session ) , and ( 7 ) cognitive restructuring ( session 3 , from session 8 to last session ) . among 113 patients , 98 patients ( 86.7% ) completed cbt , and almost all of the patients ( n = 109 ) finished all the exercise kinds , even when they were absent from a few sessions . we allowed patients to use antidepressants and benzodiazepines during cbt , because our study was based in a clinical setting and there is some evidence for combined pharmacologic / cbt therapy.11,25 patients did not participate in any other structured psychotherapy while attending group cbt . the principal therapist conducted the mood- and anxiety - disorder sections of the structured clinical interview for the dsm - iv at baseline , for the sad diagnosis , and any mood and anxiety comorbidities . the patients were assessed with self - report questionnaires at baseline , post - treatment , and by mail at the 1-year follow - up . our primary outcome was the total social phobia scale / social interaction anxiety scale ( sps / sias)26 score at the 1-year follow - up . sufficient internal consistency , reliability , and discrimination , as well as predictive and concurrent validity have been demonstrated for both original and japanese versions.27 cronbach s alphas of our sample for sps / sias were 0.88/0.600.88 . the pretreatment and 1-year follow - up scores on sps / sias were compared using paired t - tests to quantify outcomes from the cbt program . furthermore , to examine the outcomes of the cbt program across various aspects of the disorder , pre- and posttreatment scores were compared for sps , sias , and fq - sp using paired t - tests , and pretreatment and 1-year follow - up were compared for fq - sp using paired t - tests . to show the magnitude of the treatment effect , we calculated the effect size ( m pretest all statistical analyses for these treatment outcomes were conducted twice : once based on the intention - to - treat ( itt ) principle and once among the completers only . we conducted mixed - model analyses to detect the baseline predictors of treatment outcome with the 1-year follow - up sps / sias score as a dependent variable and the baseline demographic and clinical variables ( sex , age , marital status , educational status , employment status , onset , duration of sad , current mood disorder , current anxiety disorder , antidepressant use at baseline , benzodiazepine use at baseline , number of treatment sessions , severity ) as variables . from july 2003 to august 2010 , 113 outpatients with sad were enrolled in the group - based cbt program at the department of psychiatry , nagoya city university hospital , japan . this study was conducted as a single - arm , naturalistic , follow - up study in a routine japanese clinical setting . we followed the cbt manual for sad written by andrews et al,23 and we modified and improved the program according to clark and wells model.24 treatment was conducted in groups of three patients led by one principal therapist and one cotherapist , and were scheduled for 120 minutes once per week . the program included ( 1 ) psychoeducation about sad ( session 1 ) , ( 2 ) introduction about the individual cognitive behavioral model of sad ( session 2 ) , ( 3 ) experiments to drop safety behavior and self - focused attention ( from session 8 to last session ) , ( 4 ) attention training to shift focus away from themselves to the task or the external social situation ( sessions 4 and 5 ) , ( 5 ) video feedback of role - playing in anxious situations to modify their self - image ( sessions 6 and 7 ) , ( 6 ) in vivo exposure using behavioral experiments to test the patient s catastrophic predictions ( from session 8 to last session ) , and ( 7 ) cognitive restructuring ( session 3 , from session 8 to last session ) . among 113 patients , 98 patients ( 86.7% ) completed cbt , and almost all of the patients ( n = 109 ) finished all the exercise kinds , even when they were absent from a few sessions . we allowed patients to use antidepressants and benzodiazepines during cbt , because our study was based in a clinical setting and there is some evidence for combined pharmacologic / cbt therapy.11,25 patients did not participate in any other structured psychotherapy while attending group cbt . the principal therapist conducted the mood- and anxiety - disorder sections of the structured clinical interview for the dsm - iv at baseline , for the sad diagnosis , and any mood and anxiety comorbidities . the patients were assessed with self - report questionnaires at baseline , post - treatment , and by mail at the 1-year follow - up . our primary outcome was the total social phobia scale / social interaction anxiety scale ( sps / sias)26 score at the 1-year follow - up . sufficient internal consistency , reliability , and discrimination , as well as predictive and concurrent validity have been demonstrated for both original and japanese versions.27 cronbach s alphas of our sample for sps / sias were 0.88/0.600.88 . sufficient internal consistency , reliability , and discrimination , as well as predictive and concurrent validity have been demonstrated for both original and japanese versions.27 cronbach s alphas of our sample for sps / sias were 0.88/0.600.88 . the pretreatment and 1-year follow - up scores on sps / sias furthermore , to examine the outcomes of the cbt program across various aspects of the disorder , pre- and posttreatment scores were compared for sps , sias , and fq - sp using paired t - tests , and pretreatment and 1-year follow - up were compared for fq - sp using paired t - tests . to show the magnitude of the treatment effect , we calculated the effect size ( m pretest all statistical analyses for these treatment outcomes were conducted twice : once based on the intention - to - treat ( itt ) principle and once among the completers only . we conducted mixed - model analyses to detect the baseline predictors of treatment outcome with the 1-year follow - up sps / sias score as a dependent variable and the baseline demographic and clinical variables ( sex , age , marital status , educational status , employment status , onset , duration of sad , current mood disorder , current anxiety disorder , antidepressant use at baseline , benzodiazepine use at baseline , number of treatment sessions , severity ) as variables . as a result of chi - square tests for categorical variables , onset of sad and sps total score at baseline showed p < 0.05 , but no other major differences were observed between completers and dropouts . of the 113 patients who were enrolled , 98 completed treatment and 70 finished the 1-year follow - up . an examination of the change in symptom measures ( sps , sias , and fq - sp ) between pre- and posttreatment and between pre- and 1-year follow - ups revealed significant improvements not only for the completers but also for the itt samples ( all p < 0.05 ) . the effect sizes for the total sps / sias scores at the 1-year follow - up , which was our primary outcome , were 0.68 ( 95% ci 0.410.95)/0.76 ( 95% ci , 0.491.03 ) in the itt sample and 0.77 ( 0.421.10)/0.84 ( 0.491.18 ) in completers . based on the itt sample analyses , effect sizes for assessment at posttreatment were sps 0.64 ( 0.370.90 ) , sias 0.76 ( 0.491.03 ) , and fq - sp 0.66 ( 0.390.93 ) , and at 1-year follow - up fq - sp was 0.76 ( 0.481.02 ) . effect sizes for treatment completers at posttreatment were sps 0.81 ( 95% ci , 0.461.15 ) , sias 0.76 ( 0.491.10 ) , and fq - sp 0.81 ( 0.471.15 ) , and the effect size of the fq - sp at 1-year follow up was 0.96 ( 0.611.31 ) , indicating a greater change than that in the itt sample . a significant difference was found for sias in the older age - group at baseline ( p = 0.019 ) , a lower severity on sps ( p = 0.000 ) , and late onset of sad for both sps ( p = 0.001 ) and sias ( p = 0.000 ) as predictors of good treatment outcome . as a result of chi - square tests for categorical variables , onset of sad and sps total score at baseline showed p < 0.05 , but no other major differences were observed between completers and dropouts . of the 113 patients who were enrolled , 98 completed treatment and 70 finished the 1-year follow - up . an examination of the change in symptom measures ( sps , sias , and fq - sp ) between pre- and posttreatment and between pre- and 1-year follow - ups revealed significant improvements not only for the completers but also for the itt samples ( all p < 0.05 ) . the effect sizes for the total sps / sias scores at the 1-year follow - up , which was our primary outcome , were 0.68 ( 95% ci 0.410.95)/0.76 ( 95% ci , 0.491.03 ) in the itt sample and 0.77 ( 0.421.10)/0.84 ( 0.491.18 ) in completers . based on the itt sample analyses , effect sizes for assessment at posttreatment were sps 0.64 ( 0.370.90 ) , sias 0.76 ( 0.491.03 ) , and fq - sp 0.66 ( 0.390.93 ) , and at 1-year follow - up fq - sp was 0.76 ( 0.481.02 ) . effect sizes for treatment completers at posttreatment were sps 0.81 ( 95% ci , 0.461.15 ) , sias 0.76 ( 0.491.10 ) , and fq - sp 0.81 ( 0.471.15 ) , and the effect size of the fq - sp at 1-year follow up was 0.96 ( 0.611.31 ) , indicating a greater change than that in the itt sample . a significant difference was found for sias in the older age - group at baseline ( p = 0.019 ) , a lower severity on sps ( p = 0.000 ) , and late onset of sad for both sps ( p = 0.001 ) and sias ( p = 0.000 ) as predictors of good treatment outcome . although we focused on patients with the generalized subtype , who have more severe symptoms than those with the nongeneralized subtype , the effect sizes were as large as those in a meta - analysis conducted in western countries13 and our previous study at posttreatment . according to the effect - size calculation , our treatment program had significant effects at posttreatment that were maintained until the 1-year follow - up . thus , accumulating evidence for a positive effect of cbt in japan is a matter of urgency , and we hope our study contributes to this purpose . group cbt is more cost - effective than individual cbt in this regard , and we would like to diffuse this effective treatment for sad in japan . a number of studies have examined the role of particular variables in predicting the response to treatment ; however , results have been inconsistent and inconclusive.20 the severity of comorbid depression,31,32 symptomatic severity,31 avoidant personality disorder,33 and expectancy32 have been suggested as possible follow - up predictors for group cbt . although some demographic variables ( female , married , higher education ) were possible follow - up predictors in a study34 that conducted individual cbt , and the aforementioned demographic variables were not statistically significant in our group cbt study , we believe that suitable characteristics of patients are different between group cbt and individual therapy . we found that older age , late onset of sad , and less severe symptoms on sps were possible baseline treatment predictors for a good outcome . first , this study was conducted in a routine japanese clinical setting as a single - arm , naturalistic , follow - up study . we are unable to consider dose effects of medications on cbt ; however , use of medication at baseline was not a significant predictor of treatment outcomes in the present study . because this study was conducted as routine japanese clinical work , follow - up assessments done by post- and self - reporting versions of this35 have not been validated in japan to date . despite these limitations , this study provided evidence of long - term efficacy of group cbt for japanese patients with generalized sad . although there is still room for improvement , our results favor the use of cbt for generalized sad in japan . although we focused on patients with the generalized subtype , who have more severe symptoms than those with the nongeneralized subtype , the effect sizes were as large as those in a meta - analysis conducted in western countries13 and our previous study at posttreatment . according to the effect - size calculation , our treatment program had significant effects at posttreatment that were maintained until the 1-year follow - up . thus , accumulating evidence for a positive effect of cbt in japan is a matter of urgency , and we hope our study contributes to this purpose . group cbt is more cost - effective than individual cbt in this regard , and we would like to diffuse this effective treatment for sad in japan . a number of studies have examined the role of particular variables in predicting the response to treatment ; however , results have been inconsistent and inconclusive.20 the severity of comorbid depression,31,32 symptomatic severity,31 avoidant personality disorder,33 and expectancy32 have been suggested as possible follow - up predictors for group cbt . although some demographic variables ( female , married , higher education ) were possible follow - up predictors in a study34 that conducted individual cbt , and the aforementioned demographic variables were not statistically significant in our group cbt study , we believe that suitable characteristics of patients are different between group cbt and individual therapy . we found that older age , late onset of sad , and less severe symptoms on sps were possible baseline treatment predictors for a good outcome . first , this study was conducted in a routine japanese clinical setting as a single - arm , naturalistic , follow - up study . we are unable to consider dose effects of medications on cbt ; however , use of medication at baseline was not a significant predictor of treatment outcomes in the present study . because this study was conducted as routine japanese clinical work , follow - up assessments done by post- and self - reporting versions of this35 have not been validated in japan to date . despite these limitations , this study provided evidence of long - term efficacy of group cbt for japanese patients with generalized sad . although there is still room for improvement , our results favor the use of cbt for generalized sad in japan . group cbt resulted in improvements in japanese patients with generalized sad , and these improvements were maintained for up to 1 year after group cbt . we showed that older age at baseline , late onset , and lower severity of sad were predictors of good outcome at 1-year follow - up for group cbt .

intracranial vertebral artery dissecting aneurysms ( vadans ) are rare , with an incidence of 0.001% to 0.0015% in the general population2324 ) . ruptured vadans are associated with dismal clinical outcomes with rebleeding rates as high as 71.4% and mortality rates of 46.7% following rebleeding21 ) . patients are especially likely to present with rebleeding in the first 24 hours , and there is a high risk of rebleeding within the first week after the initial ictus if patients remain untreated . therefore , the early treatment of vadans with concurrent sah is imperative to the improvement of clinical outcomes8112223 ) . conventional surgical treatments for vadans include proximal occlusion of the ipsilateral vertebral artery ( va ) and trapping the affected va segment with or without bypass surgery . although proximal occlusion of the va has lower postoperative complications and is a simpler surgical procedure13 ) , some reports state that regrowth or rebleeding of the vadan can occur even after the surgical procedure due to the maintenance of blood flow from the contralateral va1111328 ) . trapping of the affected segment has been accepted as an appropriate management strategy to prevent rebleeding . however , ischemic complications and lower cranial nerve palsy may occur following the blockade of va blood flow and the perforating arteries that arise from the va . moreover , because the operative corridor of this area is narrow and deep , there are considerable operative risks that often result in incomplete trapping and revascularization failure when attempting bypass surgery3 ) . endovascular treatment ( evt ) as a means of proximal occlusion or trapping of the va is being used more frequently as the first - line treatment for vadans due to several advantages , including easy accessibility , relatively minimal invasiveness , and rapid treatment transition following diagnostic angiography9 ) . although these treatments are effective and safe with a low risk of complications , they still have ischemic risks comparable to surgical measures . nevertheless , recent attempts to use stent - assisted coil embolization and stent - only techniques with advanced intracranial stents produced satisfactory results , including both the safe obliteration of dissected aneurysms and the preservation of va and posterior inferior cerebellar artery ( pica ) patency . however , there is still an insufficient understanding of evt for treating vadans , especially those with accompanying pica involvement , bilateral presentation , and va dominance . the present study aimed to establish an endovascular therapeutic strategy relevant to the angioarchitecture of vadans that cause subarachnoid hemorrhages . from june 2006 to december 2013 , 725 patients with spontaneously ruptured intracranial aneurysms were surgically or endovascularly treated at our institution . of these patients , sah was confirmed by computed tomography ( ct ) scans , and contrast - enhanced three - dimensional ct angiography ( 3d - cta ) was performed to assess cerebral vasculature anomalies . this retrospective analysis included 9 men and 14 women whose age ranged from 39 to 72 years ( mean 54.2 years ) . two female patients had bilateral vadans , one of which eventually resulted in an sah and the other of which required a staged subsequent evt due to the fear of a future rupture resulting from hemodynamic augmentation after occlusion of the ruptured va . the clinical status of the patients at admission was recorded according to the hunt - hess grading ( hhg ) system , and the fisher grading ( fg ) system was used to evaluate the amount of blood on ct . the diagnosis of dissection was based on classical angiographic findings , such as fusiform dilatation , the " pearl and string " sign , subintimal flap , and irregular luminal stenosis . the procedure performed depended on patient status , location of the dissection , composition of the related vessels , and collateral circulation . evt was performed under general anesthesia in all patients except for one who required instantaneous sacrifice of the affected va due to a worsening clinical status . the collateral circulation was evaluated based on an elaborate analysis of the arterial and venous anatomy . when the quality of the image was poor , a diagnostic dsa was performed again under general anesthesia prior to evt . electrolytically detachable coils ( target detachable coil , boston scientific , fremont , ca , usa ) were used for arterial occlusion . stents ( neuroform , boston scientific , fremont , ca , usa and enterprise , cordis endovascular , miami lakes , fl , usa ) were deployed when trapping of the va would be expected to create an ischemic incident or when flow preservation of a neighboring pica on the dissecting va was necessary . once the dissecting segment was secured , either with the deconstructive or reconstructive evt , management for the sah followed standard of care . heparinization was discontinued immediately after evt and anti - platelet agents were orally administered for 6 months after evt when stents were used . the clinical and radiological data from our 23 patients are summarized in table 1 , 2 . ten patients ( 45% ) had poor clinical grades ( hhg iv or v ) and 16 patients ( 70% ) had intraventricular or intraparenchymal hemorrhages ( fg iv ) . four patients had ruptured aneurysms on the dominant side of the va , whereas 2 patients had ruptured aneurysms on the non - dominant va . fourteen aneurysms were located in a supra - pica area , 1 in an infra - pica area , 5 in a pica - involved segment , and 3 in an irrelevant pica area . the diagnosis of dissection , based on classical angiographic findings , was recorded as fusiform dilatation in 5 patients , " pearl and string " sign in 14 patients , and irregular luminal stenosis in 4 patients . there were 6 patients with extracranial origins of the pica ( ecopica ) lesions , and 1 patient exhibited a double origin of the pica lesion . eighteen patients ( 78% ) were treated with an internal trapping ( partial or total ) procedure using detachable coils , including 12 of 14 patients with supra - pica lesions , 1 patient with an infra - pica lesion , 1 of 3 patients with non - pica lesions , and 4 of 5 patients with pica - involved lesions . two patients with pica - involved lesions had partial internal trapping of the ruptured va segment with pica preservation , and were treated with surgical trapping for the remaining dissected segment after occipital artery ( oa)-pica bypass surgery the following day . two of the 5 patients with pica - involved lesions were treated using a stent procedure , which included consecutive stent - assisted coiling and stent - assisted trapping . one patient with a pica - involved lesion had internal trapping of the ruptured segment of the va under local anesthesia due to a poor clinical condition , therefore , the pica origin could not be determined . three of the 4 patients with ruptured dissecting aneurysms on the dominant va had stent - assisted coiling procedures to preserve the va flow . one patient had ipsilateral va trapping after an oa - pica bypass to preserve the pica flow because the contralateral va was not severely hypoplastic . ventricular or lumbar cerebrospinal fluid drainage occurred in 17 patients ( 74% ) with acute hydrocephalus or thick intraventricular hematoma , and 2 patients ( 9% ) required a permanent ventriculoperitoneal shunt for delayed communicating hydrocephalus . clinical outcomes were favorable for 15 of 23 patients ( 65% ) ; 13 patients ( 17% ) had good recovery , and 2 patients ( 4% ) had moderate disability . outcomes were unfavorable for 8 patients ( 35% ) ; 4 patients ( 17% ) had severe disability , 1 patient ( 4% ) entered a persistent vegetative state , and 3 patients ( 13% ) died . all 3 patient deaths could be attributed to the severity of the initial clinical status ( hhg iv ) . the complete occlusion of the dissected arterial and aneurysm segment was achieved in 21 of 23 patients ( 91% ) . two patients ( 1 with stent - assisted coiling and 1 with internal trapping ) had near - complete occlusions on angiography , and one required additional endovascular coiling during the follow - up period . there were 5 complications in our study : 2 patients developed rebleeding at the end of the coiling procedure , 1 patient developed symptomatic medullary infarction , and 2 patients experienced recanalization of the vadan after the first evt . rebleeding during the endovascular procedure was managed with an additional compact coil insertion or rescue stent placement and reversal of the anticoagulant . a 52-year - old woman presented with decreased consciousness after a sudden headache and vomiting . a brain ct indicated a dense sah and 3d - cta revealed dilatation and narrowing of the left va , indicating a possible dissection . an emergency cerebral dsa was conducted , and a vertebral angiogram ( vag ) indicated that the left va dissection produced multiple dilatations and stenosis ( fig . 1c ) and the distal vadan was hypothesized as the source of the sah . under general anesthesia , the ruptured distal vadan 1d , e ) ; the proximal part of the vadan was then surgically trapped after oa - pica bypass surgery the next day ( fig . 1f , g ) . a postoperative angiogram demonstrated a completely occluded vadan and well - preserved pica flow ( fig . a brain ct indicated a dense sah , and 3d - cta revealed a dilatation and narrowing of the right va , indicating a possible dissection . the proximal vadan included the origin of the pica , and the central vadan was hypothesized as the source of the sah ( fig . , the ruptured central vadan was internally trapped ; the proximal vadan , including the pica origin , then was managed with stent placement from the va to the pica ( fig . 2c ) . after the stent insertion , tighter coil packing of the ruptured portion of the vadan occurred . a postoperative vag demonstrated a completely occluded vadan and well - preserved pica flow through the stent , and the patient recovered well with no neurologic deficits ( fig . the final angiogram revealed a complete occlusion of the vadan and patent pica flow ( fig . 2f ) . a 44-year - old woman presented with a sudden onset of headache , vomiting , and disturbance of consciousness . a brain ct revealed an sah , and a 3d - cta revealed bilateral vadans ( fig . 3b ) , and the left vadan had a bleb - like lesion , which was considered to be the source of the sah ( fig . staged intervention was performed 1 week later . in order to preserve the flow , the right vadan was treated using an endovascular stent - within - a - stent construct with 2 self - expandable intracranial stents . although the control angiography revealed that the stents failed to completely exclude the aneurysm , definite stagnation of the contrast inside the aneurysm was achieved ( fig . the patient 's postoperative course was uneventful , and she was discharged in excellent neurological condition except for mild diplopia . the right va was remodeled and reconstituted , and the blood flow was preserved normally ( fig . a 52-year - old woman presented with decreased consciousness after a sudden headache and vomiting . a brain ct indicated a dense sah and 3d - cta revealed dilatation and narrowing of the left va , indicating a possible dissection . an emergency cerebral dsa was conducted , and a vertebral angiogram ( vag ) indicated that the left va dissection produced multiple dilatations and stenosis ( fig . 1c ) and the distal vadan was hypothesized as the source of the sah . under general anesthesia , the ruptured distal vadan 1d , e ) ; the proximal part of the vadan was then surgically trapped after oa - pica bypass surgery the next day ( fig . 1f , g ) . a postoperative angiogram demonstrated a completely occluded vadan and well - preserved pica flow ( fig . a brain ct indicated a dense sah , and 3d - cta revealed a dilatation and narrowing of the right va , indicating a possible dissection . the proximal vadan included the origin of the pica , and the central vadan was hypothesized as the source of the sah ( fig . , the ruptured central vadan was internally trapped ; the proximal vadan , including the pica origin , then was managed with stent placement from the va to the pica ( fig . 2c ) . after the stent insertion , tighter coil packing of the ruptured portion of the vadan occurred . a postoperative vag demonstrated a completely occluded vadan and well - preserved pica flow through the stent , and the patient recovered well with no neurologic deficits ( fig . the final angiogram revealed a complete occlusion of the vadan and patent pica flow ( fig . 2f ) . a 44-year - old woman presented with a sudden onset of headache , vomiting , and disturbance of consciousness . a brain ct revealed an sah , and a 3d - cta revealed bilateral vadans ( fig . 3b ) , and the left vadan had a bleb - like lesion , which was considered to be the source of the sah ( fig . 3d , e ) immediately . staged intervention was performed 1 week later . in order to preserve the flow , the right vadan was treated using an endovascular stent - within - a - stent construct with 2 self - expandable intracranial stents . although the control angiography revealed that the stents failed to completely exclude the aneurysm , definite stagnation of the contrast inside the aneurysm was achieved ( fig . the patient 's postoperative course was uneventful , and she was discharged in excellent neurological condition except for mild diplopia . the right va was remodeled and reconstituted , and the blood flow was preserved normally ( fig . a 52-year - old woman presented with decreased consciousness after a sudden headache and vomiting . a brain ct indicated a dense sah and 3d - cta revealed dilatation and narrowing of the left va , indicating a possible dissection . an emergency cerebral dsa was conducted , and a vertebral angiogram ( vag ) indicated that the left va dissection produced multiple dilatations and stenosis ( fig . 1c ) and the distal vadan was hypothesized as the source of the sah . under general anesthesia , the ruptured distal vadan 1d , e ) ; the proximal part of the vadan was then surgically trapped after oa - pica bypass surgery the next day ( fig . 1f , g ) . a postoperative angiogram demonstrated a completely occluded vadan and well - preserved pica flow ( fig . a brain ct indicated a dense sah , and 3d - cta revealed a dilatation and narrowing of the right va , indicating a possible dissection . the proximal vadan included the origin of the pica , and the central vadan was hypothesized as the source of the sah ( fig . , the ruptured central vadan was internally trapped ; the proximal vadan , including the pica origin , then was managed with stent placement from the va to the pica ( fig . 2c ) . after the stent insertion , tighter coil packing of the ruptured portion of the vadan occurred . a postoperative vag demonstrated a completely occluded vadan and well - preserved pica flow through the stent , and the patient recovered well with no neurologic deficits ( fig . the final angiogram revealed a complete occlusion of the vadan and patent pica flow ( fig . a 44-year - old woman presented with a sudden onset of headache , vomiting , and disturbance of consciousness . a brain ct revealed an sah , and a 3d - cta revealed bilateral vadans ( fig . 3b ) , and the left vadan had a bleb - like lesion , which was considered to be the source of the sah ( fig . 3d , e ) immediately . staged intervention was performed 1 week later . in order to preserve the flow , the right vadan was treated using an endovascular stent - within - a - stent construct with 2 self - expandable intracranial stents . although the control angiography revealed that the stents failed to completely exclude the aneurysm , definite stagnation of the contrast inside the aneurysm was achieved ( fig . the patient 's postoperative course was uneventful , and she was discharged in excellent neurological condition except for mild diplopia . a 6-month follow - up vag demonstrated that the left va remained occluded . the right va was remodeled and reconstituted , and the blood flow was preserved normally ( fig . the undefined neck and fragile aneurysm wall of vadans create an innate pathologic nature , including technical difficulties and risks of rupture when applying a microsurgical neck clip or performing endovascular coil embolization33 ) . to prevent the ruptured vadan from rebleeding , surgical or endovascular proximal occlusion and external or internal trapping are safe and effective management strategies . however , these destructive techniques increase the risk of ischemic complications resulting from occlusion of the va , pica , and perforating arteries , which have a significant impact on the clinical outcome3 ) . revascularization and reconstructive treatments , including bypass surgery , stent - assisted coiling , and multiple stenting are necessary to prevent and minimize ischemic complications . to achieve the best treatment results , which include complete obliteration and preserved blood flow patency , precise treatment planning should occur following careful inspection of angiographic architecture in each individual case . we considered the angioarchitecture of vadans in relation to the pica location , va dominancy , and bilateral lesions , as described in fig . we designed the treatment strategy to preserve pica flow patency if needed , because the pica is the major artery supplying the posterior inferior cerebellar hemisphere , inferior portion of the vermis , and lower medulla . the vadans were divided into 3 categories , according to the location of the vadan from the origin of the pica : supra - pica , infra - pica , and pica - involved . twelve of the 14 patients who had supra - pica lesions with sufficient contralateral va flow were treated with internal trapping , and the affected segments were successfully occluded . therefore , longer sections of internal trapping in the distal va segment were more prone to the fatal occlusion of perforators that supply the medulla oblongata73033 ) . however , we attempted to occlude the affected ruptured vadan segments over as long a section as possible . due to the underlying nature of dissection , we hypothesized that the actual dissected vessel wall may be longer than the angiographic feature , and that the complete occlusion of the dissected site could therefore prevent rebleeding . fortunately , there was no medullary infarction or any ischemia resulting from the occlusion of the perforating arteries when trapping occurred in cases of supra - pica lesions . of the 12 cases where internal trapping was used , 1 case involved stent - assisted trapping . in this case , the dissected section was just distal to the pica , so we decided to deploy the stent from the va to the pica in order to preserve pica flow . several authors have reported the efficacy and safety of va - pica stenting with no stent occlusion , and midterm follow - up angiography revealed that pica flow was patent without stenosis or occlusion412 ) . there was only 1 infra - pica vadan case that was treated with internal trapping in this study . similar to our results , other studies have found that infra - pica lesions are relatively rare compared to supra - pica lesions , and internal trapping has provided reliable management in cases with sufficient blood flow from the contralateral va92633 ) . some authors argue that pica occlusion does not result in severe disability , due to sufficient collaterals ; however , the ideal treatment for a pica - involved aneurysm is trapping and pica reconstruction30 ) . therefore , endovascular segmental occlusion using coils , followed by pica - to - pica side anastomosis or oa - pica revascularization , has been performed in many cases52630 ) . in our study , 2 patients had partial trapping using coils to reduce the rebleeding risk immediately after diagnostic angiography , which was followed by surgical trapping after the oa - pica bypass . we applied the stent - assisted technique , according to our extensive experience , to pica - involved vadans . two stent - assisted coiling procedures and 1 stent - assisted trapping procedure were performed . the clinical outcomes were favorable for these patients , and postoperative angiography indicated complete occlusion . stent - assisted coiling has several advantages : 1 ) maintaining the patency of the parent artery , and therefore preventing ischemic complications ; 2 ) forming an aneurysmal neck , which allows coil placement ; 3 ) altering hemodynamics and inducing thrombosis into the aneurysmal sac ; and 4 ) attaching the intimal flap to the vessel wall and reducing the size of pseudoaneurysms2631 ) . notably , the perforating arteries supplying the lateral surface of the brainstem originate from the va not the pica20 ) . consequently , there are risks of lateral medullary infarction as a result of internal trapping of the va , even when the ecopica is preserved . in the present study , of the 6 vadans with ecopicas , 5 were supra - pica cases and 1 was a pica - involved case . we had concerns regarding potential brainstem infarction , but these events did not occur . although ecopicas were observed relatively often and had anatomical features that should be considered when treating vadan , we did not find evidence that these features affected treatment or prognosis . when the affected segment is on the dominant va , trapping and proximal occlusion can cause fatal ischemia . therefore , the treatment strategy should be planned to preserve va and basilar artery patency during and after treatment . theoretically , the balloon occlusion test ( bot ) provides valuable information regarding collateral flows , but bot is not always recommended during the acute bleeding stage26 ) . we treated 3 cases with stent - assisted coiling for a total of 4 lesions of the dominant va without bot . all 3 vadans were successfully occluded , and the dominant va flows were preserved . vadans sometimes occur bilaterally1425 ) . the known pathogenesis for bilateral presentation is spontaneous dissection , which involves pre - existing vessel wall defects that typically present with symmetric distribution33 ) . these characteristics predispose the arteries to an increased risk of contralateral vadan rupture via hemodynamic changes resulting from the occlusion of ipsilateral ruptured va segments . we performed subsequent staged evt on contralateral unruptured aneurysms as a strategy to avoid future rupture . although there was no angiographic result of vadans that involved the anterior spinal artery ( asa ) in this series , asa is one of considerable anatomical factors in treating vadans . the asa arises from near the termination of va bilaterally in 45.5% , and the bilateral asas joins together at a distance to the vbj from 1.6 mm to 7.1 mm ( mean 4.8 mm)19 ) . it has a number of small perforating branches that supply the anterior surface of the medulla and anterior two thirds of the upper cervical cord32 ) , and occlusion of the asa results in infarction of medial or lateral part of medulla oblongata and spinal cord iscehmia19 ) . because of presence of anastomoses among the perforators of va , pica , asa , and ba , occlusion does not always lead to ischemia of brain stem and spinal cord10 ) . however we can not sure that the frequency and the number of their anastomoses are sufficient enough to avoid ischemia , the result of occlusion is uncertain19 ) . therefore , in cases of vadans associated with asa involvement , proximal occlusion or multiple stenting are preferred to consider for preventing ischemia of brain stem and spinal cord . several potential technique related complications of evt have been reported , including aneurysm rupture , va branch occlusion , hemodynamic compromise of the vertebrobasilar system resulting from vertebral artery occlusion , and recanalization of occluded vessels1527 ) . in the present study , mackay et al.17 ) proposed that stent - assisted coiling of the dissection might cause a dissection rupture or lead to recanalization if the coil migrates through the dissected vessel wall . rebleeding during the endovascular procedure was successfully managed with additional compact coil insertion or rescue stent placement combined with a reversal of the anticoagulant . there were 5 complications recorded in this study : 2 patients developed rebleeding at the end of the coiling procedure , 1 patient developed symptomatic medullary infarction , and 2 patients experienced recanalization of the vadan after the first evt . taha et al.27 ) reported 8% recanalization during the perioperative period after complete internal trapping . therefore , we propose that follow - up angiography is necessary during the perioperative period even when evt is considered successful . the prediction of patient outcomes following a ruptured vadan is important because these events are associated with high morbidity and mortality . previous studies have suggested the initial stroke severity , including national institute of health stroke scale score , and patient age as prognostic factors26 ) . in the present study , of the 10 patients with poor initial clinical grade ( hhg iv or v ) , clinical outcomes were favorable in 3 patients ( e.g. , good recovery , moderate disability ) and unfavorable in 7 patients ( e.g. , severe disability , persistent vegetative state , and death ) . conversely , among the 16 patients with an initial fg iv , 8 patients ( 50% ) experienced favorable clinical outcomes and 8 patients ( 50% ) had unfavorable outcomes . our findings therefore demonstrate that the initial clinical state is a good predictor of clinical outcome , and initial hhg scores are better predictors than fg scores . however , given that most of treatment options were used in this study and considering low incidence of the ruptured vadan , this study could strengthen the fact of usefulness of evts for vadans . second , this study was a retrospective study in which the data were acquired through a retrospective chart review . thus , it is thought that a prospective study using a larger number of patients will be necessary to determine the safety and effectiveness of evts for vadans with the concept of prompt application of treatment . ruptured vadans should be treated as soon as possible because of the high risk of rebleeding and mortality . evt has been demonstrated as safe and effective for ruptured vadans because of its easy accessibility , minimal invasiveness , and rapid treatment potential after diagnostic angiography . recent advancements in stent - assisted techniques , including stent - assisted coiling , enable both destructive ( internal trapping ) and reconstructive ( preservation of va and pica patency ) treatments . the careful selection of an appropriate evt technique following the meticulous inspection of the angioarchitecture , including the location of the pica origin , va dominancy , and bilateral lesions , is mandatory for successful treatment outcomes with low complication risks .

objectiveintracranial ruptured vertebral artery dissecting aneurysms ( vadans ) are associated with high morbidity and mortality when left untreated due to the high likelihood of rebleeding . the present study aimed to establish an endovascular therapeutic strategy that focuses specifically on the angioarchitecture of ruptured vadans.methodstwenty-three patients with ruptured vadan received endovascular treatment ( evt ) over 7 years . the patient group included 14 women ( 60.9% ) and 9 men ( 39.1% ) between the ages of 39 and 72 years ( mean age 54.2 years ) . clinical data and radiologic findings were retrospectively analyzed.resultsfour patients had aneurysms on the dominant vertebral artery . fourteen ( 61% ) aneurysms were located distal to the posterior inferior cerebellar artery ( pica ) . six ( 26% ) patients had an extracranial origin of the pica on the ruptured va , and 2 patients ( 9% ) had bilateral vadans . eighteen patients ( 78% ) were treated with internal coil trapping . two patients ( 9% ) required an adjunctive bypass procedure . seven patients ( 30% ) required stent - supported endovascular procedures . two patients experienced intra - procedural rupture during evt , one of which was associated with a focal medullary infarction . two patients ( 9% ) exhibited recanalization of the vadan during follow - up , which required additional coiling . no recurrent hemorrhage was observed during the follow - up period.conclusionevt of ruptured vadans based on angioarchitecture is a feasible and effective armamentarium to prevent fatal hemorrhage recurrence with an acceptable low risk of procedural complications . clinical outcomes depend mainly on the pre - procedural clinical state of the patient . radiologic follow - up is necessary to prevent hemorrhage recurrence after evt .

INTRODUCTION MATERIALS AND METHODS RESULTS Illustrative cases Case 1 Case 2 Case 3 DISCUSSION CONCLUSION

intracranial vertebral artery dissecting aneurysms ( vadans ) are rare , with an incidence of 0.001% to 0.0015% in the general population2324 ) . ruptured vadans are associated with dismal clinical outcomes with rebleeding rates as high as 71.4% and mortality rates of 46.7% following rebleeding21 ) . patients are especially likely to present with rebleeding in the first 24 hours , and there is a high risk of rebleeding within the first week after the initial ictus if patients remain untreated . conventional surgical treatments for vadans include proximal occlusion of the ipsilateral vertebral artery ( va ) and trapping the affected va segment with or without bypass surgery . although proximal occlusion of the va has lower postoperative complications and is a simpler surgical procedure13 ) , some reports state that regrowth or rebleeding of the vadan can occur even after the surgical procedure due to the maintenance of blood flow from the contralateral va1111328 ) . trapping of the affected segment has been accepted as an appropriate management strategy to prevent rebleeding . endovascular treatment ( evt ) as a means of proximal occlusion or trapping of the va is being used more frequently as the first - line treatment for vadans due to several advantages , including easy accessibility , relatively minimal invasiveness , and rapid treatment transition following diagnostic angiography9 ) . although these treatments are effective and safe with a low risk of complications , they still have ischemic risks comparable to surgical measures . nevertheless , recent attempts to use stent - assisted coil embolization and stent - only techniques with advanced intracranial stents produced satisfactory results , including both the safe obliteration of dissected aneurysms and the preservation of va and posterior inferior cerebellar artery ( pica ) patency . the present study aimed to establish an endovascular therapeutic strategy relevant to the angioarchitecture of vadans that cause subarachnoid hemorrhages . this retrospective analysis included 9 men and 14 women whose age ranged from 39 to 72 years ( mean 54.2 years ) . two female patients had bilateral vadans , one of which eventually resulted in an sah and the other of which required a staged subsequent evt due to the fear of a future rupture resulting from hemodynamic augmentation after occlusion of the ruptured va . the clinical status of the patients at admission was recorded according to the hunt - hess grading ( hhg ) system , and the fisher grading ( fg ) system was used to evaluate the amount of blood on ct . the diagnosis of dissection was based on classical angiographic findings , such as fusiform dilatation , the " pearl and string " sign , subintimal flap , and irregular luminal stenosis . the procedure performed depended on patient status , location of the dissection , composition of the related vessels , and collateral circulation . the collateral circulation was evaluated based on an elaborate analysis of the arterial and venous anatomy . stents ( neuroform , boston scientific , fremont , ca , usa and enterprise , cordis endovascular , miami lakes , fl , usa ) were deployed when trapping of the va would be expected to create an ischemic incident or when flow preservation of a neighboring pica on the dissecting va was necessary . ten patients ( 45% ) had poor clinical grades ( hhg iv or v ) and 16 patients ( 70% ) had intraventricular or intraparenchymal hemorrhages ( fg iv ) . four patients had ruptured aneurysms on the dominant side of the va , whereas 2 patients had ruptured aneurysms on the non - dominant va . fourteen aneurysms were located in a supra - pica area , 1 in an infra - pica area , 5 in a pica - involved segment , and 3 in an irrelevant pica area . there were 6 patients with extracranial origins of the pica ( ecopica ) lesions , and 1 patient exhibited a double origin of the pica lesion . eighteen patients ( 78% ) were treated with an internal trapping ( partial or total ) procedure using detachable coils , including 12 of 14 patients with supra - pica lesions , 1 patient with an infra - pica lesion , 1 of 3 patients with non - pica lesions , and 4 of 5 patients with pica - involved lesions . two patients with pica - involved lesions had partial internal trapping of the ruptured va segment with pica preservation , and were treated with surgical trapping for the remaining dissected segment after occipital artery ( oa)-pica bypass surgery the following day . two of the 5 patients with pica - involved lesions were treated using a stent procedure , which included consecutive stent - assisted coiling and stent - assisted trapping . one patient with a pica - involved lesion had internal trapping of the ruptured segment of the va under local anesthesia due to a poor clinical condition , therefore , the pica origin could not be determined . three of the 4 patients with ruptured dissecting aneurysms on the dominant va had stent - assisted coiling procedures to preserve the va flow . ventricular or lumbar cerebrospinal fluid drainage occurred in 17 patients ( 74% ) with acute hydrocephalus or thick intraventricular hematoma , and 2 patients ( 9% ) required a permanent ventriculoperitoneal shunt for delayed communicating hydrocephalus . clinical outcomes were favorable for 15 of 23 patients ( 65% ) ; 13 patients ( 17% ) had good recovery , and 2 patients ( 4% ) had moderate disability . outcomes were unfavorable for 8 patients ( 35% ) ; 4 patients ( 17% ) had severe disability , 1 patient ( 4% ) entered a persistent vegetative state , and 3 patients ( 13% ) died . all 3 patient deaths could be attributed to the severity of the initial clinical status ( hhg iv ) . the complete occlusion of the dissected arterial and aneurysm segment was achieved in 21 of 23 patients ( 91% ) . two patients ( 1 with stent - assisted coiling and 1 with internal trapping ) had near - complete occlusions on angiography , and one required additional endovascular coiling during the follow - up period . there were 5 complications in our study : 2 patients developed rebleeding at the end of the coiling procedure , 1 patient developed symptomatic medullary infarction , and 2 patients experienced recanalization of the vadan after the first evt . rebleeding during the endovascular procedure was managed with an additional compact coil insertion or rescue stent placement and reversal of the anticoagulant . a brain ct indicated a dense sah and 3d - cta revealed dilatation and narrowing of the left va , indicating a possible dissection . 1c ) and the distal vadan was hypothesized as the source of the sah . under general anesthesia , the ruptured distal vadan 1d , e ) ; the proximal part of the vadan was then surgically trapped after oa - pica bypass surgery the next day ( fig . a brain ct indicated a dense sah , and 3d - cta revealed a dilatation and narrowing of the right va , indicating a possible dissection . the proximal vadan included the origin of the pica , and the central vadan was hypothesized as the source of the sah ( fig . , the ruptured central vadan was internally trapped ; the proximal vadan , including the pica origin , then was managed with stent placement from the va to the pica ( fig . after the stent insertion , tighter coil packing of the ruptured portion of the vadan occurred . a postoperative vag demonstrated a completely occluded vadan and well - preserved pica flow through the stent , and the patient recovered well with no neurologic deficits ( fig . the final angiogram revealed a complete occlusion of the vadan and patent pica flow ( fig . 3b ) , and the left vadan had a bleb - like lesion , which was considered to be the source of the sah ( fig . in order to preserve the flow , the right vadan was treated using an endovascular stent - within - a - stent construct with 2 self - expandable intracranial stents . 1c ) and the distal vadan was hypothesized as the source of the sah . under general anesthesia , the ruptured distal vadan 1d , e ) ; the proximal part of the vadan was then surgically trapped after oa - pica bypass surgery the next day ( fig . a brain ct indicated a dense sah , and 3d - cta revealed a dilatation and narrowing of the right va , indicating a possible dissection . the proximal vadan included the origin of the pica , and the central vadan was hypothesized as the source of the sah ( fig . , the ruptured central vadan was internally trapped ; the proximal vadan , including the pica origin , then was managed with stent placement from the va to the pica ( fig . after the stent insertion , tighter coil packing of the ruptured portion of the vadan occurred . a postoperative vag demonstrated a completely occluded vadan and well - preserved pica flow through the stent , and the patient recovered well with no neurologic deficits ( fig . the final angiogram revealed a complete occlusion of the vadan and patent pica flow ( fig . a brain ct revealed an sah , and a 3d - cta revealed bilateral vadans ( fig . 3b ) , and the left vadan had a bleb - like lesion , which was considered to be the source of the sah ( fig . in order to preserve the flow , the right vadan was treated using an endovascular stent - within - a - stent construct with 2 self - expandable intracranial stents . the patient 's postoperative course was uneventful , and she was discharged in excellent neurological condition except for mild diplopia . a brain ct indicated a dense sah and 3d - cta revealed dilatation and narrowing of the left va , indicating a possible dissection . under general anesthesia , the ruptured distal vadan 1d , e ) ; the proximal part of the vadan was then surgically trapped after oa - pica bypass surgery the next day ( fig . a brain ct indicated a dense sah , and 3d - cta revealed a dilatation and narrowing of the right va , indicating a possible dissection . the proximal vadan included the origin of the pica , and the central vadan was hypothesized as the source of the sah ( fig . , the ruptured central vadan was internally trapped ; the proximal vadan , including the pica origin , then was managed with stent placement from the va to the pica ( fig . after the stent insertion , tighter coil packing of the ruptured portion of the vadan occurred . the final angiogram revealed a complete occlusion of the vadan and patent pica flow ( fig . a 44-year - old woman presented with a sudden onset of headache , vomiting , and disturbance of consciousness . a brain ct revealed an sah , and a 3d - cta revealed bilateral vadans ( fig . 3b ) , and the left vadan had a bleb - like lesion , which was considered to be the source of the sah ( fig . in order to preserve the flow , the right vadan was treated using an endovascular stent - within - a - stent construct with 2 self - expandable intracranial stents . the patient 's postoperative course was uneventful , and she was discharged in excellent neurological condition except for mild diplopia . a 6-month follow - up vag demonstrated that the left va remained occluded . to prevent the ruptured vadan from rebleeding , surgical or endovascular proximal occlusion and external or internal trapping are safe and effective management strategies . however , these destructive techniques increase the risk of ischemic complications resulting from occlusion of the va , pica , and perforating arteries , which have a significant impact on the clinical outcome3 ) . revascularization and reconstructive treatments , including bypass surgery , stent - assisted coiling , and multiple stenting are necessary to prevent and minimize ischemic complications . we considered the angioarchitecture of vadans in relation to the pica location , va dominancy , and bilateral lesions , as described in fig . we designed the treatment strategy to preserve pica flow patency if needed , because the pica is the major artery supplying the posterior inferior cerebellar hemisphere , inferior portion of the vermis , and lower medulla . the vadans were divided into 3 categories , according to the location of the vadan from the origin of the pica : supra - pica , infra - pica , and pica - involved . twelve of the 14 patients who had supra - pica lesions with sufficient contralateral va flow were treated with internal trapping , and the affected segments were successfully occluded . due to the underlying nature of dissection , we hypothesized that the actual dissected vessel wall may be longer than the angiographic feature , and that the complete occlusion of the dissected site could therefore prevent rebleeding . fortunately , there was no medullary infarction or any ischemia resulting from the occlusion of the perforating arteries when trapping occurred in cases of supra - pica lesions . in this case , the dissected section was just distal to the pica , so we decided to deploy the stent from the va to the pica in order to preserve pica flow . several authors have reported the efficacy and safety of va - pica stenting with no stent occlusion , and midterm follow - up angiography revealed that pica flow was patent without stenosis or occlusion412 ) . there was only 1 infra - pica vadan case that was treated with internal trapping in this study . in our study , 2 patients had partial trapping using coils to reduce the rebleeding risk immediately after diagnostic angiography , which was followed by surgical trapping after the oa - pica bypass . the clinical outcomes were favorable for these patients , and postoperative angiography indicated complete occlusion . stent - assisted coiling has several advantages : 1 ) maintaining the patency of the parent artery , and therefore preventing ischemic complications ; 2 ) forming an aneurysmal neck , which allows coil placement ; 3 ) altering hemodynamics and inducing thrombosis into the aneurysmal sac ; and 4 ) attaching the intimal flap to the vessel wall and reducing the size of pseudoaneurysms2631 ) . consequently , there are risks of lateral medullary infarction as a result of internal trapping of the va , even when the ecopica is preserved . in the present study , of the 6 vadans with ecopicas , 5 were supra - pica cases and 1 was a pica - involved case . when the affected segment is on the dominant va , trapping and proximal occlusion can cause fatal ischemia . we treated 3 cases with stent - assisted coiling for a total of 4 lesions of the dominant va without bot . all 3 vadans were successfully occluded , and the dominant va flows were preserved . the known pathogenesis for bilateral presentation is spontaneous dissection , which involves pre - existing vessel wall defects that typically present with symmetric distribution33 ) . these characteristics predispose the arteries to an increased risk of contralateral vadan rupture via hemodynamic changes resulting from the occlusion of ipsilateral ruptured va segments . although there was no angiographic result of vadans that involved the anterior spinal artery ( asa ) in this series , asa is one of considerable anatomical factors in treating vadans . the asa arises from near the termination of va bilaterally in 45.5% , and the bilateral asas joins together at a distance to the vbj from 1.6 mm to 7.1 mm ( mean 4.8 mm)19 ) . it has a number of small perforating branches that supply the anterior surface of the medulla and anterior two thirds of the upper cervical cord32 ) , and occlusion of the asa results in infarction of medial or lateral part of medulla oblongata and spinal cord iscehmia19 ) . because of presence of anastomoses among the perforators of va , pica , asa , and ba , occlusion does not always lead to ischemia of brain stem and spinal cord10 ) . several potential technique related complications of evt have been reported , including aneurysm rupture , va branch occlusion , hemodynamic compromise of the vertebrobasilar system resulting from vertebral artery occlusion , and recanalization of occluded vessels1527 ) . in the present study , mackay et al.17 ) proposed that stent - assisted coiling of the dissection might cause a dissection rupture or lead to recanalization if the coil migrates through the dissected vessel wall . rebleeding during the endovascular procedure was successfully managed with additional compact coil insertion or rescue stent placement combined with a reversal of the anticoagulant . there were 5 complications recorded in this study : 2 patients developed rebleeding at the end of the coiling procedure , 1 patient developed symptomatic medullary infarction , and 2 patients experienced recanalization of the vadan after the first evt . therefore , we propose that follow - up angiography is necessary during the perioperative period even when evt is considered successful . the prediction of patient outcomes following a ruptured vadan is important because these events are associated with high morbidity and mortality . in the present study , of the 10 patients with poor initial clinical grade ( hhg iv or v ) , clinical outcomes were favorable in 3 patients ( e.g. , good recovery , moderate disability ) and unfavorable in 7 patients ( e.g. conversely , among the 16 patients with an initial fg iv , 8 patients ( 50% ) experienced favorable clinical outcomes and 8 patients ( 50% ) had unfavorable outcomes . our findings therefore demonstrate that the initial clinical state is a good predictor of clinical outcome , and initial hhg scores are better predictors than fg scores . however , given that most of treatment options were used in this study and considering low incidence of the ruptured vadan , this study could strengthen the fact of usefulness of evts for vadans . ruptured vadans should be treated as soon as possible because of the high risk of rebleeding and mortality . evt has been demonstrated as safe and effective for ruptured vadans because of its easy accessibility , minimal invasiveness , and rapid treatment potential after diagnostic angiography . recent advancements in stent - assisted techniques , including stent - assisted coiling , enable both destructive ( internal trapping ) and reconstructive ( preservation of va and pica patency ) treatments . the careful selection of an appropriate evt technique following the meticulous inspection of the angioarchitecture , including the location of the pica origin , va dominancy , and bilateral lesions , is mandatory for successful treatment outcomes with low complication risks .

in mri , spatial information is obtained from the object using magnetic field gradients , which link the larmor frequency of the excited spins to their spatial location . thus , the received signal is the continuous fourier transform of the object 's proton density(1)s(k ) = (x ) eikx dx , where the k - space position k can be calculated from the time course of the applied gradients . in practice , the proton density is further modulated by spin relaxation , off - resonance effects , and other mechanisms all neglected here . it is well known that objects with compact support have a fourier transform with nonlimited support . for example , the fourier transform of a rectangle is composed of sinc functions in each dimension . because only a single location of the fourier space can be measured at a time , it is impossible to fully sample such fourier transform by travelling the mri k - space with magnetic field gradients . hence , there are two experimental restrictions for mri . first , the continuous fourier transform is sampled discretely , which can be seen as a multiplication with a comb - function in frequency space . in image space , this corresponds to a convolution with a reciprocally spaced comb - function and leads to periodic object copies with a spacing inverse to the sample distance in k - space . second , the fourier transform is sampled only within a finite region around the k - space center with all other information missing . in the conventional case , a discrete fourier transformation of the finitely measured data is performed to reconstruct an image . this strategy implicitly assumes that the fourier transform is zero everywhere outside the sampled region . it is clear that the assumption is not very appropriate for finite objects , although the corresponding reconstruction totally complies with all data measured . in fact , any solution that coincides at the sampling positions is a valid reconstruction , because the finite sampling pattern opens degrees of freedom from the null space of the projection evoked by finite sampling . unfortunately , however , the procedure corresponds to a multiplication of the true object 's fourier transform with a rect - function ( in case of cartesian sampling ) which , in image space , results in a convolution of the true object with a sinc - function . this effect is well known as truncation artifact or gibbs ringing and mainly appears as an oscillating overshoot of the image intensity near discontinuities [ 1 , 2 ] . although the problem may be reduced by increasing the measured k - space , many practical applications still rely on acquisitions with a relatively low - matrix resolution in at least one image dimension , and therefore suffer from respective artifacts . so far , various methods have been developed to ameliorate image disturbances due to finite sampling [ 25 ] . however , in the majority of mri applications and , in particular , for most commercially available mri systems , only a simple data filtering is routinely employed . in this case , visual reduction of the ringing artifacts is achieved by a smearing of the intensity oscillations , which leads to an undesired loss of image resolution . alternative methods attempt to extrapolate the measured data and thereby avoid a sharp cut - off in k - space [ 69 ] . a key difference to the filtering approach is that the actually measured data is not changed but supplemented with synthetic data a reasonable strategy as the measured data is not incorrect but only incomplete . this can be achieved by exploiting a priori knowledge about the true object and , consequently , all extrapolation techniques rely on certain assumptions , where the existing methods follow different strategies . in this regard , the present work demonstrates that also the very unspecific assumption of a piecewise - constant object can be utilized to successfully extrapolate data in k - space and concomitantly reduce the ringing artifacts without compromising image resolution . figure 1 compares the one - dimensional profile of a rectangle reconstructed by fourier transformation from only 96 fourier samples to that of the original function . it clearly illustrates severe ringing artifacts , although the true function is piecewise constant and free of any oscillations . such oscillations can be quantified using the total variation ( tv ) , which sums the modulus of jumps between all neighboring pixels of a reconstructed image i(x , y)(2)tv(i ) = y=0n x=0n|i(x , y)i(x1 , y)| + |i(x , y)i(x , y1)|.the tv concept was initially introduced to image processing by rudin et al . for denoising applications because noise patterns create a high tv value relative to that of a noise - free image , and they become particularly reduced when modifying the image in such a way that the tv value is minimized . as a specific property , edges are preserved during this procedure , and thus tv minimization emerged as one the most popular denoising techniques . in recent years , the tv concept is attracting strong interest in the field of compressed sensing because for specific sampling techniques , the tv value can be utilized to identify and to remove artifacts from undersampling , offering a remarkable reduction of the measurement time . in a similar manner , truncation artifacts lead to an increased tv value relative to that of the true object , so that the tv may also be taken as a measure of the artifact strength for finite k - space sampling , which has been recognized by landi et al . as well . therefore , the proposed idea is to add a set of synthetic frequencies v to the measured data y , which is specifically chosen such that the tv value of the image reconstructed from the combination of the measured and synthetic data is minimized(3)v = argminv tv({vy } ) , where denotes the discrete fourier transformation . interestingly , by searching for the set of synthetic frequencies v , the unmeasured k - space data is recovered to a certain degree if the assumption of a piecewise - constant object is appropriate . estimation of the synthetic data can be achieved by minimizing ( 3 ) with a nonlinear numerical optimization technique . the present proof - of - principle implementation used the cg - descent algorithm , which is a recent variant of the nonlinear conjugate gradient method that allows to rather efficiently solve large - scale problems . the algorithm can be used in a black - box manner , requiring only the evaluation of a cost function and its gradient for given estimate vectors v. the cost function is needed to quantify the goodness of a given estimate ( i.e. , it is small for a good estimate and large otherwise ) , and for the problem defined in ( 3 ) it simply has the form(4)(v ) = tv({vy } ) .the gradient of the cost function corresponds to the derivative of this function with respect to all components of the estimate vector v. because the discrete fourier transformation is a unitary operation , it can be evaluated conveniently by calculating the gradient of the tv term in the image domain ( i.e. , estimating a vector that describes how the tv value changes for modifications of the individual pixels ) , followed by an inverse fourier transformation to the frequency domain . calculation of the tv value according to ( 2 ) uses only the first - order derivative of the image with respect to its x- and y - directions . this value is minimized if an image consists of areas with constant signal intensity , so that the extrapolation procedure yields a solution primarily with constant areas . while desirable for truly flat objects like numerical phantoms , it tends to create images with a slightly blocky or patchy appearance for real - world objects . therefore , it is advisable to additionally include second - order derivatives into the tv term , which then allows for intensity gradients in the images and yields more naturally looking solutions(5)tv2(i ) = y=0n x=0n (|i(x , y)i(x1 , y)| + |i(x , y)i(x , y1)| ) + ( 1)(|i(x1 , y)2i(x , y ) + i(x + 1 , y)| + |i(x , y1)2i(x , y ) + i(x , y + 1)| + |i(x , y)i(x1 , y)i(x , y1 ) + i(x1 , y1)|).here , is a weighting factor which can be used to tune the images between a slightly more blocky looking and a slightly smoother appearance . for the reconstructions presented , it was set to = 0.77 based on the considerations by geman and yang . in practice , while the aforementioned approach is still able to reduce visible truncation artifacts under these circumstances , it does not reduce image noise because the measured k - space data remains unchanged . on the other hand , an additional denoising may be achieved by loosing the fixed bound on the measured data , that is by introducing a data fitting term . in this case , the algorithm not only adds synthetic frequencies to obtain a tv minimization , but is also allowed to find a solution that slightly diverges from the measured data , which yields an effective edge - preserving denoising . therefore , the estimate vector v has to be extended such that it contains both synthesized frequencies as well as frequencies from the measured part of k - space , which is indicated by writing vd instead . in the denoising case , the cost function takes the form(6)(vd ) = vdy22 + tv({vd } ) , where denotes an operation that calculates the residual between the measured values and the corresponding entries of the estimate , which are now contained in the vector vd . further , is a weighting factor that allows to select the desired denoising strength . while a low weight permits considerable divergences from the measured values and , thus , leads to an effective removal of noise , it can also cause a loss of object detail if selected too low . therefore , the weight has to be adjusted with respect to the signal - to - noise ratio of the measurement sequence , where a reasonable strategy is to estimate a fixed value once for each protocol by computing a set of test images with different values and selecting the value yielding the desired degree of denoising . although the basic physical quantity measured by mri , that is , the spin - density modulated by relaxation or saturation effects , should be real - valued and nonnegative in theory , inherent experimental phase variations usually cause the observed object to be complex - valued . moreover , modern mri systems often use multiple receive coils with complex - valued sensitivity profiles , yielding differently modulated views of the object . as a consequence , spatially varying transitions between the real and imaginary component occur as well as local intensity changes , which conflict with the assumption of a piecewise - constant quantity and prevent a direct application of the tv constraint . therefore , some mechanism is required to cope with the phase variations and the multicoil scenario . in this proof - of - principle study , phase variations were removed in a preprocessing step by performing a fourier transformation of the data from each coil and calculating the sum - of - squares of all channels in the image domain . subsequently , an inverse fourier transformation of the sum - of - squares data was performed to obtain a combined data set with real - valued and nonnegative values in the image domain , which enables a calculation of the tv value using only the real part of the image . while this simple technique turned out to be sufficient for demonstrating a removal of truncation artifacts by tv - constrained data extrapolation , routine applications will probably require a more sophisticated procedure , in particular when combined with advanced techniques such as parallel imaging and when using complex coil configurations with more localized sensitivities of the individual receiver elements . simulations were performed with the shepp - logan phantom , which is composed of several ellipses . because the fourier transform of a single ellipse is given by a bessel function , an analytical fourier transform of the phantom is obtained by a superposition of respective bessel functions . truncation artifacts can be studied by evaluating the noncompact analytical transform at the sampling positions along the trajectory , here yielding a matrix of 96 96 fourier samples . all simulations and processing of experimental data were done offline using an in - house software package written in c / c++ . mri experiments were conducted at 2.9 t ( siemens magnetom tim trio , erlangen , germany ) with use of a receive only 12-channel head coil equipped with hardware signal combiners , yielding four receiver channels with different combinations of the coil elements . measurements were performed for a water phantom and human brain in vivo , where written informed consent was obtained from all subjects prior to each examination . for demonstration purposes , the image acquisitions were done with a simple slice - selective spin - echo sequence at a 200 200 mm field of view , covered by a 96 96 acquisition matrix . different sequence settings were used to obtain data sets with low and high level of noise , where the latter was achieved by reducing the flip angle and slice thickness while increasing the receiver bandwidth . further , one data set was acquired with a slice - selective gradient - echo sequence , which allowed for the rapid measurement of a full 288 288 acquisition matrix . all images were reconstructed on a 288 288 matrix corresponding to an extrapolation factor of 3 . the proposed algorithm was run for a fixed number of 3000 iterations , which takes about 2 - 3 minutes on a standard microprocessor . in cases where an additional data fitting term was used , the weighting factor was adjusted manually to yield a reasonable solution as judged by visual inspection . zero - padded solutions with and without filtering were calculated for comparison . here , a simple lanczos sigma filter , that is , multiplication with a sinc - function , was applied , where the window width was selected such that the sinc - function 's first null coincides with the border of the measured k - space . although other filters might perform better , it serves to demonstrate the general problem related to data filtering . in addition , a two - dimensional version of the extrapolation method described by constable and henkelman was implemented with a window width of p = 2 for the edge - preserving sigma filter . in our implementation , the filter parameter was selected according to = c i , where i denotes the intensity of the pixel to be filtered and c is a global coefficient that was set to c = 0.1 based on visual inspection . finally , all images were magnified and cropped to improve the visibility of the artifacts . figure 2 shows different reconstructions of the shepp - logan phantom ( left column ) together with the respective fourier transforms ( right column ) . it is clearly visible that the zero - padded solution ( zero ) suffers from severe ringing artifacts around all edges of the phantom . the extent of the measured k - space can be seen in its fourier transform . most ringing artifacts disappear after filtering ( filter ) , however , at the expense of a significant loss of image resolution . in contrast , the image reconstructed with the proposed method ( tv ) is neither affected by ringing artifacts nor by blurring , and it presents with considerably sharper edges relative to the zero - padded solution . its fourier transform reveals that the measured data has been properly extrapolated into the uncovered areas of k - space . for comparison , a full reconstruction from 288 288 samples is shown in the bottom row ( full ) . figure 3 demonstrates the application of the method to experimental data obtained for a phantom ( left column ) and a human brain in vivo ( right column ) in comparison to zero - padded ( zero ) and filtered zero - padded solutions ( filter ) . again , the ringing artifacts obtained for zero padding ( indicated by arrows ) are significantly reduced when using tv - constrained data extrapolation with only first - order ( tv ) or additionally second - order derivatives ( tv2 ) . the blocky appearance of the tv reconstruction becomes much more smoother for the tv2 approach , although both solutions ( tv and tv2 ) look somewhat more blocky than the zero - padding solution . in figure 4 , the proposed approach ( tv2 ) is compared to a 2d version of the extrapolation method by constable and henkelman ( comp ) for the shepp - logan phantom ( left column ) and an experimental study of the human brain in vivo ( right column ) . it can be seen that the performance of the proposed extrapolation method is slightly better for the simulated data , while both approaches yield an effective suppression of ringing artifacts for the experimental data ( note that the alternative method leads to a slight denoising of the image ) . however , in our implementation , the algorithm by constable and henkelman tends to be sensitive to the parameter selection for the initial filter that is used to detect true edges of the object , whereas a selection of respective parameters is not needed in the tv - based approach . figure 5 shows reconstructions of the shepp - logan phantom from noisy data using zero - padding ( zero ) , the proposed extrapolation approach ( tv ) , and its combination with denoising ( tvdns ) . while the basic extrapolation approach leads to a reduction of truncation artifacts also for noisy data , it does not reduce the noise patterns . however , when extending the tv penalty to the measured data , the method effectively flattens noise patterns in addition to the suppression of ringing artifacts . finally , corresponding reconstructions from experimental data with a high degree of noise are shown in figure 6 . here , a combination of first- and second - order derivatives was used for the tv calculation . as in the simulations , the proposed method leads to a reduction of truncation artifacts ( tv2 ) , while the extension to data fitting yields an additional edge - preserving denoising ( tv2dns ) . both simulations and experiments demonstrate that tv - constrained data extrapolation effectively reduces truncation artifacts due to finitely sampled mri acquisitions . usually , the images exhibit a more blocky appearance compared to zero - padding . however , it should be noted that the smoothness observed for zero - padding originates to a significant degree from the convolution with the sinc - function . as a consequence , a sharp edge of the object is mapped as a rather smooth pattern , which might appear more familiar to the viewer than a blocky image , but strictly represents an image artifact . hence , the extrapolation technique may even lead to a slight gain of resolution due to a sharpening of the point - spread function , following from the reciprocity property of the fourier transformation . this effect can be best seen in figure 6 when comparing the borders of the dark brain vessels obtained for zero padding ( zero ) with the proposed method ( tv2 ) . first , the method is based on the assumption that the true object is piecewise constant , which is only approximately valid for real - world objects . in the presence of additional experimental effects like flow artifacts hence , the extrapolation performance depends on the object 's conformance with the assumption that it is piecewise - constant . moreover , if the true object contains strongly varying patterns , the algorithm may erroneously soften such patterns by supplementing respective high frequencies . on the other hand , in the majority of cases , the assumption of a piecewise - constant object seems to be more appropriate than that of all conventional reconstructions , namely , a fourier transform of the object that is zero outside the sampled k - space area . second , the proposed method synthesizes only a finite number of additional frequencies , whereas an infinite number of k - space samples would be required to completely eliminate all truncation effects . in practice , however , it turned out that there is no perceivable benefit of extrapolating by a factor of higher than three . the reason is that the method yields an implicit filtering of the extrapolated data : assuming that the extrapolation procedure would recover the unmeasured k - space samples exactly , then a new truncation effect would arise at the extended border and again lead to ringing artifacts in image space ( though with a higher oscillation frequency ) . because this would increment the tv value , the method automatically lowers outer frequencies during the extrapolation procedure to prevent the upcoming of new ringing artifacts . hence , the extrapolated values diverge categorically from the true frequencies which , in this case , is a rather desirable feature as the prime target is to reduce visually annoying ringing artifacts rather than to gain super - resolution . third , if a completely artifact - free reconstruction of the object would be available , then respective frequency samples could be calculated with a discrete fourier transformation of the given image . interestingly , these samples would diverge from the experimentally measured frequencies , because image pixels are discrete and , thus , the fourier transform of the image is periodic such that outer frequencies from neighboring copies ( of the true object 's noncompact fourier transform ) overlap . this is different from the experimental situation where the object is continuous and the outer frequencies are missing instead of overlapping . consequently , an artifact - free discrete reconstruction can only be obtained if the samples used for the reconstruction specifically diverge from the measured frequencies . a complete artifact removal , therefore , requires to alter the measured frequencies instead of keeping them unchanged . unfortunately , the information how the samples have to be adjusted is not available , so that in practice a data fitting term might be the best solution when a complete removal of ringing artifacts is needed . however , this might cause a loss of object detail as described in the theory section . the modulus function in the tv formula ( 2 ) has a fundamental role for the success of tv - based image processing . because subtraction of neighboring pixels performed before taking the modulus can be seen as applying a difference operator to the estimate , tv minimization yields a solution with minimum l1-norm in the difference basis . due to the specific character of the modulus function , this solution tends to be sparse in the difference basis : it has few large jumps and most differences between neighboring pixels are near zero , which directly translates into a piecewise - constant image ( and explains the edge preserving character of tv - based denoising ) . if the modulus would be replaced by a square function , then the optimizer would try to find a minimum l2-norm solution with minimal jumps between all neighboring pixels . this corresponds to a globally smooth image , which is usually undesired due to a loss of sharp edges . while it is rather simple to obtain a minimum l2-norm solution as its cost function is strictly convex , finding a minimum l1-norm solution is much more challenging ; and many optimization algorithms fail if directly applied to the tv problem . one major reason is that the derivative of the modulus function is just 1 , which does not help to guess a reasonable step size toward the function 's minimum . however , it turned out that the cg - descent algorithm is capable to handle the problem as it comprises a powerful line - search procedure , but it is probably not the optimal method for finding the solution . in particular , the convergence tends to be somewhat sensitive to the scaling of the data . in order to ensure convergence , it was , therefore , necessary to introduce a scaling factor that limits the modification strength for each iteration and to run the algorithm in turn for a high number of iterations ( e.g. , 3000 iterations as arbitrarily chosen here ) nevertheless , this issue should not be seen as a drawback of the proposed extrapolation approach itself , but rather as a technical aspect of the optimization method utilized in this proof - of - principle study . employing a dedicated algorithm for tv minimization should render a scaling factor unnecessary and although such enterprise promises reconstructions in a fraction of the current processing time , it is outside the scope of the present study . the present work demonstrates that the simple assumption of a piecewise - constant object can be exploited to extrapolate measured data in k - space . this allows for a significant reduction of ringing artifacts that arise from data truncation in k - space . in contrast to commonly used filtering approaches , the method does not degrade the spatial resolution of the reconstructed image and rather leads to a mild resolution enhancement due to sharpening of the point - spread function . if the measured data is seriously contaminated by noise , an extended approach offers edge - preserving denoising by slightly altering the measured data in addition to supplementing synthetic data . both variants can be implemented as a pure postprocessing procedure and are also applicable for partial fourier acquisitions . while the current implementation suffers from a relatively high computational load , the use of a dedicated tv optimization algorithm promises a processing speed suitable for routine applications .

the finite sampling of k - space in mri causes spurious image artifacts , known as gibbs ringing , which result from signal truncation at the border of k - space . the effect is especially visible for acquisitions at low resolution and commonly reduced by filtering at the expense of image blurring . the present work demonstrates that the simple assumption of a piecewise - constant object can be exploited to extrapolate the data in k - space beyond the measured part . the method allows for a significant reduction of truncation artifacts without compromising resolution . the assumption translates into a total variation minimization problem , which can be solved with a nonlinear optimization algorithm . in the presence of substantial noise , a modified approach offers edge - preserving denoising by allowing for slight deviations from the measured data in addition to supplementing data . the effectiveness of these methods is demonstrated with simulations as well as experimental data for a phantom and human brain in vivo .

1. INTRODUCTION 2. THEORY 3. METHODS 4. RESULTS 5. DISCUSSION 6. CONCLUSION

in mri , spatial information is obtained from the object using magnetic field gradients , which link the larmor frequency of the excited spins to their spatial location . thus , the received signal is the continuous fourier transform of the object 's proton density(1)s(k ) = (x ) eikx dx , where the k - space position k can be calculated from the time course of the applied gradients . because only a single location of the fourier space can be measured at a time , it is impossible to fully sample such fourier transform by travelling the mri k - space with magnetic field gradients . first , the continuous fourier transform is sampled discretely , which can be seen as a multiplication with a comb - function in frequency space . in image space , this corresponds to a convolution with a reciprocally spaced comb - function and leads to periodic object copies with a spacing inverse to the sample distance in k - space . second , the fourier transform is sampled only within a finite region around the k - space center with all other information missing . in the conventional case , a discrete fourier transformation of the finitely measured data is performed to reconstruct an image . it is clear that the assumption is not very appropriate for finite objects , although the corresponding reconstruction totally complies with all data measured . in fact , any solution that coincides at the sampling positions is a valid reconstruction , because the finite sampling pattern opens degrees of freedom from the null space of the projection evoked by finite sampling . unfortunately , however , the procedure corresponds to a multiplication of the true object 's fourier transform with a rect - function ( in case of cartesian sampling ) which , in image space , results in a convolution of the true object with a sinc - function . this effect is well known as truncation artifact or gibbs ringing and mainly appears as an oscillating overshoot of the image intensity near discontinuities [ 1 , 2 ] . although the problem may be reduced by increasing the measured k - space , many practical applications still rely on acquisitions with a relatively low - matrix resolution in at least one image dimension , and therefore suffer from respective artifacts . in this case , visual reduction of the ringing artifacts is achieved by a smearing of the intensity oscillations , which leads to an undesired loss of image resolution . alternative methods attempt to extrapolate the measured data and thereby avoid a sharp cut - off in k - space [ 69 ] . a key difference to the filtering approach is that the actually measured data is not changed but supplemented with synthetic data a reasonable strategy as the measured data is not incorrect but only incomplete . this can be achieved by exploiting a priori knowledge about the true object and , consequently , all extrapolation techniques rely on certain assumptions , where the existing methods follow different strategies . in this regard , the present work demonstrates that also the very unspecific assumption of a piecewise - constant object can be utilized to successfully extrapolate data in k - space and concomitantly reduce the ringing artifacts without compromising image resolution . figure 1 compares the one - dimensional profile of a rectangle reconstructed by fourier transformation from only 96 fourier samples to that of the original function . such oscillations can be quantified using the total variation ( tv ) , which sums the modulus of jumps between all neighboring pixels of a reconstructed image i(x , y)(2)tv(i ) = y=0n x=0n|i(x , y)i(x1 , y)| + |i(x , y)i(x , y1)|.the tv concept was initially introduced to image processing by rudin et al . for denoising applications because noise patterns create a high tv value relative to that of a noise - free image , and they become particularly reduced when modifying the image in such a way that the tv value is minimized . in recent years , the tv concept is attracting strong interest in the field of compressed sensing because for specific sampling techniques , the tv value can be utilized to identify and to remove artifacts from undersampling , offering a remarkable reduction of the measurement time . in a similar manner , truncation artifacts lead to an increased tv value relative to that of the true object , so that the tv may also be taken as a measure of the artifact strength for finite k - space sampling , which has been recognized by landi et al . therefore , the proposed idea is to add a set of synthetic frequencies v to the measured data y , which is specifically chosen such that the tv value of the image reconstructed from the combination of the measured and synthetic data is minimized(3)v = argminv tv({vy } ) , where denotes the discrete fourier transformation . interestingly , by searching for the set of synthetic frequencies v , the unmeasured k - space data is recovered to a certain degree if the assumption of a piecewise - constant object is appropriate . estimation of the synthetic data can be achieved by minimizing ( 3 ) with a nonlinear numerical optimization technique . the present proof - of - principle implementation used the cg - descent algorithm , which is a recent variant of the nonlinear conjugate gradient method that allows to rather efficiently solve large - scale problems . the algorithm can be used in a black - box manner , requiring only the evaluation of a cost function and its gradient for given estimate vectors v. the cost function is needed to quantify the goodness of a given estimate ( i.e. , it is small for a good estimate and large otherwise ) , and for the problem defined in ( 3 ) it simply has the form(4)(v ) = tv({vy } ) .the gradient of the cost function corresponds to the derivative of this function with respect to all components of the estimate vector v. because the discrete fourier transformation is a unitary operation , it can be evaluated conveniently by calculating the gradient of the tv term in the image domain ( i.e. this value is minimized if an image consists of areas with constant signal intensity , so that the extrapolation procedure yields a solution primarily with constant areas . therefore , it is advisable to additionally include second - order derivatives into the tv term , which then allows for intensity gradients in the images and yields more naturally looking solutions(5)tv2(i ) = y=0n x=0n (|i(x , y)i(x1 , y)| + |i(x , y)i(x , y1)| ) + ( 1)(|i(x1 , y)2i(x , y ) + i(x + 1 , y)| + |i(x , y1)2i(x , y ) + i(x , y + 1)| + |i(x , y)i(x1 , y)i(x , y1 ) + i(x1 , y1)|).here , is a weighting factor which can be used to tune the images between a slightly more blocky looking and a slightly smoother appearance . in practice , while the aforementioned approach is still able to reduce visible truncation artifacts under these circumstances , it does not reduce image noise because the measured k - space data remains unchanged . on the other hand , an additional denoising may be achieved by loosing the fixed bound on the measured data , that is by introducing a data fitting term . in this case , the algorithm not only adds synthetic frequencies to obtain a tv minimization , but is also allowed to find a solution that slightly diverges from the measured data , which yields an effective edge - preserving denoising . therefore , the estimate vector v has to be extended such that it contains both synthesized frequencies as well as frequencies from the measured part of k - space , which is indicated by writing vd instead . in the denoising case , the cost function takes the form(6)(vd ) = vdy22 + tv({vd } ) , where denotes an operation that calculates the residual between the measured values and the corresponding entries of the estimate , which are now contained in the vector vd . while a low weight permits considerable divergences from the measured values and , thus , leads to an effective removal of noise , it can also cause a loss of object detail if selected too low . as a consequence , spatially varying transitions between the real and imaginary component occur as well as local intensity changes , which conflict with the assumption of a piecewise - constant quantity and prevent a direct application of the tv constraint . in this proof - of - principle study , phase variations were removed in a preprocessing step by performing a fourier transformation of the data from each coil and calculating the sum - of - squares of all channels in the image domain . subsequently , an inverse fourier transformation of the sum - of - squares data was performed to obtain a combined data set with real - valued and nonnegative values in the image domain , which enables a calculation of the tv value using only the real part of the image . while this simple technique turned out to be sufficient for demonstrating a removal of truncation artifacts by tv - constrained data extrapolation , routine applications will probably require a more sophisticated procedure , in particular when combined with advanced techniques such as parallel imaging and when using complex coil configurations with more localized sensitivities of the individual receiver elements . because the fourier transform of a single ellipse is given by a bessel function , an analytical fourier transform of the phantom is obtained by a superposition of respective bessel functions . truncation artifacts can be studied by evaluating the noncompact analytical transform at the sampling positions along the trajectory , here yielding a matrix of 96 96 fourier samples . all simulations and processing of experimental data were done offline using an in - house software package written in c / c++ . mri experiments were conducted at 2.9 t ( siemens magnetom tim trio , erlangen , germany ) with use of a receive only 12-channel head coil equipped with hardware signal combiners , yielding four receiver channels with different combinations of the coil elements . measurements were performed for a water phantom and human brain in vivo , where written informed consent was obtained from all subjects prior to each examination . for demonstration purposes , the image acquisitions were done with a simple slice - selective spin - echo sequence at a 200 200 mm field of view , covered by a 96 96 acquisition matrix . different sequence settings were used to obtain data sets with low and high level of noise , where the latter was achieved by reducing the flip angle and slice thickness while increasing the receiver bandwidth . further , one data set was acquired with a slice - selective gradient - echo sequence , which allowed for the rapid measurement of a full 288 288 acquisition matrix . the proposed algorithm was run for a fixed number of 3000 iterations , which takes about 2 - 3 minutes on a standard microprocessor . here , a simple lanczos sigma filter , that is , multiplication with a sinc - function , was applied , where the window width was selected such that the sinc - function 's first null coincides with the border of the measured k - space . in addition , a two - dimensional version of the extrapolation method described by constable and henkelman was implemented with a window width of p = 2 for the edge - preserving sigma filter . it is clearly visible that the zero - padded solution ( zero ) suffers from severe ringing artifacts around all edges of the phantom . the extent of the measured k - space can be seen in its fourier transform . most ringing artifacts disappear after filtering ( filter ) , however , at the expense of a significant loss of image resolution . its fourier transform reveals that the measured data has been properly extrapolated into the uncovered areas of k - space . for comparison , a full reconstruction from 288 288 samples is shown in the bottom row ( full ) . figure 3 demonstrates the application of the method to experimental data obtained for a phantom ( left column ) and a human brain in vivo ( right column ) in comparison to zero - padded ( zero ) and filtered zero - padded solutions ( filter ) . in figure 4 , the proposed approach ( tv2 ) is compared to a 2d version of the extrapolation method by constable and henkelman ( comp ) for the shepp - logan phantom ( left column ) and an experimental study of the human brain in vivo ( right column ) . it can be seen that the performance of the proposed extrapolation method is slightly better for the simulated data , while both approaches yield an effective suppression of ringing artifacts for the experimental data ( note that the alternative method leads to a slight denoising of the image ) . while the basic extrapolation approach leads to a reduction of truncation artifacts also for noisy data , it does not reduce the noise patterns . however , when extending the tv penalty to the measured data , the method effectively flattens noise patterns in addition to the suppression of ringing artifacts . finally , corresponding reconstructions from experimental data with a high degree of noise are shown in figure 6 . here , a combination of first- and second - order derivatives was used for the tv calculation . as in the simulations , the proposed method leads to a reduction of truncation artifacts ( tv2 ) , while the extension to data fitting yields an additional edge - preserving denoising ( tv2dns ) . both simulations and experiments demonstrate that tv - constrained data extrapolation effectively reduces truncation artifacts due to finitely sampled mri acquisitions . however , it should be noted that the smoothness observed for zero - padding originates to a significant degree from the convolution with the sinc - function . as a consequence , a sharp edge of the object is mapped as a rather smooth pattern , which might appear more familiar to the viewer than a blocky image , but strictly represents an image artifact . hence , the extrapolation technique may even lead to a slight gain of resolution due to a sharpening of the point - spread function , following from the reciprocity property of the fourier transformation . first , the method is based on the assumption that the true object is piecewise constant , which is only approximately valid for real - world objects . in the presence of additional experimental effects like flow artifacts hence , the extrapolation performance depends on the object 's conformance with the assumption that it is piecewise - constant . on the other hand , in the majority of cases , the assumption of a piecewise - constant object seems to be more appropriate than that of all conventional reconstructions , namely , a fourier transform of the object that is zero outside the sampled k - space area . second , the proposed method synthesizes only a finite number of additional frequencies , whereas an infinite number of k - space samples would be required to completely eliminate all truncation effects . the reason is that the method yields an implicit filtering of the extrapolated data : assuming that the extrapolation procedure would recover the unmeasured k - space samples exactly , then a new truncation effect would arise at the extended border and again lead to ringing artifacts in image space ( though with a higher oscillation frequency ) . because this would increment the tv value , the method automatically lowers outer frequencies during the extrapolation procedure to prevent the upcoming of new ringing artifacts . third , if a completely artifact - free reconstruction of the object would be available , then respective frequency samples could be calculated with a discrete fourier transformation of the given image . consequently , an artifact - free discrete reconstruction can only be obtained if the samples used for the reconstruction specifically diverge from the measured frequencies . the modulus function in the tv formula ( 2 ) has a fundamental role for the success of tv - based image processing . because subtraction of neighboring pixels performed before taking the modulus can be seen as applying a difference operator to the estimate , tv minimization yields a solution with minimum l1-norm in the difference basis . due to the specific character of the modulus function , this solution tends to be sparse in the difference basis : it has few large jumps and most differences between neighboring pixels are near zero , which directly translates into a piecewise - constant image ( and explains the edge preserving character of tv - based denoising ) . this corresponds to a globally smooth image , which is usually undesired due to a loss of sharp edges . one major reason is that the derivative of the modulus function is just 1 , which does not help to guess a reasonable step size toward the function 's minimum . however , it turned out that the cg - descent algorithm is capable to handle the problem as it comprises a powerful line - search procedure , but it is probably not the optimal method for finding the solution . in order to ensure convergence , it was , therefore , necessary to introduce a scaling factor that limits the modification strength for each iteration and to run the algorithm in turn for a high number of iterations ( e.g. the present work demonstrates that the simple assumption of a piecewise - constant object can be exploited to extrapolate measured data in k - space . this allows for a significant reduction of ringing artifacts that arise from data truncation in k - space . in contrast to commonly used filtering approaches , the method does not degrade the spatial resolution of the reconstructed image and rather leads to a mild resolution enhancement due to sharpening of the point - spread function . if the measured data is seriously contaminated by noise , an extended approach offers edge - preserving denoising by slightly altering the measured data in addition to supplementing synthetic data . both variants can be implemented as a pure postprocessing procedure and are also applicable for partial fourier acquisitions . while the current implementation suffers from a relatively high computational load , the use of a dedicated tv optimization algorithm promises a processing speed suitable for routine applications .

cd4 t cells reactive against insulin were identified in islets and the peri - pancreatic lymph nodes of pre - diabetic / early diabetic mice and t cell hybridomas were generated . forty - two t cell hybridomas ( 7% of total ) from 6 different fusions , were reactive to insulin and/or to the b:9 - 23 peptide ( 617 hybridomas were screened of which 32 were further characterized ) . a number of t cell hybridomas , the conventional type a , responded to insulin as well as to the b:9 - 23 peptide . several t cells hybridomas , the typeb , only recognized the peptide b:9 - 23 but not the insulin molecule ( fig . the distribution of type a and type b t cells was equal : among the 32 insulin - reactive cd4 t cells which were characterized , 16 responded to peptide but not to insulin ( type b ) , and an equal number , 16 , responded to both ( type a ) . however , most of type a t cells reacted weakly ( in the low m range ) with either insulin or the peptide . type a t cells had a 10100-fold lower affinity to insulin than to the b:9 - 23 peptide , while the remaining reacted equally to both ( fig . type b t cell subsets were found in the peri - pancreatic node as well as within the infiltrated islets . these results imply that type b t cells were specifically recruited to the islets together with weakly reactive type a t cells in the early disease process . to examine in detail the immune response to insulin or to the b:9 - 23 peptide , mice were immunized with one or the other and the incidence of type a and type b insulin reactive cd4 t cells were examined . when nod mice were immunized with insulin , few , if any , insulin- specific t cells were detected in the draining lymph nodes ( fig . in contrast , a prominent cd4 t cell response was found upon immunization with the b:9 - 23 peptide . the response was entirely blocked by an anti - i - a antibody ( fig . these t cells were peptide specific , because they responded only to b:9 - 23 peptide , but not to the insulin protein . of 351 t cell hybridomas generated from the draining lymph nodes of the immunized mice , the majority ( 74% ) were peptide specific , while only a small number ( 1.7% ) recognized both peptide and the insulin molecule ( fig . the remaining hybridomas were either non - responsive ( 17.7% ) or autoreactive ( 6.6% ) . the type b t cells also recognized denatured insulin , indicating that they were not recognizing an artificial determinant arising from the synthetic peptide ( supplementary fig . we next compared the insulin - reactive t cells from nod wild - type and nod mice with genetic deletions of both insulin genes but expressing a transgene encoding a mutant proinsulin7 . the mutant transgene encodes an insulin molecule containing a single tyrosine to alanine amino acid substitution at residue16 of the insulin beta - chain , referred to as b16:a - dko . in the absence of native insulin genes , the transgenic mutant insulin restored biological activity , however , the substitution abrogated recognition by insulin - specific t cells7 . unlike nod mice , b16:a - dko mice immunized with insulin generated a t cell response specific to insulin protein ( fig . immunization of b16:a - dko mice with the b:9 - 23 peptide generated a peptide response and a weak response to insulin ( fig . type a hybridomas generated from b16:a - dko mice immunized with insulin displayed a much greater sensitivity for insulin compared to hybridomas derived from nod mice ( fig . this indicates that in the nod mouse the high affinity t cells to insulin are normally deleted . t cells found in peripheral tissue are less reactive , reflecting that they escaped negative selection and bear the stamp of tolerance . the explanation for the different recognition of the b:9 - 23 peptide - i - a complex between the two sets of t cells is presently unclear . initial studies indicate that in contrast to the hel system , h2-dm did not affect the development of type a or type b peptide - mhc ( pmhc ) complexes ( data not shown ) . we have searched extensively for changes in the peptide or for post - translational modifications that may account for recognition by one or the other set of t cells and have failed to identify any ( data not shown ) . both type a and type b cd4 t cells reacted to purified i - a molecules incubated with the identical b:9 - 23 peptide ( supplementary fig . the differential recognition of the b:9 - 23 peptide by the two t cells types most likely reflects different conformational states of the pmhc complex . in support of this interpretation , type a and type b t cells recognized differently a covalently - linked pmhc complex . m12.c3 cells engineered to express i - a with the b:9 - 23 peptide covalently tethered15 activated the type a t cells , including those that reacted weakly with insulin , but not the type b t cells ( fig . 1j and supplementary table 1 ) . thus , the covalently - linked complex was presented in a state or a conformation that allowed the tcr of type a t cells to recognize it , but not the tcr of type b t cells . it is probable that the intracellular pathway by which insulin reaches the class ii molecule and the site within the apc are the two critical factors in generating a unique complex that t cells recognize . to determine if type b insulin t cells are diabetogenic , eleven primary t cell lines were generated from the draining lymph nodes of b:9 - 23 immunized nod mice , expanded and adoptively transferred into nod.scid recipients . type b , meaning they recognized the b:9 - 23 peptide , but not the insulin molecule ( supplementary fig . none of them recognized the covalently linked pmhc complex on m12.c3.g79 - 23 cells ( data not shown ) . all t cell lines induced extensive insulitis , with 7 of 11 lines also inducing diabetes ( fig . a number of these lines also accelerated diabetes in young nod mice ( supplementary fig . 3a d ) . the extent of disease transfer varied among the clones and was irrespective of their cytokine profiles ( fig . thus , the peptide reactive , type b t cells could damage the islets and induce the same histopathology as other diabetogenic t cells , suggesting that they participate in the autoimmune process . in order to address where insulin peptide presentation leading to type b t cells activation occurs , antigen presenting cells ( apc ) from various tissues were examined . we could not detect antigen presentation to t cells by apcs from spleen , inguinal or peri - pancreatic lymph nodes from pre - diabetic mice , suggesting that circulating insulin was not the source of peptide . in addition , neither dendritic cells ( dc ) nor epithelial cells isolated from thymi activated type a hybridomas , indicating that presentation in the thymus is quite low . of all tissues examined the only apcs that presented insulin peptides to either the islets of langerhans harbor an average of about 10 dc per islet , most of which are constitutively expressing pmhc complexes derived from beta cell proteins , irrespective of the diabetic status , islet inflammation or beta cell death28 . whole islets cell fractions containing dc , or purified intra - islet dc from nod.rag1 mice constitutively presented antigen to both type a and type b t cells in the absence of inflammation or overt cellular death ( fig . furthermore , when nit-1 insulinoma cells , which do not express mhc class ii molecules , were incubated with purified splenic dc , uptake of insulin granules by dc was detected by electron microscopy ( fig . these dc were capable of activating both the type a and type b insulin - reactive t cells ( fig . 3e , f ) , showing that both peptide - mhc conformers are presented by apc within close contact to beta cells . the secretory granules of beta cells can constitute a source of the b:9 - 23 peptide , due to the fact each granule is comprised of about 10 insulin molecules , as well as catabolic products from its processing29 . purified secretory granules from primary beta cells incubated with splenic dc led to robust activation of both whether these granules contain insulin peptides , we generated a monoclonal antibody that specifically binds to the b:9 - 23 peptide but not to insulin or proinsulin molecules ( supplementary fig . specific granule staining was observed in whole islets or purified beta cells from nod or nod.rag1 . 4b , e ) , but not by an excess of insulin or proinsulin ( supplementary fig . 4 ) . cells containing b:9 - 23 positive granules represented a subset of total beta cells . the majority of granules inside beta cells were positive for insulin but negative for b:9 - 23 and did not contain lamp-1 ( fig . in contrast , all vesicles containing the b:9 - 23 peptide in beta cells were positive for lamp-1 , and only very few of these vesicles ( less that 10% ) , were also positive for insulin ( supplementary fig . confocal microscopy showed that the majority of dc in the examined islets had taken up b:9 - 23 positive and/or insulin granules ( fig . screening of purified islet dc showed that about 80% of them contained insulin positive granules with an average of 7 granules per dc . many of these dc stained positive for both insulin containing vesicles and b:9 - 23 containing vesicles . granules within islet dc co - localized with lamp-1 and i - a ( fig . 4 g ) , consistent with trafficking to cellular compartments involved in antigen processing and presentation . these observations suggest that the b:9 - 23 peptides presented by i - a could be derived from beta cell vesicles containing peptides of the insulin -chain and/or from the processing of insulin granules within dc . evidence for granules within intra - islet dc under physiological conditions was presented previously by electron microscopy26 . in addition , granules isolated from nit-1 cells contained 8.8 moles of anti - insulin - reactive material and 12 moles of anti-9 - 23 reactive material . initial mass spectrometry studies on purified secretory granules showed they contained c - peptide ( data not shown ) , but also peptides from the b:9 - 23 segment , which included peptides centered on the 12 - 21 residues ( vealylvgge ) of the insulin -chain ( supplementary fig . these observations suggest that peptides immunogenic for type b t cells are produced during the physiological processing of mature insulin molecules , and are not dependent on any prior inflammation . cd4 t cells reactive against insulin were identified in islets and the peri - pancreatic lymph nodes of pre - diabetic / early diabetic mice and t cell hybridomas were generated . forty - two t cell hybridomas ( 7% of total ) from 6 different fusions , were reactive to insulin and/or to the b:9 - 23 peptide ( 617 hybridomas were screened of which 32 were further characterized ) . a number of t cell hybridomas , the conventional type a , responded to insulin as well as to the b:9 - 23 peptide . several t cells hybridomas , the typeb , only recognized the peptide b:9 - 23 but not the insulin molecule ( fig . the distribution of type a and type b t cells was equal : among the 32 insulin - reactive cd4 t cells which were characterized , 16 responded to peptide but not to insulin ( type b ) , and an equal number , 16 , responded to both ( type a ) . however , most of type a t cells reacted weakly ( in the low m range ) with either insulin or the peptide . type a t cells had a 10100-fold lower affinity to insulin than to the b:9 - 23 peptide , while the remaining reacted equally to both ( fig . type b t cell subsets were found in the peri - pancreatic node as well as within the infiltrated islets . these results imply that type b t cells were specifically recruited to the islets together with weakly reactive to examine in detail the immune response to insulin or to the b:9 - 23 peptide , mice were immunized with one or the other and the incidence of type a and type b insulin reactive cd4 t cells were examined . when nod mice were immunized with insulin , few , if any , insulin- specific t cells were detected in the draining lymph nodes ( fig . in contrast , a prominent cd4 t cell response was found upon immunization with the b:9 - 23 peptide . the response was entirely blocked by an anti - i - a antibody ( fig . these t cells were peptide specific , because they responded only to b:9 - 23 peptide , but not to the insulin protein . of 351 t cell hybridomas generated from the draining lymph nodes of the immunized mice , the majority ( 74% ) were peptide specific , while only a small number ( 1.7% ) recognized both peptide and the insulin molecule ( fig . the remaining hybridomas were either non - responsive ( 17.7% ) or autoreactive ( 6.6% ) . type b t cells also recognized denatured insulin , indicating that they were not recognizing an artificial determinant arising from the synthetic peptide ( supplementary fig . we next compared the insulin - reactive t cells from nod wild - type and nod mice with genetic deletions of both insulin genes but expressing a transgene encoding a mutant proinsulin7 . the mutant transgene encodes an insulin molecule containing a single tyrosine to alanine amino acid substitution at residue16 of the insulin beta - chain , referred to as b16:a - dko . in the absence of native insulin genes , the transgenic mutant insulin restored biological activity , however , the substitution abrogated recognition by insulin - specific t cells7 . unlike nod mice , b16:a - dko mice immunized with insulin generated a t cell response specific to insulin protein ( fig . immunization of b16:a - dko mice with the b:9 - 23 peptide generated a peptide response and a weak response to insulin ( fig . type a hybridomas generated from b16:a - dko mice immunized with insulin displayed a much greater sensitivity for insulin compared to hybridomas derived from nod mice ( fig . this indicates that in the nod mouse the high affinity t cells to insulin are normally deleted . t cells found in peripheral tissue are less reactive , reflecting that they escaped negative selection and bear the stamp of tolerance . the explanation for the different recognition of the b:9 - 23 peptide - i - a complex between the two sets of t cells is presently unclear . initial studies indicate that in contrast to the hel system , h2-dm did not affect the development of type a or type b peptide - mhc ( pmhc ) complexes ( data not shown ) . we have searched extensively for changes in the peptide or for post - translational modifications that may account for recognition by one or the other set of t cells and have failed to identify any ( data not shown ) . both type a and type b cd4 t cells reacted to purified i - a molecules incubated with the identical b:9 - 23 peptide ( supplementary fig . the differential recognition of the b:9 - 23 peptide by the two t cells types most likely reflects different conformational states of the pmhc complex . in support of this interpretation , type a and type b t cells recognized differently a covalently - linked pmhc complex . m12.c3 cells engineered to express i - a with the b:9 - 23 peptide covalently tethered15 activated the type a t cells , including those that reacted weakly with insulin , but not the type b t cells ( fig . thus , the covalently - linked complex was presented in a state or a conformation that allowed the tcr of type a t cells to recognize it , but not the tcr of type b t cells . it is probable that the intracellular pathway by which insulin reaches the class ii molecule and the site within the apc are the two critical factors in generating a unique complex that t cells recognize . to determine if type b insulin t cells are diabetogenic , eleven primary t cell lines were generated from the draining lymph nodes of b:9 - 23 immunized nod mice , expanded and adoptively transferred into nod.scid recipients . type b , meaning they recognized the b:9 - 23 peptide , but not the insulin molecule ( supplementary fig . none of them recognized the covalently linked pmhc complex on m12.c3.g79 - 23 cells ( data not shown ) . all t cell lines induced extensive insulitis , with 7 of 11 lines also inducing diabetes ( fig . a number of these lines also accelerated diabetes in young nod mice ( supplementary fig . d ) . the extent of disease transfer varied among the clones and was irrespective of their cytokine profiles ( fig . thus , the peptide reactive , type b t cells could damage the islets and induce the same histopathology as other diabetogenic t cells , suggesting that they participate in the autoimmune process . in order to address where insulin peptide presentation leading to type b t cells activation occurs , antigen presenting cells ( apc ) from various tissues were examined . we could not detect antigen presentation to t cells by apcs from spleen , inguinal or peri - pancreatic lymph nodes from pre - diabetic mice , suggesting that circulating insulin was not the source of peptide . in addition , neither dendritic cells ( dc ) nor epithelial cells isolated from thymi activated type a hybridomas , indicating that presentation in the thymus is quite low . of all tissues examined the only apcs that presented insulin peptides to either type a or the islets of langerhans harbor an average of about 10 dc per islet , most of which are constitutively expressing pmhc complexes derived from beta cell proteins , irrespective of the diabetic status , islet inflammation or beta cell death28 . whole islets cell fractions containing dc , or purified intra - islet dc from nod.rag1 mice constitutively presented antigen to both type a and type b t cells in the absence of inflammation or overt cellular death ( fig . furthermore , when nit-1 insulinoma cells , which do not express mhc class ii molecules , were incubated with purified splenic dc , uptake of insulin granules by dc was detected by electron microscopy ( fig . these dc were capable of activating both the type a and type b insulin - reactive t cells ( fig . 3e , f ) , showing that both peptide - mhc conformers are presented by apc within close contact to beta cells . the secretory granules of beta cells can constitute a source of the b:9 - 23 peptide , due to the fact each granule is comprised of about 10 insulin molecules , as well as catabolic products from its processing29 . purified secretory granules from primary beta cells incubated with splenic dc led to robust activation of both whether these granules contain insulin peptides , we generated a monoclonal antibody that specifically binds to the b:9 - 23 peptide but not to insulin or proinsulin molecules ( supplementary fig . specific granule staining was observed in whole islets or purified beta cells from nod or nod.rag1 . 4b , e ) , but not by an excess of insulin or proinsulin ( supplementary fig . the majority of granules inside beta cells were positive for insulin but negative for b:9 - 23 and did not contain lamp-1 ( fig . in contrast , all vesicles containing the b:9 - 23 peptide in beta cells were positive for lamp-1 , and only very few of these vesicles ( less that 10% ) , were also positive for insulin ( supplementary fig . confocal microscopy showed that the majority of dc in the examined islets had taken up b:9 - 23 positive and/or insulin granules ( fig . screening of purified islet dc showed that about 80% of them contained insulin positive granules with an average of 7 granules per dc . many of these dc stained positive for both insulin containing vesicles and b:9 - 23 containing vesicles . granules within islet dc co - localized with lamp-1 and i - a ( fig . 4 g ) , consistent with trafficking to cellular compartments involved in antigen processing and presentation . these observations suggest that the b:9 - 23 peptides presented by i - a could be derived from beta cell vesicles containing peptides of the insulin -chain and/or from the processing of insulin granules within dc . evidence for granules within intra - islet dc under physiological conditions was presented previously by electron microscopy26 . in addition , granules isolated from nit-1 cells contained 8.8 moles of anti - insulin - reactive material and 12 moles of anti-9 - 23 reactive material . initial mass spectrometry studies on purified secretory granules showed they contained c - peptide ( data not shown ) , but also peptides from the b:9 - 23 segment , which included peptides centered on the 12 - 21 residues ( vealylvgge ) of the insulin -chain ( supplementary fig . 6 ) . these observations suggest that peptides immunogenic for type b t cells are produced during the physiological processing of mature insulin molecules , and are not dependent on any prior inflammation . one set had the unique feature of being specific to exogenous insulin peptide , but unreactive to insulin . the peptide that activated these type b t cells was normally derived from the insulin secretory granule and was loaded on mhc class ii in the intra - islet dc . these t cells were capable of transferring diabetes and were found spontaneously in infiltrated islets , indicating their participation in the autoimmune process . the two other sets of t cells were insulin and peptide - reactive t cells , but differed in their sensitivity to insulin . type a t cells , identified in the nod or in the b16:a - dko mice , respectively . in the b16:a - dko mice type a t cells were readily found after insulin immunization and showed a higher response to the insulin molecule or the b:9 - 23 peptide compared to the strong insulin reactivity found in b16:a - dko mice supports the conclusion reached by others912 that native insulin expression in the thymus influences the selection of insulin - reactive t cells , and that partial tolerance or unresponsiveness to the insulin molecule exists in nod mice . thus , the few insulin reactive t cells found in nod mice had an imprint of tolerance , with weak reactivity to insulin , which might explain why they escaped negative selection . the frequency of type b t cells is markedly greater than type a t cells in the precursor population of nod mice . however , because both type a and type b t cells were isolated from the islets , we can not conclude which set is the major participant , or if they act synergistically in disease development . more than one factor can influence the diabetogenicity of transferred t cell cells , such as time in culture and expression of adhesion molecules30 . due to this variability , strict comparisons between the diabetogenicity of type a and type b t cells were not done systematically . the b:9 - 23 peptide could be generated following dc handling of insulin upon capture of the insulin granules or from uptake of the granules that already contained the peptide . the finding that islet dc contain insulin granules and b:9 - 23 peptide , and the observation that feeding dc with purified secretory granules results in strong presentation explains another conundrum concerning insulin presentation . there is a relatively poor yield of peptide selected by mhc molecules after protein processing31 . furthermore , due to the low binding affinity of the b:9 - 23 peptide for i - a and the rapid dissociation of the peptide from i - a ( ref 18 ) , a high concentration of insulin within the apc is required for effective presentation to t cells . the concentration of insulin in blood and tissue is in the low m level . in antigen presentation assays , the concentration of insulin or b:9 - 23 peptide required for presentation is in the low m level which is unphysiological in the whole mouse . if all these aspects are taken into consideration , it is most likely that the islet dc have two essential roles : to migrate into the pancreatic draining lymph node to initiate the sensitization process32 ; and to present antigen to sensitized t cells in the effector stage of diabetogenesis . collectively , these results suggest that self - reactive type b t cells and low affinity type a t cells escape negative selection and give rise to pathogenic t cells in autoimmune diabetes . a situation akin to insulin autoimmunity may also be found in other endocrine autoimmune diseases such as in autoimmune thyroiditis in which dc are found surrounding the follicles , some of which have class ii - positive epithelial cells33,34 .

besides the genetic framework , there are two critical requirements for the development of tissue - specific autoimmune diseases . first , autoreactive t cells need to escape thymic negative selection . second , they need to find suitable conditions for autoantigen presentation and activation in the target tissue . we show here that these two conditions are fulfilled in diabetic nod mice . a set of autoreactive cd4 + t cells specific for an insulin peptide , with the noteworthy feature of not recognizing the insulin protein when processed by the antigen presenting cells ( apc ) escape thymic control , participate in diabetes and can cause disease . we also find that apcs situated in close contact with the beta cells in the islets of langerhans bear vesicles with the antigenic insulin peptides and activate the peptide - specific t cells . these findings may be relevant for other cases of endocrine autoimmunity .

RESULTS Insulin-reactive CD4 Mice respond to immunization with B:9-23 peptide Differential recognition of covalent Peptide-MHC complexes Type B T cells cause diabetes Presentation of B:9-23 by islet DC DISCUSSION Supplementary Material

several t cells hybridomas , the typeb , only recognized the peptide b:9 - 23 but not the insulin molecule ( fig . the distribution of type a and type b t cells was equal : among the 32 insulin - reactive cd4 t cells which were characterized , 16 responded to peptide but not to insulin ( type b ) , and an equal number , 16 , responded to both ( type a ) . however , most of type a t cells reacted weakly ( in the low m range ) with either insulin or the peptide . type a t cells had a 10100-fold lower affinity to insulin than to the b:9 - 23 peptide , while the remaining reacted equally to both ( fig . these results imply that type b t cells were specifically recruited to the islets together with weakly reactive type a t cells in the early disease process . to examine in detail the immune response to insulin or to the b:9 - 23 peptide , mice were immunized with one or the other and the incidence of type a and type b insulin reactive cd4 t cells were examined . when nod mice were immunized with insulin , few , if any , insulin- specific t cells were detected in the draining lymph nodes ( fig . in contrast , a prominent cd4 t cell response was found upon immunization with the b:9 - 23 peptide . these t cells were peptide specific , because they responded only to b:9 - 23 peptide , but not to the insulin protein . of 351 t cell hybridomas generated from the draining lymph nodes of the immunized mice , the majority ( 74% ) were peptide specific , while only a small number ( 1.7% ) recognized both peptide and the insulin molecule ( fig . the type b t cells also recognized denatured insulin , indicating that they were not recognizing an artificial determinant arising from the synthetic peptide ( supplementary fig . we next compared the insulin - reactive t cells from nod wild - type and nod mice with genetic deletions of both insulin genes but expressing a transgene encoding a mutant proinsulin7 . the mutant transgene encodes an insulin molecule containing a single tyrosine to alanine amino acid substitution at residue16 of the insulin beta - chain , referred to as b16:a - dko . in the absence of native insulin genes , the transgenic mutant insulin restored biological activity , however , the substitution abrogated recognition by insulin - specific t cells7 . unlike nod mice , b16:a - dko mice immunized with insulin generated a t cell response specific to insulin protein ( fig . immunization of b16:a - dko mice with the b:9 - 23 peptide generated a peptide response and a weak response to insulin ( fig . this indicates that in the nod mouse the high affinity t cells to insulin are normally deleted . t cells found in peripheral tissue are less reactive , reflecting that they escaped negative selection and bear the stamp of tolerance . the explanation for the different recognition of the b:9 - 23 peptide - i - a complex between the two sets of t cells is presently unclear . initial studies indicate that in contrast to the hel system , h2-dm did not affect the development of type a or type b peptide - mhc ( pmhc ) complexes ( data not shown ) . we have searched extensively for changes in the peptide or for post - translational modifications that may account for recognition by one or the other set of t cells and have failed to identify any ( data not shown ) . both type a and type b cd4 t cells reacted to purified i - a molecules incubated with the identical b:9 - 23 peptide ( supplementary fig . the differential recognition of the b:9 - 23 peptide by the two t cells types most likely reflects different conformational states of the pmhc complex . in support of this interpretation , type a and type b t cells recognized differently a covalently - linked pmhc complex . m12.c3 cells engineered to express i - a with the b:9 - 23 peptide covalently tethered15 activated the type a t cells , including those that reacted weakly with insulin , but not the type b t cells ( fig . thus , the covalently - linked complex was presented in a state or a conformation that allowed the tcr of type a t cells to recognize it , but not the tcr of type b t cells . it is probable that the intracellular pathway by which insulin reaches the class ii molecule and the site within the apc are the two critical factors in generating a unique complex that t cells recognize . to determine if type b insulin t cells are diabetogenic , eleven primary t cell lines were generated from the draining lymph nodes of b:9 - 23 immunized nod mice , expanded and adoptively transferred into nod.scid recipients . type b , meaning they recognized the b:9 - 23 peptide , but not the insulin molecule ( supplementary fig . none of them recognized the covalently linked pmhc complex on m12.c3.g79 - 23 cells ( data not shown ) . all t cell lines induced extensive insulitis , with 7 of 11 lines also inducing diabetes ( fig . a number of these lines also accelerated diabetes in young nod mice ( supplementary fig . thus , the peptide reactive , type b t cells could damage the islets and induce the same histopathology as other diabetogenic t cells , suggesting that they participate in the autoimmune process . in order to address where insulin peptide presentation leading to type b t cells activation occurs , antigen presenting cells ( apc ) from various tissues were examined . we could not detect antigen presentation to t cells by apcs from spleen , inguinal or peri - pancreatic lymph nodes from pre - diabetic mice , suggesting that circulating insulin was not the source of peptide . in addition , neither dendritic cells ( dc ) nor epithelial cells isolated from thymi activated type a hybridomas , indicating that presentation in the thymus is quite low . of all tissues examined the only apcs that presented insulin peptides to either the islets of langerhans harbor an average of about 10 dc per islet , most of which are constitutively expressing pmhc complexes derived from beta cell proteins , irrespective of the diabetic status , islet inflammation or beta cell death28 . whole islets cell fractions containing dc , or purified intra - islet dc from nod.rag1 mice constitutively presented antigen to both type a and type b t cells in the absence of inflammation or overt cellular death ( fig . these dc were capable of activating both the type a and type b insulin - reactive t cells ( fig . 3e , f ) , showing that both peptide - mhc conformers are presented by apc within close contact to beta cells . the secretory granules of beta cells can constitute a source of the b:9 - 23 peptide , due to the fact each granule is comprised of about 10 insulin molecules , as well as catabolic products from its processing29 . purified secretory granules from primary beta cells incubated with splenic dc led to robust activation of both whether these granules contain insulin peptides , we generated a monoclonal antibody that specifically binds to the b:9 - 23 peptide but not to insulin or proinsulin molecules ( supplementary fig . specific granule staining was observed in whole islets or purified beta cells from nod or nod.rag1 . the majority of granules inside beta cells were positive for insulin but negative for b:9 - 23 and did not contain lamp-1 ( fig . in contrast , all vesicles containing the b:9 - 23 peptide in beta cells were positive for lamp-1 , and only very few of these vesicles ( less that 10% ) , were also positive for insulin ( supplementary fig . these observations suggest that the b:9 - 23 peptides presented by i - a could be derived from beta cell vesicles containing peptides of the insulin -chain and/or from the processing of insulin granules within dc . these observations suggest that peptides immunogenic for type b t cells are produced during the physiological processing of mature insulin molecules , and are not dependent on any prior inflammation . several t cells hybridomas , the typeb , only recognized the peptide b:9 - 23 but not the insulin molecule ( fig . however , most of type a t cells reacted weakly ( in the low m range ) with either insulin or the peptide . type a t cells had a 10100-fold lower affinity to insulin than to the b:9 - 23 peptide , while the remaining reacted equally to both ( fig . these results imply that type b t cells were specifically recruited to the islets together with weakly reactive to examine in detail the immune response to insulin or to the b:9 - 23 peptide , mice were immunized with one or the other and the incidence of type a and type b insulin reactive cd4 t cells were examined . when nod mice were immunized with insulin , few , if any , insulin- specific t cells were detected in the draining lymph nodes ( fig . in contrast , a prominent cd4 t cell response was found upon immunization with the b:9 - 23 peptide . these t cells were peptide specific , because they responded only to b:9 - 23 peptide , but not to the insulin protein . of 351 t cell hybridomas generated from the draining lymph nodes of the immunized mice , the majority ( 74% ) were peptide specific , while only a small number ( 1.7% ) recognized both peptide and the insulin molecule ( fig . type b t cells also recognized denatured insulin , indicating that they were not recognizing an artificial determinant arising from the synthetic peptide ( supplementary fig . we next compared the insulin - reactive t cells from nod wild - type and nod mice with genetic deletions of both insulin genes but expressing a transgene encoding a mutant proinsulin7 . the mutant transgene encodes an insulin molecule containing a single tyrosine to alanine amino acid substitution at residue16 of the insulin beta - chain , referred to as b16:a - dko . in the absence of native insulin genes , the transgenic mutant insulin restored biological activity , however , the substitution abrogated recognition by insulin - specific t cells7 . unlike nod mice , b16:a - dko mice immunized with insulin generated a t cell response specific to insulin protein ( fig . immunization of b16:a - dko mice with the b:9 - 23 peptide generated a peptide response and a weak response to insulin ( fig . type a hybridomas generated from b16:a - dko mice immunized with insulin displayed a much greater sensitivity for insulin compared to hybridomas derived from nod mice ( fig . this indicates that in the nod mouse the high affinity t cells to insulin are normally deleted . t cells found in peripheral tissue are less reactive , reflecting that they escaped negative selection and bear the stamp of tolerance . the explanation for the different recognition of the b:9 - 23 peptide - i - a complex between the two sets of t cells is presently unclear . initial studies indicate that in contrast to the hel system , h2-dm did not affect the development of type a or type b peptide - mhc ( pmhc ) complexes ( data not shown ) . we have searched extensively for changes in the peptide or for post - translational modifications that may account for recognition by one or the other set of t cells and have failed to identify any ( data not shown ) . both type a and type b cd4 t cells reacted to purified i - a molecules incubated with the identical b:9 - 23 peptide ( supplementary fig . the differential recognition of the b:9 - 23 peptide by the two t cells types most likely reflects different conformational states of the pmhc complex . in support of this interpretation , type a and type b t cells recognized differently a covalently - linked pmhc complex . m12.c3 cells engineered to express i - a with the b:9 - 23 peptide covalently tethered15 activated the type a t cells , including those that reacted weakly with insulin , but not the type b t cells ( fig . thus , the covalently - linked complex was presented in a state or a conformation that allowed the tcr of type a t cells to recognize it , but not the tcr of type b t cells . it is probable that the intracellular pathway by which insulin reaches the class ii molecule and the site within the apc are the two critical factors in generating a unique complex that t cells recognize . to determine if type b insulin t cells are diabetogenic , eleven primary t cell lines were generated from the draining lymph nodes of b:9 - 23 immunized nod mice , expanded and adoptively transferred into nod.scid recipients . type b , meaning they recognized the b:9 - 23 peptide , but not the insulin molecule ( supplementary fig . none of them recognized the covalently linked pmhc complex on m12.c3.g79 - 23 cells ( data not shown ) . all t cell lines induced extensive insulitis , with 7 of 11 lines also inducing diabetes ( fig . thus , the peptide reactive , type b t cells could damage the islets and induce the same histopathology as other diabetogenic t cells , suggesting that they participate in the autoimmune process . in order to address where insulin peptide presentation leading to type b t cells activation occurs , antigen presenting cells ( apc ) from various tissues were examined . in addition , neither dendritic cells ( dc ) nor epithelial cells isolated from thymi activated type a hybridomas , indicating that presentation in the thymus is quite low . of all tissues examined the only apcs that presented insulin peptides to either type a or the islets of langerhans harbor an average of about 10 dc per islet , most of which are constitutively expressing pmhc complexes derived from beta cell proteins , irrespective of the diabetic status , islet inflammation or beta cell death28 . whole islets cell fractions containing dc , or purified intra - islet dc from nod.rag1 mice constitutively presented antigen to both type a and type b t cells in the absence of inflammation or overt cellular death ( fig . these dc were capable of activating both the type a and type b insulin - reactive t cells ( fig . 3e , f ) , showing that both peptide - mhc conformers are presented by apc within close contact to beta cells . the secretory granules of beta cells can constitute a source of the b:9 - 23 peptide , due to the fact each granule is comprised of about 10 insulin molecules , as well as catabolic products from its processing29 . purified secretory granules from primary beta cells incubated with splenic dc led to robust activation of both whether these granules contain insulin peptides , we generated a monoclonal antibody that specifically binds to the b:9 - 23 peptide but not to insulin or proinsulin molecules ( supplementary fig . specific granule staining was observed in whole islets or purified beta cells from nod or nod.rag1 . the majority of granules inside beta cells were positive for insulin but negative for b:9 - 23 and did not contain lamp-1 ( fig . these observations suggest that the b:9 - 23 peptides presented by i - a could be derived from beta cell vesicles containing peptides of the insulin -chain and/or from the processing of insulin granules within dc . initial mass spectrometry studies on purified secretory granules showed they contained c - peptide ( data not shown ) , but also peptides from the b:9 - 23 segment , which included peptides centered on the 12 - 21 residues ( vealylvgge ) of the insulin -chain ( supplementary fig . these observations suggest that peptides immunogenic for type b t cells are produced during the physiological processing of mature insulin molecules , and are not dependent on any prior inflammation . one set had the unique feature of being specific to exogenous insulin peptide , but unreactive to insulin . the peptide that activated these type b t cells was normally derived from the insulin secretory granule and was loaded on mhc class ii in the intra - islet dc . these t cells were capable of transferring diabetes and were found spontaneously in infiltrated islets , indicating their participation in the autoimmune process . the two other sets of t cells were insulin and peptide - reactive t cells , but differed in their sensitivity to insulin . type a t cells , identified in the nod or in the b16:a - dko mice , respectively . in the b16:a - dko mice type a t cells were readily found after insulin immunization and showed a higher response to the insulin molecule or the b:9 - 23 peptide compared to the strong insulin reactivity found in b16:a - dko mice supports the conclusion reached by others912 that native insulin expression in the thymus influences the selection of insulin - reactive t cells , and that partial tolerance or unresponsiveness to the insulin molecule exists in nod mice . thus , the few insulin reactive t cells found in nod mice had an imprint of tolerance , with weak reactivity to insulin , which might explain why they escaped negative selection . the frequency of type b t cells is markedly greater than type a t cells in the precursor population of nod mice . however , because both type a and type b t cells were isolated from the islets , we can not conclude which set is the major participant , or if they act synergistically in disease development . due to this variability , strict comparisons between the diabetogenicity of type a and type b t cells were not done systematically . the b:9 - 23 peptide could be generated following dc handling of insulin upon capture of the insulin granules or from uptake of the granules that already contained the peptide . the finding that islet dc contain insulin granules and b:9 - 23 peptide , and the observation that feeding dc with purified secretory granules results in strong presentation explains another conundrum concerning insulin presentation . furthermore , due to the low binding affinity of the b:9 - 23 peptide for i - a and the rapid dissociation of the peptide from i - a ( ref 18 ) , a high concentration of insulin within the apc is required for effective presentation to t cells . in antigen presentation assays , the concentration of insulin or b:9 - 23 peptide required for presentation is in the low m level which is unphysiological in the whole mouse . if all these aspects are taken into consideration , it is most likely that the islet dc have two essential roles : to migrate into the pancreatic draining lymph node to initiate the sensitization process32 ; and to present antigen to sensitized t cells in the effector stage of diabetogenesis . collectively , these results suggest that self - reactive type b t cells and low affinity type a t cells escape negative selection and give rise to pathogenic t cells in autoimmune diabetes . a situation akin to insulin autoimmunity may also be found in other endocrine autoimmune diseases such as in autoimmune thyroiditis in which dc are found surrounding the follicles , some of which have class ii - positive epithelial cells33,34 .

bladder cancer is one of the most frequent malignancies around the world ( 1 ) . in the united states , the estimated number of new cases for 2014 is 69,000 while the estimated number of deaths is approximately 15,000 ( 2 ) . evidence suggests that the activation of the telomerase enzyme is a pivotal step in the development of bladder cancer ; furthermore , somatic mutations in tert promoters were identified in 55.6% of the bladder cancers ( 3 ) . the addition of hexameric ttaggg repeats to telomeres , located at the ends of chromosomal dna ( 4 ) , plays a critical role in counteracting the end - replication loss and consequent dna damage repair , leading to genome instability , chromosomal fusions and rearrangements ( 5 ) . tert and clptm1l were located in chromosome 5p15.33 , which was regularly suggested to mediate the telomerase function . moreover , rs401681 ( located in 27 kb from the tert and the intron 13 of clptm1l ) is one of the most widely studied snps , which has been reported to be associated with an increased risk of many cancer types ( e.g. prostate and lung cancers ) via gwas ( 6 - 9 ) . nevertheless , a decreased risk of colorectal cancer and melanoma was identified by some studies on the major ( c ) allele of rs401681 ( 10 , 11 ) . although several studies have paid attention to the relationship between rs401681 polymorphism and bladder cancer susceptibility ( 1 , 9 , 12 ) , the reported data is inconclusive . thus , we conducted a meta - analysis on all eligible studies to derive a more authentic estimation of the relevance and a better understanding of its possible influence on bladder cancer risk . we performed an in silico search of the embase , pubmed and google scholar databases to retrieve articles linking rs401681 polymorphism in tert - clptm1l gene and susceptibility to bladder cancer available up to december 2014 using the combinations of tert , or clptm1-like and polymorphism , or gene , or variant , or snp and association or risk and tumor or cancer or malignancy or neoplasm or carcinoma . all the searched publications were retrieved , and we also used a hand search of references of reviewed articles or original studies on this point to uncover additional studies . the search was limited to english language literatures , and all relevant studies were reviewed . only the first published study was selected when overlapping studies existed . for republished studies , only the one with the largest sample size articles which met the following criteria were included : ( 1 ) parameters about the rs401681 polymorphism and bladder cancer risk are evaluated ; ( 3 ) race and numbers of affected and unaffected subjects are reported ; ( 5 ) sufficient data for calculating an odds ratio ( or ) with 95 percent confidence interval ( 95%ci ) in additive model is available ; ( 6 ) sufficient data for detail genotype frequencies in asian population is available . exclusion criteria were : ( 1 ) studies on the subjects of family cancer risks or cancer - prone disposition ; ( 2 ) the study which has no usable data reported or contains duplicated data ; ( 3 ) abstract , comment , review and editorial ; ( 4 ) when multiple publications reported the same or to overlapping patients , we retained only the largest study to avoid duplication of information . three investigators ( meng zhang , xun wu and wei lu ) independently extracted data in a standardized form and have reached a consensus of all publications . for each eligible study , the following information was recorded : the name of the first author , the publication year , ethnicity , source of controls , minor allele frequency ( maf ) , genotype frequency and/or additive or and 95% ci and the number of the cases and controls . the detail information about the genotype frequency in asian population was provided by three case - control studies . the association between rs401681 polymorphism and bladder cancer risk was evaluated under four genetic models . we evaluated the association between rs401681 polymorphism and bladder cancer risk by using crude or with 95% ci in overall population . the heterogeneity of the individual studies was evaluated by q test ( for the association between rs401681 polymorphism and bladder cancer risk in asian ) . if the p value of q test was 0.05 , the fixed effects model was used to pool the data ; otherwise , random effects model will be selected . however , the test for heterogeneity does not have enough power for selecting the effects model for the pooling analysis of the association between rs401681 polymorphism and overall bladder cancer risk . thus , random effects model was selected for all the analyses . both funnel plot and egger s test were applied to evaluate the publication bias ( p<0.10 was considered representative of statistical significance ) . we used stata software ( version 12.0 , stata corp ) to perform all statistical tests and for any test or model , p<0.05 was considered to be statistically significant . further , the four genetic models : allele contrast ( t vs. c ) , homozygote ( tt vs. cc ) , recessive ( tt vs. tc / cc ) , and dominant ( tt / tc vs. cc ) models were used to evaluate the association between polymorphism and bladder cancer risk in asian population group . we performed an in silico search of the embase , pubmed and google scholar databases to retrieve articles linking rs401681 polymorphism in tert - clptm1l gene and susceptibility to bladder cancer available up to december 2014 using the combinations of tert , or clptm1-like and polymorphism , or gene , or variant , or snp and association or risk and tumor or cancer or malignancy or neoplasm or carcinoma . all the searched publications were retrieved , and we also used a hand search of references of reviewed articles or original studies on this point to uncover additional studies . the search was limited to english language literatures , and all relevant studies were reviewed . only the first published study was selected when overlapping studies existed . for republished studies , only the one with the largest sample size articles which met the following criteria were included : ( 1 ) parameters about the rs401681 polymorphism and bladder cancer risk are evaluated ; ( 3 ) race and numbers of affected and unaffected subjects are reported ; ( 5 ) sufficient data for calculating an odds ratio ( or ) with 95 percent confidence interval ( 95%ci ) in additive model is available ; ( 6 ) sufficient data for detail genotype frequencies in asian population is available . exclusion criteria were : ( 1 ) studies on the subjects of family cancer risks or cancer - prone disposition ; ( 2 ) the study which has no usable data reported or contains duplicated data ; ( 3 ) abstract , comment , review and editorial ; ( 4 ) when multiple publications reported the same or to overlapping patients , we retained only the largest study to avoid duplication of information . three investigators ( meng zhang , xun wu and wei lu ) independently extracted data in a standardized form and have reached a consensus of all publications . for each eligible study , the following information was recorded : the name of the first author , the publication year , ethnicity , source of controls , minor allele frequency ( maf ) , genotype frequency and/or additive or and 95% ci and the number of the cases and controls . the detail information about the genotype frequency in asian population was provided by three case - control studies . the association between rs401681 polymorphism and bladder cancer risk was evaluated under four genetic models . we evaluated the association between rs401681 polymorphism and bladder cancer risk by using crude or with 95% ci in overall population . the heterogeneity of the individual studies was evaluated by q test ( for the association between rs401681 polymorphism and bladder cancer risk in asian ) . if the p value of q test was 0.05 , the fixed effects model was used to pool the data ; otherwise , random effects model will be selected . however , the test for heterogeneity does not have enough power for selecting the effects model for the pooling analysis of the association between rs401681 polymorphism and overall bladder cancer risk . thus , random effects model was selected for all the analyses . both funnel plot and egger s test were applied to evaluate the publication bias ( p<0.10 was considered representative of statistical significance ) . we used stata software ( version 12.0 , stata corp ) to perform all statistical tests and for any test or model , p<0.05 was considered to be statistically significant . further , the four genetic models : allele contrast ( t vs. c ) , homozygote ( tt vs. cc ) , recessive ( tt vs. tc / cc ) , and dominant ( tt / tc vs. cc ) models were used to evaluate the association between polymorphism and bladder cancer risk in asian population group . we performed an in silico search of the embase , pubmed and google scholar databases to retrieve articles linking rs401681 polymorphism in tert - clptm1l gene and susceptibility to bladder cancer available up to december 2014 using the combinations of tert , or clptm1-like and polymorphism , or gene , or variant , or snp and association or risk and tumor or cancer or malignancy or neoplasm or carcinoma . all the searched publications were retrieved , and we also used a hand search of references of reviewed articles or original studies on this point to uncover additional studies . the search was limited to english language literatures , and all relevant studies were reviewed . only the first published study was selected when overlapping studies existed . for republished studies , only the one with the largest sample size articles which met the following criteria were included : ( 1 ) parameters about the rs401681 polymorphism and bladder cancer risk are evaluated ; ( 3 ) race and numbers of affected and unaffected subjects are reported ; ( 5 ) sufficient data for calculating an odds ratio ( or ) with 95 percent confidence interval ( 95%ci ) in additive model is available ; ( 6 ) sufficient data for detail genotype frequencies in asian population is available . exclusion criteria were : ( 1 ) studies on the subjects of family cancer risks or cancer - prone disposition ; ( 2 ) the study which has no usable data reported or contains duplicated data ; ( 3 ) abstract , comment , review and editorial ; ( 4 ) when multiple publications reported the same or to overlapping patients , we retained only the largest study to avoid duplication of information . three investigators ( meng zhang , xun wu and wei lu ) independently extracted data in a standardized form and have reached a consensus of all publications . for each eligible study , the following information was recorded : the name of the first author , the publication year , ethnicity , source of controls , minor allele frequency ( maf ) , genotype frequency and/or additive or and 95% ci and the number of the cases and controls . the detail information about the genotype frequency in asian population was provided by three case - control studies . the association between rs401681 polymorphism and bladder cancer risk was evaluated under four genetic models . we evaluated the association between rs401681 polymorphism and bladder cancer risk by using crude or with 95% ci in overall population . the heterogeneity of the individual studies was evaluated by q test ( for the association between rs401681 polymorphism and bladder cancer risk in asian ) . if the p value of q test was 0.05 , the fixed effects model was used to pool the data ; otherwise , random effects model will be selected . however , the test for heterogeneity does not have enough power for selecting the effects model for the pooling analysis of the association between rs401681 polymorphism and overall bladder cancer risk . thus , random effects model was selected for all the analyses . both funnel plot and egger s test were applied to evaluate the publication bias ( p<0.10 was considered representative of statistical significance ) . we used stata software ( version 12.0 , stata corp ) to perform all statistical tests and for any test or model , p<0.05 was considered to be statistically significant . further , the four genetic models : allele contrast ( t vs. c ) , homozygote ( tt vs. cc ) , recessive ( tt vs. tc / cc ) , and dominant ( tt / tc vs. cc ) models were used to evaluate the association between polymorphism and bladder cancer risk in asian population group . in total , five eligible case control studies of four publications involving 9,196 cases and 42570 controls were selected in this meta - analysis ( 1 , 6 , 9 , 12 ) . and three case - control studies including 1044 cases and 1869 controls were selected to evaluate the association between the genetic models of the polymorphism and bladder cancer risk in asian . the main characteristics of these studies are demonstrated in table 1 ( 1 , 9 , 12 ) . the distribution of rs401681[c ] allele and the genotype frequencies of asian publications among bladder cancer cases and controls are shown in table 1 and methodological quality of the included studies according to the newcastle - ottawa scale was shown in table 2 . study characteristics in an analysis of the association between rs401681 polymorphism and bladder cancer risk maf : minor allele frequency ; - : not mentioned ; population : population - based ; hospital : hospital - based . methodological quality of the included studies according to the newcastle - ottawa scale , yes ; na , not applicable ; the last item non - response rate was eliminated from this study the main results of this meta - analysis and the heterogeneity tests are shown in table 3 . rs401681[c ] allele was proved to be associated with bladder cancer risk in overall population ( per allele , or=1.132 , 95% ci : 1.0801.187 ; p<0.001 , figure 1a ) . in the stratified analysis by ethnicity , the rs401681[c ] locus conferred susceptibility to bladder cancer in asian group ( per allele , or=1.172 , 95 % ci 1.0391.322 ; p=0.010 ) and caucasian group ( per allele , or=1.125 , 95%ci=1.068 - 1.184 ; p<0.001 ) . results from stratified analysis of the rs401681 polymorphism and bladder cancer risk in asian or odds ratio , ci confidence interval odds ratio of bladder cancer risk associated with rs401681 under the additive model by fixed effects furthermore , our work also showed that rs401681 polymorphism is associated with bladder cancer risk in asian population under four models : the rs401681 polymorphism was associated with increased risk of bladder cancer in allele model ( or=3.722 , 95% ci=1.311 - 10.568 , p=0.014 ) , and decreased risk in homozygote model ( or=0.692 , 95 % ci=0.513 - 0.934 , p= 0.016 , figure 1b ) and recessive model ( or=0.728 , 95% ci=0.541 - 0.980 , p=0.036 , figure 1c ) in asian ( table 3 ) . odds ratio of bladder cancer risk associated with rs401681 under the homozygote model in asian by fixed effects odds ratio of bladder cancer risk associated with rs401681 under the recessive model in asian by fixed effects a sensitivity analysis was done to explore the influence of individual publications on the collected results by removing a single publication from the pooled analysis once at a time and no individual study influenced the pooled or value . then , we performed both begg s funnel plot and egger s test to assess the publication bias of the literature ( per allele , begg s test : t=0.97 , p= 0.406 , figure 2 ) . no obvious asymmetry was obtained from the shape of funnel plots in overall meta - analysis . funnel plot analysis to detect publication bias for the rs401681 in the involved four data sets in total , five eligible case control studies of four publications involving 9,196 cases and 42570 controls were selected in this meta - analysis ( 1 , 6 , 9 , 12 ) . and three case - control studies including 1044 cases and 1869 controls were selected to evaluate the association between the genetic models of the polymorphism and bladder cancer risk in asian . the main characteristics of these studies are demonstrated in table 1 ( 1 , 9 , 12 ) . the distribution of rs401681[c ] allele and the genotype frequencies of asian publications among bladder cancer cases and controls are shown in table 1 and methodological quality of the included studies according to the newcastle - ottawa scale was shown in table 2 . study characteristics in an analysis of the association between rs401681 polymorphism and bladder cancer risk maf : minor allele frequency ; - : not mentioned ; population : population - based ; hospital : hospital - based . methodological quality of the included studies according to the newcastle - ottawa scale , yes ; na , not applicable ; the last item non - response rate was eliminated from this study the main results of this meta - analysis and the heterogeneity tests are shown in table 3 . rs401681[c ] allele was proved to be associated with bladder cancer risk in overall population ( per allele , or=1.132 , 95% ci : 1.0801.187 ; p<0.001 , figure 1a ) . in the stratified analysis by ethnicity , the rs401681[c ] locus conferred susceptibility to bladder cancer in asian group ( per allele , or=1.172 , 95 % ci 1.0391.322 ; p=0.010 ) and caucasian group ( per allele , or=1.125 , 95%ci=1.068 - 1.184 ; p<0.001 ) . results from stratified analysis of the rs401681 polymorphism and bladder cancer risk in asian or odds ratio , ci confidence interval odds ratio of bladder cancer risk associated with rs401681 under the additive model by fixed effects furthermore , our work also showed that rs401681 polymorphism is associated with bladder cancer risk in asian population under four models : the rs401681 polymorphism was associated with increased risk of bladder cancer in allele model ( or=3.722 , 95% ci=1.311 - 10.568 , p=0.014 ) , and decreased risk in homozygote model ( or=0.692 , 95 % ci=0.513 - 0.934 , p= 0.016 , figure 1b ) and recessive model ( or=0.728 , 95% ci=0.541 - 0.980 , p=0.036 , figure 1c ) in asian ( table 3 ) . odds ratio of bladder cancer risk associated with rs401681 under the homozygote model in asian by fixed effects odds ratio of bladder cancer risk associated with rs401681 under the recessive model in asian by fixed effects a sensitivity analysis was done to explore the influence of individual publications on the collected results by removing a single publication from the pooled analysis once at a time and no individual study influenced the pooled or value . then , we performed both begg s funnel plot and egger s test to assess the publication bias of the literature ( per allele , begg s test : t=0.97 , p= 0.406 , figure 2 ) . no obvious asymmetry was obtained from the shape of funnel plots in overall meta - analysis . funnel plot analysis to detect publication bias for the rs401681 in the involved four data sets in total , five eligible case control studies of four publications involving 9,196 cases and 42570 controls were selected in this meta - analysis ( 1 , 6 , 9 , 12 ) . and three case - control studies including 1044 cases and 1869 controls were selected to evaluate the association between the genetic models of the polymorphism and bladder cancer risk in asian . the main characteristics of these studies are demonstrated in table 1 ( 1 , 9 , 12 ) . the distribution of rs401681[c ] allele and the genotype frequencies of asian publications among bladder cancer cases and controls are shown in table 1 and methodological quality of the included studies according to the newcastle - ottawa scale was shown in table 2 . study characteristics in an analysis of the association between rs401681 polymorphism and bladder cancer risk maf : minor allele frequency ; - : not mentioned ; population : population - based ; hospital : hospital - based . methodological quality of the included studies according to the newcastle - ottawa scale , yes ; na , not applicable ; the last item non - response rate was eliminated from this study the main results of this meta - analysis and the heterogeneity tests are shown in table 3 . rs401681[c ] allele was proved to be associated with bladder cancer risk in overall population ( per allele , or=1.132 , 95% ci : 1.0801.187 ; p<0.001 , figure 1a ) . in the stratified analysis by ethnicity , the rs401681[c ] locus conferred susceptibility to bladder cancer in asian group ( per allele , or=1.172 , 95 % ci 1.0391.322 ; p=0.010 ) and caucasian group ( per allele , or=1.125 , 95%ci=1.068 - 1.184 ; p<0.001 ) . results from stratified analysis of the rs401681 polymorphism and bladder cancer risk in asian or odds ratio , ci confidence interval odds ratio of bladder cancer risk associated with rs401681 under the additive model by fixed effects furthermore , our work also showed that rs401681 polymorphism is associated with bladder cancer risk in asian population under four models : the rs401681 polymorphism was associated with increased risk of bladder cancer in allele model ( or=3.722 , 95% ci=1.311 - 10.568 , p=0.014 ) , and decreased risk in homozygote model ( or=0.692 , 95 % ci=0.513 - 0.934 , p= 0.016 , figure 1b ) and recessive model ( or=0.728 , 95% ci=0.541 - 0.980 , p=0.036 , figure 1c ) in asian ( table 3 ) . odds ratio of bladder cancer risk associated with rs401681 under the homozygote model in asian by fixed effects odds ratio of bladder cancer risk associated with rs401681 under the recessive model in asian by fixed effects a sensitivity analysis was done to explore the influence of individual publications on the collected results by removing a single publication from the pooled analysis once at a time and no individual study influenced the pooled or value . then , we performed both begg s funnel plot and egger s test to assess the publication bias of the literature ( per allele , begg s test : t=0.97 , p= 0.406 , figure 2 ) . no obvious asymmetry was obtained from the shape of funnel plots in overall meta - analysis . funnel plot analysis to detect publication bias for the rs401681 in the involved four data sets chromosome 5p15.33 region contains the clptm1l and tert genes and genetic variations in this region have been associated with increased or decreased risk of multiple cancer types ( 14 , 15 ) . the rs401681 polymorphism was located in the intron 13 of clptm1l and 27 kb from the tert , which has been widely reported to be associated with an increased risk of lung , prostate and bladder cancer . rafnar et al , first conducted gwas which composed of 3,945 bladder cancer patients and 34,988 controls , and showed that the rs401681[c ] allele was associated with an increased cancer risk with a combined or of 1.12 ( 95% ci , 1.031.11 ) ( 6 ) . recently , yu et al , examined the association between snp rs401681 and bladder cancer risk in a chinese population of 367 cases and 420 controls ( 1 ) . moreover , in the present study , we confirmed that the rs401681 polymorphism was associated with bladder cancer risk that was consistent with a previous study ( 6 ) . heterogeneity and sensitivity analyses were conducted to promise the reliability of the data . to sum up , we conducted a comprehensive research for all eligible studies and provided an overview of the association between rs401681[c ] allele and bladder cancer risk , as well as the association between the four genetic models and bladder cancer risk . still , there exist several limitations in our meta - analysis that should be noted . first , the non - english literatures were excluded , which may result in publications bias . second , we have calculated the pooled ors in asian group under four genetic models ; however , since another two studies provided insufficient genotype frequencies , we were unable to calculate the pooled ors in addition to additive model . besides , ors with and without adjustment were pooled together , which might be a consideration source of heterogeneity . based on larger sample size , our meta - analysis provided a more precise estimation that rs401681[c ] is a risk factor for bladder cancer in asian and caucasian groups and rs401681 polymorphism was a risk factor for bladder cancer under allele model and a protective factor in homozygote model and recessive model in asian group . future well - designed studies are warranted to refine the investigation on this issue of interest .

objective(s):genome - wide association studies have identified a number of genetic variants of telomerase reverse transcriptase ( tert ) , cleft lip and palate transmembrane1-like ( clptm1l ) associated with the risk of bladder cancer . rs401681 polymorphism in tert - clptm1l was of special interest for bladder cancer risk , whereas the results were inconclusive.materials and methods : publications illustrating the association between rs401681 polymorphism and bladder cancer risk were collected from the embase , pubmed and google scholar . three independent reviewers worked on the data extraction . the meta - analysis was performed by stata 12.0 . the odds ratio ( or ) with 95% confidence interval ( ci ) was calculated for these data.results:six case - control studies were retrieved reporting a total of 9196 bladder cancer patients and 42570 controls . the strength of the relevance between rs401681 polymorphism and bladder cancer risk was evaluated by stata 12.0 software . rs401681[c ] allele was identified marginally associated with increased bladder cancer risk , with per allele or of 1.132 ( 95% ci=1.080 - 1.187 , pheterogeneity=0.701 ) ; in the stratified analysis by ethnicity , the increased cancer risk was revealed in asian and caucasian groups . moreover , we also revealed that rs401681 polymorphism was associated with an increased risk of bladder cancer in asian population with three publications under allele model ( or=3.722 , 95% ci=1.311 - 10.568 , p=0.014 ) , whereas a decreased risk was identified in homozygote model ( or=0.692 , 95 % ci=0.513 - 0.934 , p= 0.016 ) and recessive model ( or=0.728 , 95% ci=0.541 - 0.980 , p=0.036).conclusion : in summary , our study provided evidence that rs401681 polymorphism is associated with the risk of bladder cancer .

Introduction Materials and Methods None Search strategy Inclusion and exclusion criteria Data extraction Statistical analysis Results None Eligible studies Meta-analysis Publication bias Discussion Conclusion Conflicts of interest statement

evidence suggests that the activation of the telomerase enzyme is a pivotal step in the development of bladder cancer ; furthermore , somatic mutations in tert promoters were identified in 55.6% of the bladder cancers ( 3 ) . moreover , rs401681 ( located in 27 kb from the tert and the intron 13 of clptm1l ) is one of the most widely studied snps , which has been reported to be associated with an increased risk of many cancer types ( e.g. nevertheless , a decreased risk of colorectal cancer and melanoma was identified by some studies on the major ( c ) allele of rs401681 ( 10 , 11 ) . although several studies have paid attention to the relationship between rs401681 polymorphism and bladder cancer susceptibility ( 1 , 9 , 12 ) , the reported data is inconclusive . thus , we conducted a meta - analysis on all eligible studies to derive a more authentic estimation of the relevance and a better understanding of its possible influence on bladder cancer risk . we performed an in silico search of the embase , pubmed and google scholar databases to retrieve articles linking rs401681 polymorphism in tert - clptm1l gene and susceptibility to bladder cancer available up to december 2014 using the combinations of tert , or clptm1-like and polymorphism , or gene , or variant , or snp and association or risk and tumor or cancer or malignancy or neoplasm or carcinoma . for republished studies , only the one with the largest sample size articles which met the following criteria were included : ( 1 ) parameters about the rs401681 polymorphism and bladder cancer risk are evaluated ; ( 3 ) race and numbers of affected and unaffected subjects are reported ; ( 5 ) sufficient data for calculating an odds ratio ( or ) with 95 percent confidence interval ( 95%ci ) in additive model is available ; ( 6 ) sufficient data for detail genotype frequencies in asian population is available . for each eligible study , the following information was recorded : the name of the first author , the publication year , ethnicity , source of controls , minor allele frequency ( maf ) , genotype frequency and/or additive or and 95% ci and the number of the cases and controls . the detail information about the genotype frequency in asian population was provided by three case - control studies . the association between rs401681 polymorphism and bladder cancer risk was evaluated under four genetic models . we evaluated the association between rs401681 polymorphism and bladder cancer risk by using crude or with 95% ci in overall population . the heterogeneity of the individual studies was evaluated by q test ( for the association between rs401681 polymorphism and bladder cancer risk in asian ) . however , the test for heterogeneity does not have enough power for selecting the effects model for the pooling analysis of the association between rs401681 polymorphism and overall bladder cancer risk . further , the four genetic models : allele contrast ( t vs. c ) , homozygote ( tt vs. cc ) , recessive ( tt vs. tc / cc ) , and dominant ( tt / tc vs. cc ) models were used to evaluate the association between polymorphism and bladder cancer risk in asian population group . we performed an in silico search of the embase , pubmed and google scholar databases to retrieve articles linking rs401681 polymorphism in tert - clptm1l gene and susceptibility to bladder cancer available up to december 2014 using the combinations of tert , or clptm1-like and polymorphism , or gene , or variant , or snp and association or risk and tumor or cancer or malignancy or neoplasm or carcinoma . for republished studies , only the one with the largest sample size articles which met the following criteria were included : ( 1 ) parameters about the rs401681 polymorphism and bladder cancer risk are evaluated ; ( 3 ) race and numbers of affected and unaffected subjects are reported ; ( 5 ) sufficient data for calculating an odds ratio ( or ) with 95 percent confidence interval ( 95%ci ) in additive model is available ; ( 6 ) sufficient data for detail genotype frequencies in asian population is available . for each eligible study , the following information was recorded : the name of the first author , the publication year , ethnicity , source of controls , minor allele frequency ( maf ) , genotype frequency and/or additive or and 95% ci and the number of the cases and controls . the detail information about the genotype frequency in asian population was provided by three case - control studies . the association between rs401681 polymorphism and bladder cancer risk was evaluated under four genetic models . we evaluated the association between rs401681 polymorphism and bladder cancer risk by using crude or with 95% ci in overall population . the heterogeneity of the individual studies was evaluated by q test ( for the association between rs401681 polymorphism and bladder cancer risk in asian ) . however , the test for heterogeneity does not have enough power for selecting the effects model for the pooling analysis of the association between rs401681 polymorphism and overall bladder cancer risk . further , the four genetic models : allele contrast ( t vs. c ) , homozygote ( tt vs. cc ) , recessive ( tt vs. tc / cc ) , and dominant ( tt / tc vs. cc ) models were used to evaluate the association between polymorphism and bladder cancer risk in asian population group . we performed an in silico search of the embase , pubmed and google scholar databases to retrieve articles linking rs401681 polymorphism in tert - clptm1l gene and susceptibility to bladder cancer available up to december 2014 using the combinations of tert , or clptm1-like and polymorphism , or gene , or variant , or snp and association or risk and tumor or cancer or malignancy or neoplasm or carcinoma . for republished studies , only the one with the largest sample size articles which met the following criteria were included : ( 1 ) parameters about the rs401681 polymorphism and bladder cancer risk are evaluated ; ( 3 ) race and numbers of affected and unaffected subjects are reported ; ( 5 ) sufficient data for calculating an odds ratio ( or ) with 95 percent confidence interval ( 95%ci ) in additive model is available ; ( 6 ) sufficient data for detail genotype frequencies in asian population is available . for each eligible study , the following information was recorded : the name of the first author , the publication year , ethnicity , source of controls , minor allele frequency ( maf ) , genotype frequency and/or additive or and 95% ci and the number of the cases and controls . the detail information about the genotype frequency in asian population was provided by three case - control studies . the association between rs401681 polymorphism and bladder cancer risk was evaluated under four genetic models . we evaluated the association between rs401681 polymorphism and bladder cancer risk by using crude or with 95% ci in overall population . the heterogeneity of the individual studies was evaluated by q test ( for the association between rs401681 polymorphism and bladder cancer risk in asian ) . however , the test for heterogeneity does not have enough power for selecting the effects model for the pooling analysis of the association between rs401681 polymorphism and overall bladder cancer risk . further , the four genetic models : allele contrast ( t vs. c ) , homozygote ( tt vs. cc ) , recessive ( tt vs. tc / cc ) , and dominant ( tt / tc vs. cc ) models were used to evaluate the association between polymorphism and bladder cancer risk in asian population group . in total , five eligible case control studies of four publications involving 9,196 cases and 42570 controls were selected in this meta - analysis ( 1 , 6 , 9 , 12 ) . and three case - control studies including 1044 cases and 1869 controls were selected to evaluate the association between the genetic models of the polymorphism and bladder cancer risk in asian . the distribution of rs401681[c ] allele and the genotype frequencies of asian publications among bladder cancer cases and controls are shown in table 1 and methodological quality of the included studies according to the newcastle - ottawa scale was shown in table 2 . study characteristics in an analysis of the association between rs401681 polymorphism and bladder cancer risk maf : minor allele frequency ; - : not mentioned ; population : population - based ; hospital : hospital - based . methodological quality of the included studies according to the newcastle - ottawa scale , yes ; na , not applicable ; the last item non - response rate was eliminated from this study the main results of this meta - analysis and the heterogeneity tests are shown in table 3 . rs401681[c ] allele was proved to be associated with bladder cancer risk in overall population ( per allele , or=1.132 , 95% ci : 1.0801.187 ; p<0.001 , figure 1a ) . in the stratified analysis by ethnicity , the rs401681[c ] locus conferred susceptibility to bladder cancer in asian group ( per allele , or=1.172 , 95 % ci 1.0391.322 ; p=0.010 ) and caucasian group ( per allele , or=1.125 , 95%ci=1.068 - 1.184 ; p<0.001 ) . results from stratified analysis of the rs401681 polymorphism and bladder cancer risk in asian or odds ratio , ci confidence interval odds ratio of bladder cancer risk associated with rs401681 under the additive model by fixed effects furthermore , our work also showed that rs401681 polymorphism is associated with bladder cancer risk in asian population under four models : the rs401681 polymorphism was associated with increased risk of bladder cancer in allele model ( or=3.722 , 95% ci=1.311 - 10.568 , p=0.014 ) , and decreased risk in homozygote model ( or=0.692 , 95 % ci=0.513 - 0.934 , p= 0.016 , figure 1b ) and recessive model ( or=0.728 , 95% ci=0.541 - 0.980 , p=0.036 , figure 1c ) in asian ( table 3 ) . odds ratio of bladder cancer risk associated with rs401681 under the homozygote model in asian by fixed effects odds ratio of bladder cancer risk associated with rs401681 under the recessive model in asian by fixed effects a sensitivity analysis was done to explore the influence of individual publications on the collected results by removing a single publication from the pooled analysis once at a time and no individual study influenced the pooled or value . then , we performed both begg s funnel plot and egger s test to assess the publication bias of the literature ( per allele , begg s test : t=0.97 , p= 0.406 , figure 2 ) . funnel plot analysis to detect publication bias for the rs401681 in the involved four data sets in total , five eligible case control studies of four publications involving 9,196 cases and 42570 controls were selected in this meta - analysis ( 1 , 6 , 9 , 12 ) . and three case - control studies including 1044 cases and 1869 controls were selected to evaluate the association between the genetic models of the polymorphism and bladder cancer risk in asian . the distribution of rs401681[c ] allele and the genotype frequencies of asian publications among bladder cancer cases and controls are shown in table 1 and methodological quality of the included studies according to the newcastle - ottawa scale was shown in table 2 . study characteristics in an analysis of the association between rs401681 polymorphism and bladder cancer risk maf : minor allele frequency ; - : not mentioned ; population : population - based ; hospital : hospital - based . rs401681[c ] allele was proved to be associated with bladder cancer risk in overall population ( per allele , or=1.132 , 95% ci : 1.0801.187 ; p<0.001 , figure 1a ) . in the stratified analysis by ethnicity , the rs401681[c ] locus conferred susceptibility to bladder cancer in asian group ( per allele , or=1.172 , 95 % ci 1.0391.322 ; p=0.010 ) and caucasian group ( per allele , or=1.125 , 95%ci=1.068 - 1.184 ; p<0.001 ) . results from stratified analysis of the rs401681 polymorphism and bladder cancer risk in asian or odds ratio , ci confidence interval odds ratio of bladder cancer risk associated with rs401681 under the additive model by fixed effects furthermore , our work also showed that rs401681 polymorphism is associated with bladder cancer risk in asian population under four models : the rs401681 polymorphism was associated with increased risk of bladder cancer in allele model ( or=3.722 , 95% ci=1.311 - 10.568 , p=0.014 ) , and decreased risk in homozygote model ( or=0.692 , 95 % ci=0.513 - 0.934 , p= 0.016 , figure 1b ) and recessive model ( or=0.728 , 95% ci=0.541 - 0.980 , p=0.036 , figure 1c ) in asian ( table 3 ) . odds ratio of bladder cancer risk associated with rs401681 under the homozygote model in asian by fixed effects odds ratio of bladder cancer risk associated with rs401681 under the recessive model in asian by fixed effects a sensitivity analysis was done to explore the influence of individual publications on the collected results by removing a single publication from the pooled analysis once at a time and no individual study influenced the pooled or value . then , we performed both begg s funnel plot and egger s test to assess the publication bias of the literature ( per allele , begg s test : t=0.97 , p= 0.406 , figure 2 ) . no obvious asymmetry was obtained from the shape of funnel plots in overall meta - analysis . funnel plot analysis to detect publication bias for the rs401681 in the involved four data sets in total , five eligible case control studies of four publications involving 9,196 cases and 42570 controls were selected in this meta - analysis ( 1 , 6 , 9 , 12 ) . and three case - control studies including 1044 cases and 1869 controls were selected to evaluate the association between the genetic models of the polymorphism and bladder cancer risk in asian . the distribution of rs401681[c ] allele and the genotype frequencies of asian publications among bladder cancer cases and controls are shown in table 1 and methodological quality of the included studies according to the newcastle - ottawa scale was shown in table 2 . study characteristics in an analysis of the association between rs401681 polymorphism and bladder cancer risk maf : minor allele frequency ; - : not mentioned ; population : population - based ; hospital : hospital - based . methodological quality of the included studies according to the newcastle - ottawa scale , yes ; na , not applicable ; the last item non - response rate was eliminated from this study the main results of this meta - analysis and the heterogeneity tests are shown in table 3 . rs401681[c ] allele was proved to be associated with bladder cancer risk in overall population ( per allele , or=1.132 , 95% ci : 1.0801.187 ; p<0.001 , figure 1a ) . in the stratified analysis by ethnicity , the rs401681[c ] locus conferred susceptibility to bladder cancer in asian group ( per allele , or=1.172 , 95 % ci 1.0391.322 ; p=0.010 ) and caucasian group ( per allele , or=1.125 , 95%ci=1.068 - 1.184 ; p<0.001 ) . results from stratified analysis of the rs401681 polymorphism and bladder cancer risk in asian or odds ratio , ci confidence interval odds ratio of bladder cancer risk associated with rs401681 under the additive model by fixed effects furthermore , our work also showed that rs401681 polymorphism is associated with bladder cancer risk in asian population under four models : the rs401681 polymorphism was associated with increased risk of bladder cancer in allele model ( or=3.722 , 95% ci=1.311 - 10.568 , p=0.014 ) , and decreased risk in homozygote model ( or=0.692 , 95 % ci=0.513 - 0.934 , p= 0.016 , figure 1b ) and recessive model ( or=0.728 , 95% ci=0.541 - 0.980 , p=0.036 , figure 1c ) in asian ( table 3 ) . odds ratio of bladder cancer risk associated with rs401681 under the homozygote model in asian by fixed effects odds ratio of bladder cancer risk associated with rs401681 under the recessive model in asian by fixed effects a sensitivity analysis was done to explore the influence of individual publications on the collected results by removing a single publication from the pooled analysis once at a time and no individual study influenced the pooled or value . then , we performed both begg s funnel plot and egger s test to assess the publication bias of the literature ( per allele , begg s test : t=0.97 , p= 0.406 , figure 2 ) . funnel plot analysis to detect publication bias for the rs401681 in the involved four data sets chromosome 5p15.33 region contains the clptm1l and tert genes and genetic variations in this region have been associated with increased or decreased risk of multiple cancer types ( 14 , 15 ) . the rs401681 polymorphism was located in the intron 13 of clptm1l and 27 kb from the tert , which has been widely reported to be associated with an increased risk of lung , prostate and bladder cancer . rafnar et al , first conducted gwas which composed of 3,945 bladder cancer patients and 34,988 controls , and showed that the rs401681[c ] allele was associated with an increased cancer risk with a combined or of 1.12 ( 95% ci , 1.031.11 ) ( 6 ) . recently , yu et al , examined the association between snp rs401681 and bladder cancer risk in a chinese population of 367 cases and 420 controls ( 1 ) . moreover , in the present study , we confirmed that the rs401681 polymorphism was associated with bladder cancer risk that was consistent with a previous study ( 6 ) . to sum up , we conducted a comprehensive research for all eligible studies and provided an overview of the association between rs401681[c ] allele and bladder cancer risk , as well as the association between the four genetic models and bladder cancer risk . based on larger sample size , our meta - analysis provided a more precise estimation that rs401681[c ] is a risk factor for bladder cancer in asian and caucasian groups and rs401681 polymorphism was a risk factor for bladder cancer under allele model and a protective factor in homozygote model and recessive model in asian group .

molecular chaperones are essential for the maintenance of cellular homeostasis by facilitating various functions including degradation of proteins , translocation , folding of co - translational products , and protein complex assembly . many molecular chaperones are also known as heat shock proteins ( hsp ) and their expression is upregulated in response to stress . the large and varied heat shock protein family has usually been classified into major classes defined by molecular weight . the 70 kda family of proteins is the most abundant and thoroughly studied family of heat shock proteins . hsp70 functions both as a holdase ( binding and holding onto unfolded polypeptides by withdrawing aggregation - prone species ) as well as a refoldase ( assisting non - native proteins to fold to the native state ) . the highly conserved hsp70 structure is typically composed of an n - terminal atpase domain ( 44 kda ) , a c - terminal domain containing a -sandwich subdomain , which is the substrate binding domain ( 1518 kda , ) followed by an -helical subdomain ( 10 kda ) [ 7 , 8 ] . hsp70 protein activity occurs through the interaction of hydrophobic peptide segments of other proteins with its substrate binding domain ( sbd ) in an atp - dependent manner . the hsp70 atpase cycle switches between the low affinity atp - bound state and rapid substrate - exchange rates and the high affinity adp - bound state with low substrate - exchange rates . hsp40 is a co - chaperone and is required to stimulate the atpase activity of hsp70 and results in hsp70-adp with a high affinity for substrate . in order for the nucleotide binding cleft to be opened , the release of adp is facilitated by a nucleotide - exchange factor , such as bag-1 protein . the atpase cycle is completed when atp binds the atpase domain of hsp70 resulting in a conformational change in the substrate binding domain and bound substrates are released . the eevd motif present at the end of the c - terminus of hsp70 members is involved in binding to the tetratricopeptide repeat ( tpr ) domains of hsp70/hsp90 organizing protein ( hop ) and carboxy terminus of hsp70 interacting protein ( chip ) . the eevd motif has been suggested to recruit hsp40 as it has been shown to bind and regulate the function of hsp40 [ 12 , 13 ] . all hsp40s contain a j - domain that is required for facilitating the cellular activity of hsp70 proteins through its interaction with their partner hsp70s . the j - domain has a conserved histidine - proline - aspartic acid ( hpd ) tripeptide ; variations within the hpd motif are known to abolish the stimulation of hsp70 atpase activity by hsp40 [ 14 , 15 ] . type i hsp40s are highly conserved and contain a glycine - phenylalanine ( g / f ) and cysteine rich region that contains four motifs of cxxcxgxg , a glycine / methionine rich region , a carboxy - terminal peptide binding domain and a dimerization domain [ 16 , 17 ] . type i hsp40s also have a peptide - binding region at the c - terminus which aids in the binding to non - native polypeptides , in turn allowing hsp40 proteins to transport substrates to hsp70 proteins for folding [ 18 , 19 ] . type ii hsp40s contain the j - domain and the g / f rich region , along with the peptide - binding region at the c - terminus [ 2022 ] . the highly specialized type iii hsp40s possess only the conserved j - domain which can occur anywhere on its sequence and primarily functions to recruit hsp70 to a particular location . the african trypanosome , trypanosoma brucei , is a blood - borne unicellular parasitic protozoan and the etiological agent of both human and animal african trypanosomiasis . african trypanosomiasis is described as one of the neglected tropical diseases crippling economic development and causing death in africa 's poorest and most marginalized communities . due to a lack of new treatments against human african trypanosomiasis and emergence of resistance to older drugs , human treatments currently available are connected with high levels of toxicity and resistance . heat shock proteins have been emerging as prospective drug targets ( hsp70s as drug targets reviewed in ) . drugs causing cellular stress resulting in the induction of heat shock proteins anti - heat shock protein drugs used in combination with current drugs could therefore synergistically improve the effectiveness of available drugs . parasitic heat shock proteins have been revealed as drug targets , including malarial drug target p. falciparum hsp90 and trypanosomal t. evansi hsp90 [ 29 , 30 ] . amongst the known hsp90 proteins , pfhsp90 was shown to have the highest atpase activity , and its inhibition by geldanamycin ( ga ) was stronger than seen for human hsp90 . semi - synthetic inhibitor 17-(allylamino)-17-demethoxygeldanamycin ( 17-aag ) has been shown to curb growth of the p. falciparum parasite and to inhibit the growth of the t. evansi parasite by specifically binding and inhibiting hsp90 . 17-aag could likely be effective against infection caused by t. brucei due to the sequence similarity between tbhsp90 and tehsp90 . sera from patients infected with trypanosomes were screened to identify diagnostic antigens , and although tbhsp70 was identified as a candidate , by itself , it demonstrated inadequate specificity and sensitivity in diagnosis of trypanosomiasis . it may however be useful as a diagnostic antigen if used in conjunction with other immunogenic proteins . of the leishmanial and trypanosomal hsp70s and hsp40s , a number of important and well - known studies have been performed on t. cruzi . cytoplasmic tchsp70b demonstrated high atpase activity in comparison to human hsp70 and was shown to be heat inducible . in addition to stimulating the immune response , tchsp70b is one of the immunodominant antigens in t. cruzi infected individuals [ 31 , 33 ] . a type i hsp40 , tcj2 , was able to stimulate the already high basal atpase activity of tchsp70b by 1.5 fold , whilst the type iii hsp40 tcj1 showed no significant stimulation . furthermore , tcj2 was shown to complement the well - characterized saccharomyces cerevisiae type i hsp40 , ydj1 , and was subsequently suggested to be involved in cytoprotection . tchsp70b , displaying typical chaperone properties , is homologous to t. brucei hsp70 , and tcj1 and tcj2 are homologous to tbj1 and tbj2 , respectively . genome annotation revealed t. brucei to possess a complement of 65 hsp40 proteins and 12 hsp70 proteins . in silico analysis of tbhsp70.c indicated the presence of atypical acidic residues in the substrate binding domain as well as in the substrate binding cavity of tbhsp70.c and these may play a role in substrate discrimination . however , the residues required for association with hsp40 proteins within both the atpase and substrate binding cavity , as well as the hsp70 phosphorylation site , are well conserved . in addition the c - terminal eevd motif is absent from tbhsp70.c and it contains the sequence rieainante at the end of the protein . tbhsp70.c was predicted to be cytoplasmic . to date , tbhsp70.c has not been biochemically characterized and co - chaperones have not been identified . of the 65 hsp40s , only one of the type iii t. brucei hsp40 proteins , tbj1 , has been expressed , purified , and biochemically characterized to date . no in vitro characterization of the type i t. brucei hsp40 protein , tbj2 , has been completed ; in vivo analysis revealed that tbj2 is essential to the survival of the cell [ 38 , 39 ] . tbj2 was demonstrated to be upregulated upon inducing heat shock and to localize in the cytoplasm . according to tritrypdb , tbhsp70.c is localized in the cytoplasm and tbj2 was selected as a probable partner of tbhsp70.c . this research aims to functionally characterize tbhsp70.c and tbj2 as its potential co - chaperone . we successfully expressed and purified tbhsp70.c and revealed for the first time that tbhsp70.c and tbj2 can prevent thermal aggregation of malate dehydrogenase ( mdh ) and rhodanese and that tbj2 stimulates the atpase activity of tbhsp70.c . . a greater understanding of the hsp70-hsp40 partnerships is important as molecular chaperones have been implicated in parasite survival and growth . louis , mo usa ) , merck chemicals ( darmstadt , germany ) , biorad ( usa ) , or roche molecular biochemicals ( indianapolis , in , usa ) . the t. brucei treu927 strain was a kind donation from professor george cross ( rockefeller university , new york , usa ) . e. coli strain bb1994 ( mc4100 dnak52 sidb1::tc pdmi,1::cmr kanr ) was kindly provided by dr . prior to phylogenetic tree analysis , trypanosomatid amino acid sequences were obtained from the genedb database and other hsp70 protein sequences were retrieved from the national centre for biotechnology information ( ncbi ) , both sets of sequences in fasta format . the phylogenetic tree was generated using molecular evolutionary genetics analysis ( mega5 ) version 5.2.2 maximum likelihood analysis . the coding regions of full length tbhsp70.c and tbj2 were pcr amplified from t. b. brucei treu927 genomic dna . forward primer ( 5-ggt acc atg acc tac gaa gga-3 ) with a kpni restriction site ( underlined ) and reverse primer ( 5-gtc gac tta ctc tgt gtt tgc-3 ) with a sali restriction site ( underlined ) were used for tbhsp70.c . the forward ( 5-gaattcggatcccatatggtgaaagaaacaaaatac-3 ) ( ecori , bamhi , ndei ) and reverse ( 5-aagcttctcgaggtcgacctattgctgcgtacacg-3 ) ( hindiii , xhoi , sali ) primers were used for pcr amplification of tbj2 . the n - terminal his - tagged pqe80-tbhsp70.c and pet28a - tbj2 constructs were verified by restriction digestion analysis and dna sequencing ( data not shown ) . an overnight culture was prepared by inoculating e. coli bb1994 [ pqe80tbhsp70.c ] in 25 ml 2 yt broth supplemented with 100 g / ml ampicillin and 50 g / ml kanamycin at 30c with shaking . the overnight culture was transferred to 225 ml 2 yt broth and grown with shaking to midlog phase ( a600 0.6 ) before inducing tbhsp70.c protein expression by the addition of isopropylthiogalactoside ( iptg ) to a final concentration of 1 mm . cells were harvested ( 5000 g ; 15 min ) 3 hours after induction and resuspended in lysis buffer ( 100 mm tris - hcl , ph 8 , 300 mm nacl , 10 mm imidazole ) . cells were stored at 80c overnight and thawed with the addition of 1 mg / l lysozyme and 1 mm pmsf culminating in lysis . cellular debris was removed by centrifugation ( 12000 g ; 40 min ; 4c ) and the supernatant was incubated with nickel - charged sepharose beads in lysis buffer overnight at 4c . the bead - suspension was centrifuged ( 5000 g ; 1 min ; 4c ) and three washes were performed with wash buffer ( 100 mm tris - hcl , ph 8 , 300 mm nacl , 20 mm imidazole ) . purified protein was subsequently dialysed overnight [ 100 mm tris - hcl , ph8 , 100 mm nacl , 5% ( v / v ) glycerol , 50 mm kcl , 2 mm mgcl2 , 0.5 mm dithiothreitol ] . both e. coli bl21(de3 ) [ pet28a - tbj2 ] and bl21(de3 ) [ pet14b - tchsp70b ] cells were grown at 37c , maintaining a selective pressure using kanamycin ( 50 g / ml ) and were harvested 5 hours after induction . to remove the presence of co - purified dnak , tbj2 and tchsp70b bound to the ni - nta column were washed 5 times with the addition of 20% glycerol and 10 mm atp to the wash buffer . all of other steps of the purification were performed in the same manner as for tbhsp70.c . sodium - dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) analysis was used to evaluate the purity of the protein . anti - tbhsp70.c was produced in rabbit using the c - terminal antigen , cqrgrgvtegsgrpp at residues 648662 , from the tbhsp70.c protein sequence . western analysis was used to verify the integrity and identity of tbhsp70.c using affinity - purified anti - tbhsp70.c peptide antibody . western analysis was performed using anti - his antibody to target tbj2 and tchsp70b and anti - dnak antibody to detect dnak . antibodies were detected using the ecl advance blotting detection kit and viewed using the chemidoc eq ( biorad , usa ) . the ability of tbhsp70.c and tbj2 to suppress aggregation of bovine rhodanese ( sigma - aldrich ) was assessed spectrophotometrically . rhodanese was denatured overnight at 30c ; rhodanese denatured in denaturing buffer ( 6 m guanidine hydrochloride , 50 mm hepes , ph 7.0 , 100 mm nacl ) was added to assay buffer ( 50 mm hepes , ph 7.0 , 100 mm nacl ) to a final concentration of 1.5 m was monitored at 300 nm over a period of 40 min at room temperature using a kc junior microplate reader ( bio - tek instruments , usa ) . molecular chaperone proteins of interest were added at various concentrations to measure their ability to suppress rhodanese aggregation . tbhsp70.c , tchsp70b , and tbj2 are not aggregation prone as no significant increase in turbidity was observed when these proteins were assayed in the absence of rhodanese ; protein samples that had been inactivated by boiling for 20 minutes also displayed no chaperone activity ( data not shown ) . absorbance was plotted as percent rhodanese aggregation over 40 min subsequent to normalizing against assays with rhodanese alone . the abilities of tbhsp70.c and tbj2 to prevent thermal aggregation of mdh were analysed by spectrophotometry . the assay was initiated by adding 0.72 m mdh and the molecular chaperone proteins to assay buffer ( 50 mm tris - hcl , 100 mm nacl ; ph 7.4 ) heated to 48c . aggregation of the protein substrate was monitored by measuring the scatter of light at 360 nm over 30 min at 48c in a helios alpha db spectrophotometer with a peltier - controlled cell . hydrolysis of atp by tbhsp70.c was assessed using a modified version of the ascorbic acid / ammonium molybdate colorimetric assay to measure the release of inorganic phosphate during the reaction . the assay was performed as described in with a few modifications . tbhsp70.c and control protein tchsp70b ( 0.4 m ) were equilibrated to 37c in atpase buffer ( 25 mm hepes , ph 7.4 , 2 mm mgcl2 , 50 mm kcl , 0.5 mm dtt ) . stimulation of the basal atpase activity of both tbhsp70.c and tchsp70b by co - chaperone tbj2 was assessed at equal concentrations ( 0.4 m ) and in a 2-fold molar excess to tbhsp70.c ( 1 m ) and tchsp70b ( 1 m ) . samples were taken in triplicate at regular time intervals ( 0 , 30 , 60 , 120 , 180 , and 240 minutes ) . all assays were corrected for the spontaneous breakdown of atp observed in a control experiment in the absence of protein . any background atp hydrolysis observed for tbj2 was corrected for by subtracting this activity from the reactions containing this protein . statistical analysis was performed on data generated from both the aggregation suppression and atpase activity assays . the relationship between two variables was analysed using one - way analysis of variance ( anova ) . wild type lister 927 variant 221 t. brucei brucei bloodstream form lysates ( 10 cells / ml ) were used for the heat stress inducibility experiment . bloodstream form t. brucei lister 927 variant 221 strain trypanosome parasites were cultured in filter sterilized complete iscoves modified dulbeccos media ( imdm ) based hm1 - 9 medium [ imdm base powder , 3.6 mm sodium bicarbonate , 1 mm hypoxanthine , 1 mm sodium pyruvate , 0.16 mm thymidine , 0.05 mm bathocuprone sulphate acid , 10% ( v / v ) heat inactivated foetal bovine serum , 1.5 mm l - cysteine , 0.2 mm -mercaptoethanol , ph 7.5 ] in a humidified chamber at 37c with an atmosphere of 5% co2 . separate 25 ml culture of cells were exposed to heat shock at 42c for a period of 60 min in plugged flasks , allowing no entry of co2 . cell lysates were harvested by centrifugation at 800 g for 10 min , washed twice in pbs buffer ( 10 mm na2hpo4 , 2 mm kh2po4 , 137 mm nacl , 2.7 mm kcl ) , and repelleted prior to resuspension in sds - page loading buffer to a final cell count of 5 105 cells/l . the lysates ( 5 10 cells per lane ) were resolved on a 10% sds - page gel . differences in tbhsp70.c protein expression were detected using polyclonal rabbit anti - tbhsp70.c peptide antibody ( 1 : 5000 ) and goat anti - rabbit igg hrp - conjugated secondary antibody ( 1 : 5000 ) in subsequent western analysis . bioinformatic analysis revealed t. brucei hsp70.c to be a eukaryotic isoform that may represent a novel family of hsp70 proteins as it did not cluster phylogenetically with any of the other primary hsp70 proteins . phylogenetic tree analysis was used to establish orthology between the novel hsp70.c proteins within the tritryps and to compare their phylogenetic relationships with cytoplasmic hsp70s from the tritryps and the hsp70s from the t. brucei complement along with well - characterized eukaryotic and prokaryotic canonical hsp70 proteins ( figure 1 ) . tbhsp70.c may prove to be an anti - parasitic drug target as phylogenetic analysis suggests that tbhsp70.c clusters only with hsp70 proteins from the tritryps . not surprisingly , the hsp70.c proteins clustered together phylogenetically , tbhsp70.c formed a monophyletic clade with tbghsp70.c and a close link with tcohsp70.c , suggesting possible paralogy . the hsp70.c cluster formed a closer phylogenetic relationship with the cytoplasmic hsp70s , including inducible human hsp70b ( hshspa6 ) , constitutively expressed human hsc70 ( hshspa8 ) , bovine hsc70 and plant hsp70 than with mitochondrial hsp70s , the nef - acting hsp110s , or the dnaks ( prokaryotic hsp70 homologue ) ( figure 1 ) . recombinant tbhsp70.c was expressed and purified from an e. coli dnak - minus strain ( figure 2(a ) ) . the e. coli dnak - minus strain was used to eliminate endogenous dnak , the bacterial homologue of hsp70 . soluble tbhsp70.c was successfully purified by nickel - affinity chromatography under non - denaturing conditions and anti - tbhsp70.c peptide antibodies recognized full - length tbhsp70.c at 73 kda , confirmed by western analysis ( figure 2(a ) ) . 600 mg of protein was purified from 1 l of e. coli cells and was of sufficient purity for subsequent in vitro assays . an alternative strategy was adopted for the purifications of recombinant tchsp70b and tbj2 to eliminate co - purifying dnak due to a lack of an e. coli dnak - minus strain compatible with the pet vectors . modifications to the nickel - affinity chromatography protocol included the addition of 20% glycerol and 10 mm atp to the wash steps . the non - denaturing purification resulted in the removal of co - purified dnak , confirmed by western analysis , from both the tchsp70b ( figure 2(b ) ) and tbj2 ( figure 2(c ) ) purifications . for the tchsp70b and tbj2 purifications , 1176 mg and 900 mg of protein were purified from 1 l of e. coli cells , respectively , and the elimination of dnak warranted the use of tchsp70b and tbj2 in subsequent in vitro assays . tbhsp70.c was able to suppress the protein aggregation of chemically denatured rhodanese in a dose - dependent manner ( figure 3 ) . tchsp70b , an orthologue of bos taurus hspa8 , was used as a positive control . in addition to demonstrating typical chaperone properties [ 32 , 34 ] , the previously characterized tchsp70b , also homologous to tbhsp70 , contains an intact c - terminal eevd motif . the addition of tbhsp70.c and tchsp70b resulted in a marked increase of aggregation suppression in a dose - dependent manner ( figure 3 ) . tbj2 ( 0.5 m , 0.7 m , 1 m ) resulted in 4.1% , 8.3% , and 14.2% suppression of rhodanese aggregation , respectively . to investigate the ability of tbj2 to enhance the holdase function of tbhsp70.c , the addition of tbj2 at a submolar concentration to tbhsp70.c resulted in 27.7% suppression of rhodanese aggregation ( figure 3 ) . equal concentrations of tbj2 and tbhsp70.c resulted in 37.5% suppression of rhodanese aggregation , which was comparable to the 41.2% suppression produced by tchsp70b at equimolar concentrations to tbj2 . tbj2 in partnership with tbhsp70.c demonstrated increased aggregation suppression . at equimolar concentrations tbj2 demonstrated a synergistic effect and thus the ability to function as a co - chaperone to both tchsp70b and tbhsp70.c . mdh was used as a second substrate to establish whether tbhsp70.c and tbj2 could suppress aggregation of mdh . bsa was used as a negative control and had no effect on mdh aggregation ( data not shown ) . a dose response was evident upon the addition of various concentrations of tbhsp70.c as turbidity levels decreased with an increase in chaperone concentration ( figure 4 ) . a similar result was obtained for tbj2 where increased chaperone concentrations caused decreased turbidity and a dose - dependent suppression of mdh aggregation ( figure 4 ) . the ability of tbj2 to enhance the holdase function of tbhsp70.c was assessed by maintaining constant tbhsp70.c concentrations and varying those of tbj2 ( figure 4 ) . tbhsp70.c ( 0.5 m ) resulted in 61.8% suppression of mdh aggregation ( figure 4 ) . the addition of tbj2 at a submolar concentration ( 0.3 m ) to tbhsp70.c resulted in 66.9% mdh aggregation suppression and tbj2 at molar excess ( 0.7 m ) resulted in 85% suppression of mdh aggregation ( figure 4 ) . tbj2 concentrations of 0.3 m and 0.7 m enhanced tbhsp70.c chaperone activity ; however , the increase in turbidity was not enough to be an additive effect . equal concentrations of tbj2 and control protein tchsp70b resulted in 93.9% suppression of mdh aggregation , which appeared to be an additive effect ( figure 4 ) . tbj2 did not demonstrate the ability to co - chaperone either tbhsp70.c or tchsp70b using mdh as a substrate . tbj2 may have been binding as substrate to tbhsp70.c , thus resulting in the decreased suppression of mdh aggregation that was observed . the ability of tbj2 to stimulate the basal atpase activity of tbhsp70.c and tchsp70b was investigated . tbhsp70.c was determined to have a basal atpase activity of 7.6 nmol pi / min / mg and tchsp70b a basal atpase activity of 18.3 nmol pi / min / mg . the basal atpase activity of tbhsp70.c and tchsp70b was represented as 100% ( figure 5 ) . equal concentrations of tbj2 and tbhsp70.c resulted in a significant 1.62-fold stimulation of the basal atpase activity of tbhsp70.c ( figure 5 ) . equal concentrations of tbj2 and tchsp70b caused a slightly lower but still significant 1.39-fold stimulation of tchsp70b basal atpase activity ( figure 5 ) . tbj2 at 2-fold molar excess to tbhsp70.c and tchsp70b resulted in a significant 2.88-fold enhancement of the basal atpase activity of tbhsp70.c and a significant 2.62-fold stimulation of tchsp70b atpase activity ( figure 5 ) . tbhsp70.c , tchsp70b , and tbj2 denatured by boiling displayed no atpase activity ( data not shown ) . under conditions of stress , a cell responds by increasing the synthesis of heat shock proteins to manage the elevated levels of denatured proteins . t. b. brucei 427 v221 cells were exposed to heat shock by incubating them at 42c for one hour . recombinant tbhsp70.c purified from e. coli bb1994 cells was used as a positive control . even though there was no notable difference in the tbhsp70.c protein levels on the sds - page gel , tbhsp70.c protein expression proved to be inducible by heat stress , as shown by an increase in the top band in the western analysis ( figure 6 ) . two bands were detected by the anti - tbhsp70.c peptide antibody . the top band at 73 kda increased in intensity from 37c to 42c indicating that at 37c tbhsp70.c was expressed in bloodstream form cells and upon exposure to heat stress tbhsp70.c was upregulated . interestingly , the expression level of the non - specific 68 kda band , detected by anti - tbhsp70.c peptide antibodies , remained constant upon heat shock , suggesting it to be a separate protein and not a degradation product of tbhsp70.c ( figure 6 ) . a study of the evolutionary relationships between kinetoplastid hsp70 proteins and typical hsp70 proteins has revealed a novel and divergent group of proteins , the orthologues tbhsp70.c , tchsp70.c , and lmhsp70.c . tbj2 was selected as a probable co - chaperone of tbhsp70.c due to the presence of its functional j - domain and because of its predicted cytosolic localization ( data not shown ) . when studying molecular chaperones , the presence of dnak is a major concern due to the possibility that basal expression of bacterial or co - purified dnak could mask the chaperone activity of the t. brucei heat shock proteins under investigation . the co - purification of dnak is likely as a result of denatured exposed surfaces of the target protein interacting with dnak by acting as a substrate . using an e. coli dnak - minus strain and modifications to the purification protocol , dnak was eliminated from the tbhsp70.c and the tbj2 and tchsp70b purifications , respectively . amongst the various roles of hsp70 proteins demonstration of the holdase - function , by suppressing protein aggregation , is used to display chaperone activity [ 48 , 49 ] . mdh aggregation suppression assays are often performed in the absence of atp [ 50 , 51 ] . the addition of atp decreases the affinity of an hsp70 protein for mdh , resulting in its release and subsequent re - aggregation . a loss of affinity for substrate in the presence of atp has been demonstrated for p. falciparum hsp70 , e. colihsc66 , dnak and bovine hsp90 . the model thermolabile substrate mdh was used in this study to investigate the ability of tbhsp70.c and tbj2 , in isolation and in partnership , to suppress the heat - induced aggregation of mdh . a second substrate , rhodanese , was selected to investigate whether the substrate binding specificity of tbhsp70.c for rhodanese would differ considering the presence of fewer hydrophobic residues within rhodanese than in mdh . a recent in silico study using an e. coli dnak l484w mutant predicted that an increase in hydrogen bonds and hydrophobic interactions allows an enhanced interaction between chaperone and substrate . tbhsp70.c was shown to bind and suppress aggregation of both rhodanese and mdh , thereby displaying typical chaperone activity . however , tbhsp70.c suppressed aggregation of mdh to a greater degree than was observed for rhodanese . hsp70s generally function in cooperation with hsp40s , where hsp40 either mediates the interaction of hsp70 with its substrate or holds the substrate and recruits hsp70 to the unfolded polypeptide . hsp40 proteins are able to bind unfolded substrates independently of hsp70 proteins and prevent their aggregation [ 18 , 58 ] . once bound , the substrate is transferred to hsp70 by the j - protein ; at the same time hsp40 stabilizes the hsp70-substrate association by stimulating the atpase activity of hsp70 . a similar result was seen for the homologue of tbj2 , tcj2 , in its ability to independently suppress aggregation of mdh . the reduced chaperone activity observed in this study using rhodanese could furthermore be as a result of non - productive and aggregation - prone intermediates formed whilst rhodanese refolds . in the presence of tbhsp70.c the effect of tbj2 on both tbhsp70.c and tchsp70b was additive rather than synergistic when using mdh . a similar result was observed during the suppression of aggregation of mdh by plasmodium falciparum hsp40 and hsp70 . the ability of a type iii hsp40 protein , tbj1 , to function as a co - chaperone was supported by the findings that tbj1 assisted trypanosoma cruzi hsp70 and medicago sativa hsp70 in suppression of mdh aggregation . suppression of protein aggregation assays has also been used to successfully determine the effect of small molecule modulators on the abilities of pfhsp70 proteins to suppress the aggregation of alcohol dehydrogenase and mdh . the ability of tbhsp70.c to successfully suppress aggregation of two individual substrates was not surprising . even though some conservation has been lost in the tbhsp70.c hydrophobic arch , some residues are replaced by acidic and aliphatic residues and the hydrophobic pocket , represented by the valine residue , is well conserved ( data not shown ) . enhanced hsp70 atpase activity is specific and is modulated through the interaction of an hsp40 partner . this data would suggest that tbj2 acts as a co - chaperone by means of its j - domain interacting with the atpase domain of tbhsp70.c , either recruiting substrates to the hsp70 or merely mediating the interaction of the hsp70 with its substrate and stimulating the atpase activity of the hsp70 . however , the level of atpase activity stimulation by tbj2 is low in comparison to what has previously been observed for hsp70-hsp40 partnerships . yeast hsp40 , ydj1p , was shown to stimulate the atpase activity of yeast hsp70 , ssa1p , by 6 - 8-fold [ 18 , 61 , 62 ] . tbj2 , homologue of tcj2 , demonstrated a greater stimulation of tbhsp70.c atpase activity than was observed for tcj2-stimulated atpase activity of tchsp70b . the low stimulation of the atpase activity of tchsp70 by tcj2 was suggested to be as a result of a masking effect of the already very high basal atpase activity of tchsp70b , reported to be 100 times greater than that observed for human hsp70 . further characterization of the atpase activity of tbhsp70.c would entail investigating the activation of the atpase activity of tbhsp70.c by various substrates . maximal stimulation of the atpase activity of hsp70 proteins has been demonstrated to take place in the presence of both co - chaperone and substrate . to date , very little research has been performed on the heat shock response within t. brucei . the focus has been on investigating the effect of heat stress on t. cruzi and/or its vector , panstrongylus megistus as well as on molecular chaperones belonging to the t. cruzi parasite . a study in which t. b. brucei bloodstream form cultures were exposed to heat shock resulted in the upregulation of the expression levels of tbhsp70.c . this finding would imply that tbhsp70.c is required for protein quality control not only under normal conditions but also when the cell is placed under thermal stress . this is the first report of data showing that the expression levels of tbhsp70.c are increased by heat stress . tchsp70 mrna levels showed a very significant four - fold upregulation upon heat stress at 37c when compared to mrna levels at 28c . the ability of tbj2 to significantly enhance the basal atpase activity of tbhsp70.c suggests that tbj2 functions as a co - chaperone of tbhsp70.c . however , further experimental analyses including binding studies and co - localization will need to be performed to conclusively confirm tbj2 as co - chaperone of tbhsp70.c . furthermore , the possibility of other cytoplasmic tbj proteins functioning as co - chaperones of tbhsp70.c will need to be investigated . due to its atypical features , tbhsp70.c is likely to interact with a specific set of protein substrates in the cell . tbhsp70.c is the first t. brucei hsp70 protein to be functionally characterized , along with its probable partnership with co - chaperone tbj2 . this research opens up prospects for further studies of the yet unexplored multiple hsp70/hsp40 partnerships in t. brucei . future research would entail isolating and characterizing the remaining 11 hsp70 proteins from t. brucei to enable differentiation between unique and canonical features and furthermore investigating which of them form partnerships with the 65 hsp40 proteins . characterization of these and other chaperone / co - chaperone interactions could further enhance understanding of the cell biology of t. brucei .

the neglected tropical disease , african trypanosomiasis , is fatal and has a crippling impact on economic development . heat shock protein 70 ( hsp70 ) is an important molecular chaperone that is expressed in response to stress and hsp40 acts as its co - chaperone . these proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host . a novel cytosolic hsp70 , from trypanosoma brucei , tbhsp70.c , contains an acidic substrate binding domain and lacks the c - terminal eevd motif . the ability of a cytosolic hsp40 from trypanosoma brucei j protein 2 , tbj2 , to function as a co - chaperone of tbhsp70.c was investigated . the main objective was to functionally characterize tbhsp70.c to further expand our knowledge of parasite biology . tbhsp70.c and tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile mdh and chemically denatured rhodanese . atpase assays revealed a 2.8-fold stimulation of the atpase activity of tbhsp70.c by tbj2 . tbhsp70.c and tbj2 both demonstrated chaperone activity and tbj2 functions as a co - chaperone of tbhsp70.c . in vivo heat stress experiments indicated upregulation of the expression levels of tbhsp70.c .

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

many molecular chaperones are also known as heat shock proteins ( hsp ) and their expression is upregulated in response to stress . the highly conserved hsp70 structure is typically composed of an n - terminal atpase domain ( 44 kda ) , a c - terminal domain containing a -sandwich subdomain , which is the substrate binding domain ( 1518 kda , ) followed by an -helical subdomain ( 10 kda ) [ 7 , 8 ] . hsp70 protein activity occurs through the interaction of hydrophobic peptide segments of other proteins with its substrate binding domain ( sbd ) in an atp - dependent manner . hsp40 is a co - chaperone and is required to stimulate the atpase activity of hsp70 and results in hsp70-adp with a high affinity for substrate . the atpase cycle is completed when atp binds the atpase domain of hsp70 resulting in a conformational change in the substrate binding domain and bound substrates are released . the eevd motif present at the end of the c - terminus of hsp70 members is involved in binding to the tetratricopeptide repeat ( tpr ) domains of hsp70/hsp90 organizing protein ( hop ) and carboxy terminus of hsp70 interacting protein ( chip ) . the j - domain has a conserved histidine - proline - aspartic acid ( hpd ) tripeptide ; variations within the hpd motif are known to abolish the stimulation of hsp70 atpase activity by hsp40 [ 14 , 15 ] . type i hsp40s are highly conserved and contain a glycine - phenylalanine ( g / f ) and cysteine rich region that contains four motifs of cxxcxgxg , a glycine / methionine rich region , a carboxy - terminal peptide binding domain and a dimerization domain [ 16 , 17 ] . type i hsp40s also have a peptide - binding region at the c - terminus which aids in the binding to non - native polypeptides , in turn allowing hsp40 proteins to transport substrates to hsp70 proteins for folding [ 18 , 19 ] . type ii hsp40s contain the j - domain and the g / f rich region , along with the peptide - binding region at the c - terminus [ 2022 ] . the african trypanosome , trypanosoma brucei , is a blood - borne unicellular parasitic protozoan and the etiological agent of both human and animal african trypanosomiasis . african trypanosomiasis is described as one of the neglected tropical diseases crippling economic development and causing death in africa 's poorest and most marginalized communities . due to a lack of new treatments against human african trypanosomiasis and emergence of resistance to older drugs , human treatments currently available are connected with high levels of toxicity and resistance . drugs causing cellular stress resulting in the induction of heat shock proteins anti - heat shock protein drugs used in combination with current drugs could therefore synergistically improve the effectiveness of available drugs . tchsp70b , displaying typical chaperone properties , is homologous to t. brucei hsp70 , and tcj1 and tcj2 are homologous to tbj1 and tbj2 , respectively . in silico analysis of tbhsp70.c indicated the presence of atypical acidic residues in the substrate binding domain as well as in the substrate binding cavity of tbhsp70.c and these may play a role in substrate discrimination . in addition the c - terminal eevd motif is absent from tbhsp70.c and it contains the sequence rieainante at the end of the protein . to date , tbhsp70.c has not been biochemically characterized and co - chaperones have not been identified . no in vitro characterization of the type i t. brucei hsp40 protein , tbj2 , has been completed ; in vivo analysis revealed that tbj2 is essential to the survival of the cell [ 38 , 39 ] . according to tritrypdb , tbhsp70.c is localized in the cytoplasm and tbj2 was selected as a probable partner of tbhsp70.c . this research aims to functionally characterize tbhsp70.c and tbj2 as its potential co - chaperone . we successfully expressed and purified tbhsp70.c and revealed for the first time that tbhsp70.c and tbj2 can prevent thermal aggregation of malate dehydrogenase ( mdh ) and rhodanese and that tbj2 stimulates the atpase activity of tbhsp70.c . the coding regions of full length tbhsp70.c and tbj2 were pcr amplified from t. b. brucei treu927 genomic dna . the overnight culture was transferred to 225 ml 2 yt broth and grown with shaking to midlog phase ( a600 0.6 ) before inducing tbhsp70.c protein expression by the addition of isopropylthiogalactoside ( iptg ) to a final concentration of 1 mm . all of other steps of the purification were performed in the same manner as for tbhsp70.c . anti - tbhsp70.c was produced in rabbit using the c - terminal antigen , cqrgrgvtegsgrpp at residues 648662 , from the tbhsp70.c protein sequence . western analysis was used to verify the integrity and identity of tbhsp70.c using affinity - purified anti - tbhsp70.c peptide antibody . the ability of tbhsp70.c and tbj2 to suppress aggregation of bovine rhodanese ( sigma - aldrich ) was assessed spectrophotometrically . molecular chaperone proteins of interest were added at various concentrations to measure their ability to suppress rhodanese aggregation . tbhsp70.c , tchsp70b , and tbj2 are not aggregation prone as no significant increase in turbidity was observed when these proteins were assayed in the absence of rhodanese ; protein samples that had been inactivated by boiling for 20 minutes also displayed no chaperone activity ( data not shown ) . the abilities of tbhsp70.c and tbj2 to prevent thermal aggregation of mdh were analysed by spectrophotometry . hydrolysis of atp by tbhsp70.c was assessed using a modified version of the ascorbic acid / ammonium molybdate colorimetric assay to measure the release of inorganic phosphate during the reaction . stimulation of the basal atpase activity of both tbhsp70.c and tchsp70b by co - chaperone tbj2 was assessed at equal concentrations ( 0.4 m ) and in a 2-fold molar excess to tbhsp70.c ( 1 m ) and tchsp70b ( 1 m ) . bioinformatic analysis revealed t. brucei hsp70.c to be a eukaryotic isoform that may represent a novel family of hsp70 proteins as it did not cluster phylogenetically with any of the other primary hsp70 proteins . not surprisingly , the hsp70.c proteins clustered together phylogenetically , tbhsp70.c formed a monophyletic clade with tbghsp70.c and a close link with tcohsp70.c , suggesting possible paralogy . recombinant tbhsp70.c was expressed and purified from an e. coli dnak - minus strain ( figure 2(a ) ) . an alternative strategy was adopted for the purifications of recombinant tchsp70b and tbj2 to eliminate co - purifying dnak due to a lack of an e. coli dnak - minus strain compatible with the pet vectors . the non - denaturing purification resulted in the removal of co - purified dnak , confirmed by western analysis , from both the tchsp70b ( figure 2(b ) ) and tbj2 ( figure 2(c ) ) purifications . tbhsp70.c was able to suppress the protein aggregation of chemically denatured rhodanese in a dose - dependent manner ( figure 3 ) . in addition to demonstrating typical chaperone properties [ 32 , 34 ] , the previously characterized tchsp70b , also homologous to tbhsp70 , contains an intact c - terminal eevd motif . the addition of tbhsp70.c and tchsp70b resulted in a marked increase of aggregation suppression in a dose - dependent manner ( figure 3 ) . to investigate the ability of tbj2 to enhance the holdase function of tbhsp70.c , the addition of tbj2 at a submolar concentration to tbhsp70.c resulted in 27.7% suppression of rhodanese aggregation ( figure 3 ) . at equimolar concentrations tbj2 demonstrated a synergistic effect and thus the ability to function as a co - chaperone to both tchsp70b and tbhsp70.c . mdh was used as a second substrate to establish whether tbhsp70.c and tbj2 could suppress aggregation of mdh . a dose response was evident upon the addition of various concentrations of tbhsp70.c as turbidity levels decreased with an increase in chaperone concentration ( figure 4 ) . the ability of tbj2 to enhance the holdase function of tbhsp70.c was assessed by maintaining constant tbhsp70.c concentrations and varying those of tbj2 ( figure 4 ) . the addition of tbj2 at a submolar concentration ( 0.3 m ) to tbhsp70.c resulted in 66.9% mdh aggregation suppression and tbj2 at molar excess ( 0.7 m ) resulted in 85% suppression of mdh aggregation ( figure 4 ) . tbj2 concentrations of 0.3 m and 0.7 m enhanced tbhsp70.c chaperone activity ; however , the increase in turbidity was not enough to be an additive effect . tbj2 did not demonstrate the ability to co - chaperone either tbhsp70.c or tchsp70b using mdh as a substrate . tbj2 may have been binding as substrate to tbhsp70.c , thus resulting in the decreased suppression of mdh aggregation that was observed . the ability of tbj2 to stimulate the basal atpase activity of tbhsp70.c and tchsp70b was investigated . tbhsp70.c was determined to have a basal atpase activity of 7.6 nmol pi / min / mg and tchsp70b a basal atpase activity of 18.3 nmol pi / min / mg . the basal atpase activity of tbhsp70.c and tchsp70b was represented as 100% ( figure 5 ) . equal concentrations of tbj2 and tbhsp70.c resulted in a significant 1.62-fold stimulation of the basal atpase activity of tbhsp70.c ( figure 5 ) . equal concentrations of tbj2 and tchsp70b caused a slightly lower but still significant 1.39-fold stimulation of tchsp70b basal atpase activity ( figure 5 ) . tbj2 at 2-fold molar excess to tbhsp70.c and tchsp70b resulted in a significant 2.88-fold enhancement of the basal atpase activity of tbhsp70.c and a significant 2.62-fold stimulation of tchsp70b atpase activity ( figure 5 ) . tbhsp70.c , tchsp70b , and tbj2 denatured by boiling displayed no atpase activity ( data not shown ) . recombinant tbhsp70.c purified from e. coli bb1994 cells was used as a positive control . even though there was no notable difference in the tbhsp70.c protein levels on the sds - page gel , tbhsp70.c protein expression proved to be inducible by heat stress , as shown by an increase in the top band in the western analysis ( figure 6 ) . the top band at 73 kda increased in intensity from 37c to 42c indicating that at 37c tbhsp70.c was expressed in bloodstream form cells and upon exposure to heat stress tbhsp70.c was upregulated . interestingly , the expression level of the non - specific 68 kda band , detected by anti - tbhsp70.c peptide antibodies , remained constant upon heat shock , suggesting it to be a separate protein and not a degradation product of tbhsp70.c ( figure 6 ) . a study of the evolutionary relationships between kinetoplastid hsp70 proteins and typical hsp70 proteins has revealed a novel and divergent group of proteins , the orthologues tbhsp70.c , tchsp70.c , and lmhsp70.c . tbj2 was selected as a probable co - chaperone of tbhsp70.c due to the presence of its functional j - domain and because of its predicted cytosolic localization ( data not shown ) . when studying molecular chaperones , the presence of dnak is a major concern due to the possibility that basal expression of bacterial or co - purified dnak could mask the chaperone activity of the t. brucei heat shock proteins under investigation . the co - purification of dnak is likely as a result of denatured exposed surfaces of the target protein interacting with dnak by acting as a substrate . using an e. coli dnak - minus strain and modifications to the purification protocol , dnak was eliminated from the tbhsp70.c and the tbj2 and tchsp70b purifications , respectively . amongst the various roles of hsp70 proteins demonstration of the holdase - function , by suppressing protein aggregation , is used to display chaperone activity [ 48 , 49 ] . the model thermolabile substrate mdh was used in this study to investigate the ability of tbhsp70.c and tbj2 , in isolation and in partnership , to suppress the heat - induced aggregation of mdh . a second substrate , rhodanese , was selected to investigate whether the substrate binding specificity of tbhsp70.c for rhodanese would differ considering the presence of fewer hydrophobic residues within rhodanese than in mdh . tbhsp70.c was shown to bind and suppress aggregation of both rhodanese and mdh , thereby displaying typical chaperone activity . however , tbhsp70.c suppressed aggregation of mdh to a greater degree than was observed for rhodanese . once bound , the substrate is transferred to hsp70 by the j - protein ; at the same time hsp40 stabilizes the hsp70-substrate association by stimulating the atpase activity of hsp70 . a similar result was seen for the homologue of tbj2 , tcj2 , in its ability to independently suppress aggregation of mdh . in the presence of tbhsp70.c the effect of tbj2 on both tbhsp70.c and tchsp70b was additive rather than synergistic when using mdh . the ability of a type iii hsp40 protein , tbj1 , to function as a co - chaperone was supported by the findings that tbj1 assisted trypanosoma cruzi hsp70 and medicago sativa hsp70 in suppression of mdh aggregation . suppression of protein aggregation assays has also been used to successfully determine the effect of small molecule modulators on the abilities of pfhsp70 proteins to suppress the aggregation of alcohol dehydrogenase and mdh . the ability of tbhsp70.c to successfully suppress aggregation of two individual substrates was not surprising . even though some conservation has been lost in the tbhsp70.c hydrophobic arch , some residues are replaced by acidic and aliphatic residues and the hydrophobic pocket , represented by the valine residue , is well conserved ( data not shown ) . this data would suggest that tbj2 acts as a co - chaperone by means of its j - domain interacting with the atpase domain of tbhsp70.c , either recruiting substrates to the hsp70 or merely mediating the interaction of the hsp70 with its substrate and stimulating the atpase activity of the hsp70 . yeast hsp40 , ydj1p , was shown to stimulate the atpase activity of yeast hsp70 , ssa1p , by 6 - 8-fold [ 18 , 61 , 62 ] . tbj2 , homologue of tcj2 , demonstrated a greater stimulation of tbhsp70.c atpase activity than was observed for tcj2-stimulated atpase activity of tchsp70b . the low stimulation of the atpase activity of tchsp70 by tcj2 was suggested to be as a result of a masking effect of the already very high basal atpase activity of tchsp70b , reported to be 100 times greater than that observed for human hsp70 . further characterization of the atpase activity of tbhsp70.c would entail investigating the activation of the atpase activity of tbhsp70.c by various substrates . maximal stimulation of the atpase activity of hsp70 proteins has been demonstrated to take place in the presence of both co - chaperone and substrate . a study in which t. b. brucei bloodstream form cultures were exposed to heat shock resulted in the upregulation of the expression levels of tbhsp70.c . this finding would imply that tbhsp70.c is required for protein quality control not only under normal conditions but also when the cell is placed under thermal stress . this is the first report of data showing that the expression levels of tbhsp70.c are increased by heat stress . the ability of tbj2 to significantly enhance the basal atpase activity of tbhsp70.c suggests that tbj2 functions as a co - chaperone of tbhsp70.c . however , further experimental analyses including binding studies and co - localization will need to be performed to conclusively confirm tbj2 as co - chaperone of tbhsp70.c . furthermore , the possibility of other cytoplasmic tbj proteins functioning as co - chaperones of tbhsp70.c will need to be investigated . due to its atypical features , tbhsp70.c is likely to interact with a specific set of protein substrates in the cell . tbhsp70.c is the first t. brucei hsp70 protein to be functionally characterized , along with its probable partnership with co - chaperone tbj2 . characterization of these and other chaperone / co - chaperone interactions could further enhance understanding of the cell biology of t. brucei .

there is urgency to the problem of the need for efficacious drug treatment in the management of alzheimer s disease with psychosis ( ad+p ) . ad+p has grave consequence , particularly for caregivers , as it is associated with physical aggression , and caregivers are most often the victims.13 violent behavior that is directed towards caregivers is disruptive of care at the very least , but can also be dangerous for those charged with providing a safe environment for those suffering with disease . not only is aggressive behavior inherently troublesome for families , but perhaps in part because of the difficulty of managing these behaviors , the rates of institutionalization are much higher in ad patients who have exhibited violent behavior.4,5 notably , in one study of participants with elevated levels of physical aggression , 80% had delusions.6 this suggests that psychosis at least aggravates the clinical course of ad , and creates profound caregiver stress that compounds the difficulty of taking care of a loved one with the cognitive impairment that is a defining feature of the illness . unfortunately , the current treatment approaches to ad+p , relying mostly on agents approved for the treatment of schizophrenia , have been largely disappointing . the efficacy of the atypical antipsychotics in the treatment of behavioral and psychological symptoms of dementia derived from randomized placebo - controlled trials has been parsed many ways , owing in part to the broad range of rating scales employed as outcome measures and the spectrum of neurobehavioral syndromes associated with ad . indeed , trials that have enrolled over 5,000 patients to date have included a number of different psychometric scales of behavioral impairment , including the neuropsychiatric inventory ( npi ) ; the brief psychiatric rating scale ( bprs ) ; the cohen - mansfield agitation inventory ; the clinical global impression of change ; the clinical global impression of severity ; and the behavioral pathology in alzheimer s disease ( behave - ad ) . evaluating the efficacy of currently available treatments in ad+p is challenging , as the only available data comprises subscales distilled from comprehensive agitation inventories . the most recent and most comprehensive meta - analysis to date of randomized placebo - controlled trials in the treatment of the behavioral and psychological symptoms of dementia7 suggests a consistent superiority of atypical agents ( aripiprazole , quetiapine , olanzapine , risperidone ) over placebo as measured with the aforementioned behavioral rating scales . in fact , when atypical agents are amalgamated and their efficacy data is compared with placebo , the most recent meta - analysis reveals that antipsychotics demonstrate a convincing superiority over placebo in the treatment of behavioral disturbances . calculated mean differences and 95% confidence intervals on behavioral rating scales support this contention : bprs , 1.58 ( 2.52 to 0.65 ) ; cohen - mansfield agitation inventory , 1.84 ( 3.01 to 0.61 ) ; npi , 2.81 ( 4.35 to 1.28 ) ; clinical global impression of change , 0.32 ( 0.44 to 0.20 ) ; clinical global impression of severity , 0.19 ( 0.3 to 0.09 ) . these data , especially in light of the bprs differences , could be taken as evidence that atypical antipsychotics are efficacious in the treatment of ad+p . a careful look at these studies , however , suggests the ironic truth that amongst therapies for behavioral disturbances in ad , the atypical antipsychotics are inconsistent at best in the treatment of psychosis . positive results from the above referenced meta - analytic data on bprs total score outcomes do not necessarily reflect improvement in psychosis . the bprs , ( overall and gorham 1962 , psychological report)8 an inventory of psychopathology that has been extensively utilized in the evaluation of outcomes in schizophrenia and other psychotic illnesses , is a remarkably broad 18-item scale.9 only a few items could be interpreted with any relevance in ad+p : hostility , suspiciousness , hallucinatory behavior , and perhaps unusual thought content and uncooperativeness . although not included in the most recent meta - analysis , data on psychosis can be extracted from the majority of placebo - controlled clinical trials of antipsychotics in dementia populations by looking at psychosis subscores derived from the npi , the behave - ad , and the bprs ( table 1 ) . olanzapine has been evaluated in comparison with placebo over 636 weeks in nursing home patients and outpatients.1012 in the first study , olanzapine at a dose of 5 mg and 10 mg , but not 15 mg , was superior to placebo on the psychosis subscale total score derived from the nursing home version of the npi , the npi - nh ( table 1).10 in a second study of 1.0 mg , 2.5 mg , 5.0 mg , and 7.5 mg doses of olanzapine , only the 7.5 mg dose was superior to placebo as measured by the npi - nh psychosis subscale.11 in the clinical antipsychotics trials of intervention effectiveness - alzheimer s disease ( catie - ad ) study , an outpatient study comparing flexible doses of atypical antipsychotics ( olanzapine , quetiapine , risperidone ) to placebo , olanzapine at a mean dose of 5.5 mg was not superior to placebo on the psychosis factor of the bprs.12 two 10-week randomized placebo - controlled trials have investigated aripiprazole in the treatment of ad+p.13,14 in the first study , an outpatient study employing a flexible dose , aripiprazole at a mean dose of 10 mg demonstrated superiority to placebo on the bprs psychosis subscale but not on the npi psychosis subscale.13 in a nursing home study , comparing aripiprazole at doses of 2 mg , 5 mg , and 10 mg , only 10 mg was superior to placebo on the npi - nh psychosis subscale.14 two studies , of 6 weeks and 36 weeks duration in nursing facilities and outpatient populations , respectively , have evaluated the efficacy of quetiapine in comparison with placebo for the treatment of ad+p.12,15 no evidence of efficacy in treating psychosis was generated for quetiapine from either study on the basis of the bprs psychosis subscales . prior to catie - ad , there were four randomized placebo - controlled trials of risperidone in ad+p , ranging from 836 weeks in both the nursing home residents and outpatients.1619 a meta - analysis of these four studies revealed that risperidone was superior to placebo on the behave - ad psychosis subscale , with an effect size of 0.154 ( 95% confidence interval 0.0380.269 ) , suggesting only a modest effect of drug treatment.20 data from catie - ad also evidenced a benefit of risperidone over placebo on the bprs psychosis factor , the only agent to beat placebo on this measure.12 the data on atypical antipsychotics , while inconsistent , do suggest some utility for most agents , with some superiority for risperidone ; however , the magnitudes of treatment effects are not robust , and highly efficacious agents are desperately needed . one solution to this problem is to develop novel treatments based on the emerging neurobiology of ad+p , rather than relying on existing treatments for other psychotic conditions . psychotic symptoms in ad comprise delusions , with a reported prevalence of between 30%40%,2124 and , to a smaller extent , hallucinations , with a prevalence of 5%20%.21,23,2527 the heritability of ad+p has been firmly established28 and is estimated to be between 30%60%.29 the persistence of untreated psychosis over time in alzheimer s disease in individual patients has not been firmly established , but available evidence suggests that approximately 40%50% of psychotic individuals will continue to manifest psychotic symptoms after 1 year.30 rather than the bizarre delusions and complex network of unsubstantiated belief systems commonly seen in schizophrenia typified by logical impossibility and experiences alien to ordinary life situations,31 the delusions in ad+p generally involve rather narrow elaborations and distortions of actual experience . the delusions of ad have been largely reconciled into two categories,32,33 delusions of persecution ( eg , theft , harm , jealousy , abandonment)2,3436 and delusions of misidentification ( eg , phantom boarder , television images are animate , capgras syndrome , house is not one s home),2,3538 the latter having once been conceptualized as representing perceptual abnormalities , but actually representing ideational fixations.32,39,40 a category of autobiographical delusions,32 for instance that a dead relative is still alive , has also been suggested,41 but these may be regarded as an extension of the misidentification domain . a few theoretical models have been developed to explain the common emergence of psychosis in ad particularly focused on delusions in those otherwise not afflicted with mental illness . carefully reviewed by reeves et al32 the earliest of these ratiocinations suggested that psychosis results from a logical attempt to understand the environment in the context of degraded cognitive integrity.42 for instance , the common delusions in ad theft , jealousy , misidentification , abandonment , phantom boarder , television images are animate21,43,44 are possible narrative confabulations that fill in the gaps or normalize the experiences of those confronted with a history they have become unmoored from , and who rely on a gradually eroding apparatus for discriminating object from representation . the most intuitive support for this model comes from the most prevalent delusion in ad , that people are hiding or stealing things,4547 a potential confabulation that is entirely consistent with the primary cognitive deficit in ad , accelerated forgetting . lack of corrective judgment from other cortical structures.49 these conceptualizations , while interesting , are broad and speculative and do not really generate any testable scientific predictions . more recent work has been focused on concrete neurobiological correlates of psychotic ad , and from that a glimpse of disease pathophysiology has begun to emerge . the earliest specific reference to the problem of psychosis in the modern literature on alzheimer s disease emerges in a russian neuropsychiatric journal in 1964.50 it was another 20 years before critical observations were made that contributed to a reconceptualization of psychotic phenomena as expressions of a unique pathophysiological syndrome rather than a mere behavioral nuisance . in the context of searching for clinical signs that would predict the velocity of decline in ad , psychosis in addition to myoclonus and extrapyramidal signs was found to be an early marker heralding a hastened deterioration.51,52 this observation was quickly replicated in several studies reinforcing the prognostic value of psychosis in relationship to decline.53,54 studies focused on the exploration of risk factors for psychotic symptoms in ad have also consistently identified a greater burden of cognitive impairment as an independent risk factor for psychosis , with acceleration of decline emerging prior to the onset of psychosis.24,46,55,56 the features of accelerated cognitive impairment in ad+p suggest some degree of frontal localization . in one study of ad+p and alzheimer s disease without psychosis ( ad - p ) that compared subjects of similar duration of illness , age , and education , ad+p subjects evidenced more impairment on tasks of frontal - lobe function , including the wisconsin card sorting test and the similarities subtest of the wechsler adult intelligence scale.57 recent studies suggest that working memory performance , a test of frontal function depending largely on the dorsolateral prefrontal cortex , enjoys a unique association with psychosis in ad . early evidence for this emerged from an epidemiological study of ad+p risk factors , wherein performance on the digit span , a task of working memory , was observed to be a robust predictor of future psychosis in ad.24 impressively , a one - standard - deviation improvement in this task reduced the risk of developing hallucinations or delusions by two thirds . in our own study of cognition in ad+p , we evaluated ad and performed stepwise regression of cognitive domains in order to determine whether any cognitive domains ( memory , executive function , language ) predicted psychosis . we found that of the wide spectrum of domains of cognition interrogated in the analysis , only working memory interacted with psychosis.58 in order to replicate the finding and to determine whether working memory , beyond its mere association with the state of psychosis , might track uniquely with the accelerated decline in ad+p , we performed an analysis of cognitive data from the alzheimer s disease neuroimaging initiative ( adni ) study database . the adni database includes longitudinal cognitive data on subjects some of whom destined to become psychotic , as evidenced by psychometric ratings , over the course of the study . we hypothesized that digit - span performance , amongst available cognitive metrics , would decline more rapidly over time as a marker of the accelerated decline of ad+p . results of the analysis demonstrated that digit - span performance is one of only two cognitive measures that decline more rapidly in ad+p when compared with ad - p.59 it is not clear how impairment in working memory is etiologically related to ad+p . one possibility is that it is merely a byproduct of frontal degeneration , and not directly etiologically related ; another is that accelerated frontal degeneration engenders working memory deficits , and that failures in working memory impair successful environmental surveillance in ad , culminating in misinterpretations and psychosis . evidence of a more aggressive cognitive phenotype as a marker of virulence would predict an acceleration of overall decline and decreased survival rates in those with ad+p . it is puzzling , given the established relationship between rate of cognitive decline and mortality in ad,60,61 that most,53,6065 but not all,66 early studies did not find an increased mortality rate in ad+p . more recent studies , with larger sample sizes and an enhanced follow - up with a more powerful statistical approach ( cox proportional hazard regression analysis ) have consistently observed the predictive value of psychosis in ad survival.6770 a parsimonious hypothesis that explains the accelerated cognitive deterioration with some specificity and the early demise of patients with ad+p is that psychosis is a behavioral expression of a more - rapid neurodegenerative process that may be regional . what are the neurochemical and neuropathological hallmarks of this distinct ad syndrome ? can answers to these questions point the way forward in developing new anti - ad+p treatments ? in the search for cerebral localization of deficit patterns in ad+p , investigators have , with varying levels of success related to sample sizes and heterogeneous subject composition , availed themselves of structural and functional neuroimaging . the literature on imaging in ad+p has been recently reviewed and thoughtfully organized by murray et al71 who separated the studies by methodological approach , including computed tomography , magnetic resonance imaging ( mri ) , single - photon emission computed tomography , and [ f]-fluorodeoxyglucose position emission tomography ( pet ) studies . as the authors highlight , with some variability and inconsistency , the preponderance of imaging evidence across methodologies implicate the frontal lobes.71 a computed tomography study investigating regional brain atrophy in ad+p found that subjects with delusions demonstrated frontal and temporal patterns of asymmetric atrophy , compared with ad - p subjects.72 mri studies of ad+p have generally implicated the frontal cortex , with both orbitofrontal and medial frontal involvement.73,74 pet studies with only one exception75 have consistently shown frontal hypometabolism in ad+p relative to ad - p.7680 in an analysis of a large , well - controlled sample drawn from the adni database , we observed a specific relationship between orbitofrontal hypometabolism and ad+p.59 an early theory promulgated in an effort to explain the prevalence of psychosis in ad suggested that a preservation of dopaminergic tone in the context of early cholinergic deficits may result in a relative hyperdopaminergic state , with resultant psychosis.81 however evidence for a relative excess of dopaminergic tone has been limited . an early postmortem study of neurotransmission in psychotic ad did not identify any abnormalities in markers of dopamine signaling.82 one small postmortem study suggested a selective increase in d3 receptor availability.83 further , using [ c]-raclopride pet , a modest increase in tracer binding in the striatum was observed in a cohort of delusional patients , suggesting some increase in d3 availability.84 yet , a recent comprehensive postmortem study of monoaminergic correlates of behavioral symptoms in ad did not observe any relationship between alterations in dopamine or dopamine metabolite concentrations in nine behaviorally and neurochemically relevant brain regions and psychotic symptoms in ad.85 perturbations in serotonergic systems have been reported in postmortem studies of psychotic ad . a reduction of serotonin ( 5-ht ) has been reported in several studies across several brain regions,82,86 as have altered 5-ht6 receptor function87 and a reduction in cell counts in the dorsal raphe nucleus.88 in a recent study , a significant reduction in serotonergic tone comprising lower 5-hydroxyindoleacetic acid concentrations in the hippocampus and lower 5-ht concentrations in the superior temporal gyrus and cerebral cortex , correlated with dementia severity.85 it may be that the association observed in previous studies between psychosis in ad and reduced 5-ht may be related to regional disease severity in psychotic ad , and not an independent risk factor for the emergence of the syndrome . although currently available agents have not achieved satisfying rates of success in treating ad+p , continued exploration of perturbations in neurotransmitter systems in ad+p , together with the advent of more - sophisticated supportive therapeutics , may prove helpful in treating this condition . the classical protein pathologies in alzheimer s disease comprise extracellular amyloid - beta ( a ) plaques and intracellular neurofibrillary tangles , composed primarily of hyperphosphorylated tau proteins . as ad+p is a more - rapidly progressing disease variant , with evidence of some localization of disease to the frontal cortex , neuropathological correlation studies are critical in the search for disease etiology . in general , evidence collected from neuropathological studies supports an association of ad+p with tau , rather than a pathology.44 this is not entirely surprising , since in ad distribution of tangle pathology , rather than a pathology , correlates most closely with disease severity.89 postmortem studies consistently point to a heavier burden of tangle pathology in ad+p , with some localization in the frontal cortex.82,88,90,91 in order to explore tau pathology in ad+p , our group accessed the adni database to look for cerebrospinal fluid ( csf ) evidence of increased tau in ad+p . previous reports have linked elevations of csf total tau with the postmortem burden of neurofibrillary tangles,92,93 so we approached the data with the hypothesis that we would find elevations in total tau in ad+p relative to ad - p as a reflection of tangle pathology . indeed , we found that of the three core csf ad biomarkers ( a , total tau , phospho - tau ) , only elevation of total tau in csf was statistically significantly associated with psychosis in ad , driven by a robust association in females with ad+p.94 in a contemporaneous postmortem immunohistochemical study of the frontal cortex conducted by colleagues in the laboratory of dr robert sweet , hyperphosphorylated tau ( the main component of neurofibrillary tangles)95 was overabundant in subjects with ad+p compared with ad - p , strengthening the association of ad+p with tau.96 recently , in collaboration with dr . sweet , we conducted a biochemical analysis of phospho - tau epitopes in ad+p using a high - sensitivity enzyme - linked immunosorbent assay ( elisa ) developed in the laboratory of dr peter davies.97 the elisas utilize highly specific monoclonal antibodies to phospho - tau epitopes that are representative of a range of tau pathology , from neuritic to pretangle to mature tangles.98 our experiments were conducted on postmortem tissue samples from the superior frontal gyri of 71 subjects . as -synuclein pathology is known to contribute to a psychotic phenotype in dementia with lewy bodies ( dlb ) , we controlled for -synuclein pathology.99 we observed in women with ad+p , but not in men with ad+p , a robust association with tau pathology.100 in females , while levels of nonpathogenic total tau as measured with da31 antibody elisa did not differ between ad+p and ad - p groups , pathogenic phospho - tau species did differ significantly . ad+p females had significantly higher levels of phosphorylated tau than did ad - p females at each epitope interrogated : ser396/404 ( phf-1 ) , ser202 ( cp13 ) , thr231 ( rz3 ) , and ser199/202/thr205 ( at8 ) . males with ad+p , all of whom were not suffering with clinical dlb prior to death , nonetheless had an increase in -synuclein pathology . in males this suggests that -synuclein may make a unique contribution to ad+p , while in females tau pathology may be the source of vulnerability . previously conducted neuropathology studies have not been parsed by sex . there is evidence , however , that female sex is an independent risk factor for psychosis in ad.30,46,101,102 there is some support for biological underpinnings of this distinction from imaging studies . an mri study in ad+p evidenced reduced frontotemporal cortical thickness in women , rather than in men , with ad+p.74 additionally , cerebral perfusion patterns in ad+p have been shown to be sex - specific.103 the significance of the sex specificity of the association of ad+p with tau pathology is not yet clear , and more research is necessary to confirm this relationship . the published research in ad+p points toward an accelerated cognitive decline , with a particular burden of impairment affecting performance on frontal tasks . there is strong evidence that degeneration is accelerated in the frontal cortex , likely mediated by tau pathology , especially in females . . there may be some sex specificity to this model , and the degree to which the frontal tauopathy of ad+p is a female problem is not yet clear . however , the model , in identifying pathogenic proteins and localization , does enable translational research that may aid in illuminating in just what regions , and to what degree , tau pathology may contribute to the psychotic phenotype . animal models of psychiatric disease pose a particular challenge , in that the core of psychiatric illness is experience , something inaccessible in observations of animals that are language deficient . however , there is a long history of observing animals under conditions designed to serve as analogues of precipitants of human behavioral disorders and of studying physiological changes in these animals ; a more recent history of observing animals with physiological changes known to be associated with human disease and of studying behavioral changes . in ad , translational research relies to a large degree on the advent of transgenic mice carrying autosomal dominant mutations in human genes that are known to cause the disease , or to cause pathology associated with the disease . the mice , depending on the identity of the mutation , produce an abundance of a-driven plaque or tau - driven neurofibrillary tangle pathology and display dense cognitive deficits.104 curiously , to date , these models have only infrequently been employed to study the behavioral syndromes so common in ad . are there outcome measures in mice relevant to psychosis that could be utilized in such an exploration ? if so , could extant mouse models be employed to elucidate the neuropathological substrate responsible for ad+p ? if ad+p in humans emerges , in part , secondary to frontal neurodegeneration driven by tau , could a mouse model aid in a more - precise localization of the frontal deficits responsible ? currently employed paradigms for modeling psychosis in mice include induction of locomotor hyperactivity and disruption of sensorimotor gating , quantified with prepulse inhibition ( ppi ) of acoustic startle.105 locomotor hyperactivity , as a model of psychosis in rodents resulting from exogenous agents promoting dopamine release , is the conceptual offspring of the hyperdopaminergic hypothesis of schizophrenia;106 deficits of ppi represent an endophenotype of psychosis itself that has been associated with schizophrenia and may have broad relevance in all psychotic states.107 ppi is defined as the reduction of the intensity of motor startle in response to a startle stimulus ( for example , acoustic ) when the stimulus is preceded by a diminished , nonstartle stimulus . in a normal state , the startle induced by the startle stimulus should be significantly gated by the attention paid to the preceding stimulus . in psychosis , this ability to inhibit startle is disrupted by a failure of normal gaiting , perhaps representing a diminished capacity to monitor environmental stimuli appropriately.108 ppi of acoustic startle as a phenotypic marker of psychosis may be best described as the interface of psychosis and cognition,105,109 representing a cognitive failure that potentially influences environmental surveillance and may promote psychotic misinterpretation . the confluence of cognitive impairment and environmental misapprehension implicit in ppi deficiency makes ppi an attractive outcome measure in investigating potential animal models of ad+p . in order to develop a preclinical mouse model of ad+p , we recently selected an ad model manifesting early frontal tauopathy , the transgenic rtg(taup301l)4510 mouse,110 and characterized ppi deficits over time in relationship to tau pathology.111 we demonstrated that ppi deficits accrued over time in female rtg(taup301l)4510 mice , and that these deficits were driven by the same pathogenic hyperphosphorylated tau molecules that we had previously observed in the frontal cortex of human female ad+p subjects.100 this suggests that the rtg(taup301l)4510 mouse may be a candidate for preclinical study of ad+p biology and the development of novel therapeutics . there is more work to be done prior to settling on any animal model of ad+p . future studies involving this model will include expanding the phenotypic characterization of this mouse beyond ppi to include other cognitive and behavioral markers of psychosis , and exploring the sex specificity of the observed behavioral characteristics . the way forward in the treatment of ad+p may transcend traditional psychiatric treatments of psychosis and may involve the reduction of pathogenic proteins rather than supporting aberrant neurotransmitter systems that are the downstream effects of disease pathology . several tau reduction agents are currently in the drug development stage for the treatment of ad , and even if they do not provide a cure for the primary illness , they warrant exploration in the service of combating a particularly morbid and common manifestation of the disease .

psychotic symptoms emerging in the context of neurodegeneration as a consequence of alzheimer s disease was recognized and documented by alois alzheimer himself in his description of the first reported case of the disease . over a quarter of a century ago , in the context of attempting to develop prognostic markers of disease progression , psychosis was identified as an independent predictor of a more - rapid cognitive decline . this finding has been subsequently well replicated , rendering psychotic symptoms an important area of exploration in clinical history taking above and beyond treatment necessity as their presence has prognostic significance . further , there is now a rapidly accreting body of research that suggests that psychosis in alzheimer s disease ( ad+p ) is a heritable disease subtype that enjoys neuropathological specificity and localization . there is now hope that the elucidation of the neurobiology of the syndrome will pave the way to translational research eventuating in new treatments . to date , however , the primary treatments employed in alleviating the suffering caused by ad+p are the atypical antipsychotics . these agents are approved by the us food and drug administration for the treatment of schizophrenia , but they have only marginal efficacy in treating ad+p and are associated with troubling levels of morbidity and mortality . for clinical approaches to ad+p to be optimized , this syndrome must be disentangled from other primary psychotic disorders , and recent scientific advances must be translated into disease - specific therapeutic interventions . here we provide a review of atypical antipsychotic efficacy in ad+p , followed by an overview of critical neurobiological observations that point towards a frontal , tau - mediated model of disease , and we suggest a new preclinical animal model for future translational research .

Introduction Phenomenology in AD+P: theoretical considerations Cognitive course and mortality in AD+P: evidence for a distinct syndrome? Imaging correlates in AD+P: a predominance of frontal localization Neurotransmission in AD+P Neuropathology in AD+P: a focus on tau Future directions: a pathology-guided approach

there is urgency to the problem of the need for efficacious drug treatment in the management of alzheimer s disease with psychosis ( ad+p ) . not only is aggressive behavior inherently troublesome for families , but perhaps in part because of the difficulty of managing these behaviors , the rates of institutionalization are much higher in ad patients who have exhibited violent behavior.4,5 notably , in one study of participants with elevated levels of physical aggression , 80% had delusions.6 this suggests that psychosis at least aggravates the clinical course of ad , and creates profound caregiver stress that compounds the difficulty of taking care of a loved one with the cognitive impairment that is a defining feature of the illness . unfortunately , the current treatment approaches to ad+p , relying mostly on agents approved for the treatment of schizophrenia , have been largely disappointing . the efficacy of the atypical antipsychotics in the treatment of behavioral and psychological symptoms of dementia derived from randomized placebo - controlled trials has been parsed many ways , owing in part to the broad range of rating scales employed as outcome measures and the spectrum of neurobehavioral syndromes associated with ad . indeed , trials that have enrolled over 5,000 patients to date have included a number of different psychometric scales of behavioral impairment , including the neuropsychiatric inventory ( npi ) ; the brief psychiatric rating scale ( bprs ) ; the cohen - mansfield agitation inventory ; the clinical global impression of change ; the clinical global impression of severity ; and the behavioral pathology in alzheimer s disease ( behave - ad ) . the most recent and most comprehensive meta - analysis to date of randomized placebo - controlled trials in the treatment of the behavioral and psychological symptoms of dementia7 suggests a consistent superiority of atypical agents ( aripiprazole , quetiapine , olanzapine , risperidone ) over placebo as measured with the aforementioned behavioral rating scales . in fact , when atypical agents are amalgamated and their efficacy data is compared with placebo , the most recent meta - analysis reveals that antipsychotics demonstrate a convincing superiority over placebo in the treatment of behavioral disturbances . these data , especially in light of the bprs differences , could be taken as evidence that atypical antipsychotics are efficacious in the treatment of ad+p . a careful look at these studies , however , suggests the ironic truth that amongst therapies for behavioral disturbances in ad , the atypical antipsychotics are inconsistent at best in the treatment of psychosis . the bprs , ( overall and gorham 1962 , psychological report)8 an inventory of psychopathology that has been extensively utilized in the evaluation of outcomes in schizophrenia and other psychotic illnesses , is a remarkably broad 18-item scale.9 only a few items could be interpreted with any relevance in ad+p : hostility , suspiciousness , hallucinatory behavior , and perhaps unusual thought content and uncooperativeness . although not included in the most recent meta - analysis , data on psychosis can be extracted from the majority of placebo - controlled clinical trials of antipsychotics in dementia populations by looking at psychosis subscores derived from the npi , the behave - ad , and the bprs ( table 1 ) . olanzapine has been evaluated in comparison with placebo over 636 weeks in nursing home patients and outpatients.1012 in the first study , olanzapine at a dose of 5 mg and 10 mg , but not 15 mg , was superior to placebo on the psychosis subscale total score derived from the nursing home version of the npi , the npi - nh ( table 1).10 in a second study of 1.0 mg , 2.5 mg , 5.0 mg , and 7.5 mg doses of olanzapine , only the 7.5 mg dose was superior to placebo as measured by the npi - nh psychosis subscale.11 in the clinical antipsychotics trials of intervention effectiveness - alzheimer s disease ( catie - ad ) study , an outpatient study comparing flexible doses of atypical antipsychotics ( olanzapine , quetiapine , risperidone ) to placebo , olanzapine at a mean dose of 5.5 mg was not superior to placebo on the psychosis factor of the bprs.12 two 10-week randomized placebo - controlled trials have investigated aripiprazole in the treatment of ad+p.13,14 in the first study , an outpatient study employing a flexible dose , aripiprazole at a mean dose of 10 mg demonstrated superiority to placebo on the bprs psychosis subscale but not on the npi psychosis subscale.13 in a nursing home study , comparing aripiprazole at doses of 2 mg , 5 mg , and 10 mg , only 10 mg was superior to placebo on the npi - nh psychosis subscale.14 two studies , of 6 weeks and 36 weeks duration in nursing facilities and outpatient populations , respectively , have evaluated the efficacy of quetiapine in comparison with placebo for the treatment of ad+p.12,15 no evidence of efficacy in treating psychosis was generated for quetiapine from either study on the basis of the bprs psychosis subscales . prior to catie - ad , there were four randomized placebo - controlled trials of risperidone in ad+p , ranging from 836 weeks in both the nursing home residents and outpatients.1619 a meta - analysis of these four studies revealed that risperidone was superior to placebo on the behave - ad psychosis subscale , with an effect size of 0.154 ( 95% confidence interval 0.0380.269 ) , suggesting only a modest effect of drug treatment.20 data from catie - ad also evidenced a benefit of risperidone over placebo on the bprs psychosis factor , the only agent to beat placebo on this measure.12 the data on atypical antipsychotics , while inconsistent , do suggest some utility for most agents , with some superiority for risperidone ; however , the magnitudes of treatment effects are not robust , and highly efficacious agents are desperately needed . psychotic symptoms in ad comprise delusions , with a reported prevalence of between 30%40%,2124 and , to a smaller extent , hallucinations , with a prevalence of 5%20%.21,23,2527 the heritability of ad+p has been firmly established28 and is estimated to be between 30%60%.29 the persistence of untreated psychosis over time in alzheimer s disease in individual patients has not been firmly established , but available evidence suggests that approximately 40%50% of psychotic individuals will continue to manifest psychotic symptoms after 1 year.30 rather than the bizarre delusions and complex network of unsubstantiated belief systems commonly seen in schizophrenia typified by logical impossibility and experiences alien to ordinary life situations,31 the delusions in ad+p generally involve rather narrow elaborations and distortions of actual experience . carefully reviewed by reeves et al32 the earliest of these ratiocinations suggested that psychosis results from a logical attempt to understand the environment in the context of degraded cognitive integrity.42 for instance , the common delusions in ad theft , jealousy , misidentification , abandonment , phantom boarder , television images are animate21,43,44 are possible narrative confabulations that fill in the gaps or normalize the experiences of those confronted with a history they have become unmoored from , and who rely on a gradually eroding apparatus for discriminating object from representation . the earliest specific reference to the problem of psychosis in the modern literature on alzheimer s disease emerges in a russian neuropsychiatric journal in 1964.50 it was another 20 years before critical observations were made that contributed to a reconceptualization of psychotic phenomena as expressions of a unique pathophysiological syndrome rather than a mere behavioral nuisance . in the context of searching for clinical signs that would predict the velocity of decline in ad , psychosis in addition to myoclonus and extrapyramidal signs was found to be an early marker heralding a hastened deterioration.51,52 this observation was quickly replicated in several studies reinforcing the prognostic value of psychosis in relationship to decline.53,54 studies focused on the exploration of risk factors for psychotic symptoms in ad have also consistently identified a greater burden of cognitive impairment as an independent risk factor for psychosis , with acceleration of decline emerging prior to the onset of psychosis.24,46,55,56 the features of accelerated cognitive impairment in ad+p suggest some degree of frontal localization . in one study of ad+p and alzheimer s disease without psychosis ( ad - p ) that compared subjects of similar duration of illness , age , and education , ad+p subjects evidenced more impairment on tasks of frontal - lobe function , including the wisconsin card sorting test and the similarities subtest of the wechsler adult intelligence scale.57 recent studies suggest that working memory performance , a test of frontal function depending largely on the dorsolateral prefrontal cortex , enjoys a unique association with psychosis in ad . early evidence for this emerged from an epidemiological study of ad+p risk factors , wherein performance on the digit span , a task of working memory , was observed to be a robust predictor of future psychosis in ad.24 impressively , a one - standard - deviation improvement in this task reduced the risk of developing hallucinations or delusions by two thirds . we found that of the wide spectrum of domains of cognition interrogated in the analysis , only working memory interacted with psychosis.58 in order to replicate the finding and to determine whether working memory , beyond its mere association with the state of psychosis , might track uniquely with the accelerated decline in ad+p , we performed an analysis of cognitive data from the alzheimer s disease neuroimaging initiative ( adni ) study database . results of the analysis demonstrated that digit - span performance is one of only two cognitive measures that decline more rapidly in ad+p when compared with ad - p.59 it is not clear how impairment in working memory is etiologically related to ad+p . it is puzzling , given the established relationship between rate of cognitive decline and mortality in ad,60,61 that most,53,6065 but not all,66 early studies did not find an increased mortality rate in ad+p . more recent studies , with larger sample sizes and an enhanced follow - up with a more powerful statistical approach ( cox proportional hazard regression analysis ) have consistently observed the predictive value of psychosis in ad survival.6770 a parsimonious hypothesis that explains the accelerated cognitive deterioration with some specificity and the early demise of patients with ad+p is that psychosis is a behavioral expression of a more - rapid neurodegenerative process that may be regional . in the search for cerebral localization of deficit patterns in ad+p , investigators have , with varying levels of success related to sample sizes and heterogeneous subject composition , availed themselves of structural and functional neuroimaging . as the authors highlight , with some variability and inconsistency , the preponderance of imaging evidence across methodologies implicate the frontal lobes.71 a computed tomography study investigating regional brain atrophy in ad+p found that subjects with delusions demonstrated frontal and temporal patterns of asymmetric atrophy , compared with ad - p subjects.72 mri studies of ad+p have generally implicated the frontal cortex , with both orbitofrontal and medial frontal involvement.73,74 pet studies with only one exception75 have consistently shown frontal hypometabolism in ad+p relative to ad - p.7680 in an analysis of a large , well - controlled sample drawn from the adni database , we observed a specific relationship between orbitofrontal hypometabolism and ad+p.59 an early theory promulgated in an effort to explain the prevalence of psychosis in ad suggested that a preservation of dopaminergic tone in the context of early cholinergic deficits may result in a relative hyperdopaminergic state , with resultant psychosis.81 however evidence for a relative excess of dopaminergic tone has been limited . an early postmortem study of neurotransmission in psychotic ad did not identify any abnormalities in markers of dopamine signaling.82 one small postmortem study suggested a selective increase in d3 receptor availability.83 further , using [ c]-raclopride pet , a modest increase in tracer binding in the striatum was observed in a cohort of delusional patients , suggesting some increase in d3 availability.84 yet , a recent comprehensive postmortem study of monoaminergic correlates of behavioral symptoms in ad did not observe any relationship between alterations in dopamine or dopamine metabolite concentrations in nine behaviorally and neurochemically relevant brain regions and psychotic symptoms in ad.85 perturbations in serotonergic systems have been reported in postmortem studies of psychotic ad . a reduction of serotonin ( 5-ht ) has been reported in several studies across several brain regions,82,86 as have altered 5-ht6 receptor function87 and a reduction in cell counts in the dorsal raphe nucleus.88 in a recent study , a significant reduction in serotonergic tone comprising lower 5-hydroxyindoleacetic acid concentrations in the hippocampus and lower 5-ht concentrations in the superior temporal gyrus and cerebral cortex , correlated with dementia severity.85 it may be that the association observed in previous studies between psychosis in ad and reduced 5-ht may be related to regional disease severity in psychotic ad , and not an independent risk factor for the emergence of the syndrome . although currently available agents have not achieved satisfying rates of success in treating ad+p , continued exploration of perturbations in neurotransmitter systems in ad+p , together with the advent of more - sophisticated supportive therapeutics , may prove helpful in treating this condition . as ad+p is a more - rapidly progressing disease variant , with evidence of some localization of disease to the frontal cortex , neuropathological correlation studies are critical in the search for disease etiology . in general , evidence collected from neuropathological studies supports an association of ad+p with tau , rather than a pathology.44 this is not entirely surprising , since in ad distribution of tangle pathology , rather than a pathology , correlates most closely with disease severity.89 postmortem studies consistently point to a heavier burden of tangle pathology in ad+p , with some localization in the frontal cortex.82,88,90,91 in order to explore tau pathology in ad+p , our group accessed the adni database to look for cerebrospinal fluid ( csf ) evidence of increased tau in ad+p . indeed , we found that of the three core csf ad biomarkers ( a , total tau , phospho - tau ) , only elevation of total tau in csf was statistically significantly associated with psychosis in ad , driven by a robust association in females with ad+p.94 in a contemporaneous postmortem immunohistochemical study of the frontal cortex conducted by colleagues in the laboratory of dr robert sweet , hyperphosphorylated tau ( the main component of neurofibrillary tangles)95 was overabundant in subjects with ad+p compared with ad - p , strengthening the association of ad+p with tau.96 recently , in collaboration with dr . as -synuclein pathology is known to contribute to a psychotic phenotype in dementia with lewy bodies ( dlb ) , we controlled for -synuclein pathology.99 we observed in women with ad+p , but not in men with ad+p , a robust association with tau pathology.100 in females , while levels of nonpathogenic total tau as measured with da31 antibody elisa did not differ between ad+p and ad - p groups , pathogenic phospho - tau species did differ significantly . in males this suggests that -synuclein may make a unique contribution to ad+p , while in females tau pathology may be the source of vulnerability . there is evidence , however , that female sex is an independent risk factor for psychosis in ad.30,46,101,102 there is some support for biological underpinnings of this distinction from imaging studies . an mri study in ad+p evidenced reduced frontotemporal cortical thickness in women , rather than in men , with ad+p.74 additionally , cerebral perfusion patterns in ad+p have been shown to be sex - specific.103 the significance of the sex specificity of the association of ad+p with tau pathology is not yet clear , and more research is necessary to confirm this relationship . however , the model , in identifying pathogenic proteins and localization , does enable translational research that may aid in illuminating in just what regions , and to what degree , tau pathology may contribute to the psychotic phenotype . however , there is a long history of observing animals under conditions designed to serve as analogues of precipitants of human behavioral disorders and of studying physiological changes in these animals ; a more recent history of observing animals with physiological changes known to be associated with human disease and of studying behavioral changes . in ad , translational research relies to a large degree on the advent of transgenic mice carrying autosomal dominant mutations in human genes that are known to cause the disease , or to cause pathology associated with the disease . the mice , depending on the identity of the mutation , produce an abundance of a-driven plaque or tau - driven neurofibrillary tangle pathology and display dense cognitive deficits.104 curiously , to date , these models have only infrequently been employed to study the behavioral syndromes so common in ad . if ad+p in humans emerges , in part , secondary to frontal neurodegeneration driven by tau , could a mouse model aid in a more - precise localization of the frontal deficits responsible ? currently employed paradigms for modeling psychosis in mice include induction of locomotor hyperactivity and disruption of sensorimotor gating , quantified with prepulse inhibition ( ppi ) of acoustic startle.105 locomotor hyperactivity , as a model of psychosis in rodents resulting from exogenous agents promoting dopamine release , is the conceptual offspring of the hyperdopaminergic hypothesis of schizophrenia;106 deficits of ppi represent an endophenotype of psychosis itself that has been associated with schizophrenia and may have broad relevance in all psychotic states.107 ppi is defined as the reduction of the intensity of motor startle in response to a startle stimulus ( for example , acoustic ) when the stimulus is preceded by a diminished , nonstartle stimulus . in order to develop a preclinical mouse model of ad+p , we recently selected an ad model manifesting early frontal tauopathy , the transgenic rtg(taup301l)4510 mouse,110 and characterized ppi deficits over time in relationship to tau pathology.111 we demonstrated that ppi deficits accrued over time in female rtg(taup301l)4510 mice , and that these deficits were driven by the same pathogenic hyperphosphorylated tau molecules that we had previously observed in the frontal cortex of human female ad+p subjects.100 this suggests that the rtg(taup301l)4510 mouse may be a candidate for preclinical study of ad+p biology and the development of novel therapeutics . there is more work to be done prior to settling on any animal model of ad+p . future studies involving this model will include expanding the phenotypic characterization of this mouse beyond ppi to include other cognitive and behavioral markers of psychosis , and exploring the sex specificity of the observed behavioral characteristics . the way forward in the treatment of ad+p may transcend traditional psychiatric treatments of psychosis and may involve the reduction of pathogenic proteins rather than supporting aberrant neurotransmitter systems that are the downstream effects of disease pathology . several tau reduction agents are currently in the drug development stage for the treatment of ad , and even if they do not provide a cure for the primary illness , they warrant exploration in the service of combating a particularly morbid and common manifestation of the disease .

it is well established that prostaglandins ( pgs ) protect the gastroduodenal mucosa against physiologic [ gastric acid and pepsin ] and non - physiologic irritants ( 1 ) . the involved mechanisms by which pgs maintain the integrity of gastric mucosa are decreasing the gastric acid output and increasing the mucus and bicarbonate secretion as well as improving the gastric mucosal blood flow ( 2 ) . the major pgs produced by the human and rodent gastric mucosa are pge2 and pgi2 ( 3 ) . pharmacological studies have revealed that there are four pge2 receptors , ep1-ep4 ( 3 ) . pge2 has dual effect on gastric acid secretion in rats ( 2 ) . at low concentrations , inhibits acid secretion through activation of ep3 receptors while at high concentration , stimulates the gastric acid output by activating ep4 receptors . both ep3 and ep4receptors are expressed by parietal cells ( 4 ) . activation of ep1 receptors of pge2 in the rat s stomach leads to bicarbonate secretion ( 5 ) . the gastro - protective effect of the third gasotran- smitter , hydrogen sulfide ( h2s ) , is well established ( 6 ) . h2s and its donor [ nahs ] have been shown to protect the gastric mucosa through different pathways such as potentiating the gastric mucosal barrier [ increasing mucosal blood flow , mucus , bicarbonate secretion , etc ] and inhibiting the gastric acid output ( 6 - 8 ) . it has been shown that nahs through increasing the mucosal production of pge2 protect the rat s gastric mucosa against water immersion stress - induced gastric lesions ( 9 ) . another research has been demonstrated that endogenous and exogenous h2s increased the ph of gastric contents by upregulating the mucosal mrna expression of sodium bicarbonate cotransporter1 [ nbc1 ] in rats ( 10 ) . therefore , previous reports have shown the effects of nahs on mucosal production of pge2 and bicarbonate as well as mucus secretion , but on the best of our knowledge its effect on mucosal mrna expression of pge2 receptors has not been investigated . therefore , the present study designed to evaluate the effect of nahs on mrna expression of pge2 receptors [ ep1 , ep3 and ep4 ) in gastric mucosa in rats . to determine this effect of nahs , the gastric mucosa exposed to distention - induced gastric acid output and mucosal application of hydrochloric acid ( ph=1 ) . male wistar rats ( 150 - 200 g ) were supplied from the animal house of ahvaz jundishapur university of medical sciences . they were maintained under standard conditions of humidity , temperature ( 22 2 c ) and light / dark cycle ( 12 hr : 12 hr ) . all experiments were carried out in accordance with ethics committee of ahvaz jundishapur university of medical sciences ( ir.ajums.rec.1394.661 ) . twenty - four rats were randomly assigned into 4 groups ( 6 per group ) . fasted rats were anesthetized with a mixture of ketamine and xylasine ( 60 + 15 mg / kg , ip ) . depth of anesthesia was monitored throughout the experiment by the pedal withdrawal ( toe pinch ) reflex every 30 - 45 min . if the reflex was observed , a supplemental dose of anesthetics ( 1/3 of initial dose ) was administered to maintain adequate anesthesia . animal body temperature was controlled with a rectal thermometer and maintained at 370.5 c by using a homoeothermic blanket control system ( harvard , edenbridge , uk ) . anesthetized rats underwent a midline laparotomy , and then both the stomach and duodenum were exposed . a polyethylene catheter [ 3 mm , od ( outer diameter ) ] was inserted into the stomach through the duodenum and held in place by a ligature around the pylorus . at the beginning of each experiment , the lumen of the stomach was gently rinsed with isotonic saline ( ph=7 , 37 c ) until gastric wash out was clear . in the first set of experiments , to evaluate the effect of nahs on mucosal mrna expression of eps receptors in response to distention - induced gastric acid secretion , 12 rats were assigned into control , and nahs - treated rats . thirty min after surgical operation , isotonic saline ( 1.5 ml/100 g of body weight ; ph=7 , 37 c ) ( 11 ) was instilled into the stomach to stimulate the gastric acid output . nahs - treated rats received a single ip injection of nahs ( a h2s donor ) at 80 g / kg ( 7 ) along with gastric distention by isotonic saline . in the second set of experiments , to evaluate the effect of nahs on mucosal mrna expression of eps receptors in response to mucosal acidification 30 min after surgical operation , one ml of isotonic acidic solution ( ph=1 ) ( 12 ) was instilled into the stomach of experimental groups . nahs - treated rats were received a single ip injection of nahs at 80 g / kg ( 7 ) along with the instillation of acidic solution ( hcl ; ph=1 ) . ninety min after the instillation of isotonic saline or acidic solution , animals were sacrificed by an overdose of anesthetics , their stomachs were removed and gastric contents collected . the ph of each sample was measured with a digital ph meter ( istek ; inc , south korea ) . after that , the stomachs were opened along the greater curvature , rinsed with physiological saline and pinned out in ice - cold saline . to determine the gastric mucus wall , thirty mg of gastric mucosal tissues were quickly snap - frozen and stored in liquid nitrogen for mrna analysis . the total rna was extracted from the frozen tissue samples using rneasy plus min kit ( qiagen , qiagengmbh , hilden , germany ) . the purity and concentration of the extracted rna was determined spectrophotometerically at 260 and 280 nm wave - length ( nanodrop thermoscientific s.n : d015 ) . the cdna was synthesized from 1 g of the total rna using quantitect reverse transcription kit ( qiagen , qiagengmbh , hilden , germany ) according to the manufacturers instruction . the mrna levels of prostaglandin e2 receptors [ ep1 , ep3 , and ep4 ] , and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) were measured by quantitative real - time pcr ( qrt - pcr ) using a lightcycler 96 system ( roche diagnostics ) . the specific primers ( bioneer , daejeon , south korea ) for measurement of target genes and gapdh were used and the lengths for amplified products were as follows : gapdh ( forward : 5-tgctggtgctgagtatgtcgtg-3 and 5-ggagatgatgacccttttgg-3 , 101 bp ) ; ep1(forward : 5-tggcacagatacaggggatg-3and reverse : 5-gtgggacgtgaatccagaact-3 , 145 bp ) ; ep3 ( forward : 5-gtgtgtactgtccgtctgct-3and reverse : 5-tcaggttgttcatcatctggca-3,230 bp ) ; and ep4 ( forward : 5-gttctggcagagacggttca-3 and reverse : 5-aagttctcagcgaggtggtg-3 , 234 bp ) . all pcr amplifications were performed in duplicate reactions and in final volume of 20 l containing 2l cdna , 0.8 l of specific primers , 10 l of master mix sybr green(takara sybr _ premix ex taqtm ii , tli rnaseh plus , bulk[takara , bio inc , shiga , japan ] ) , 6.4 l ddh2o using the following protocol : pre - incubation at 95 c for 1 min to activate dna taq polymerase and 40 two - step cycles with denaturation at 95 c for 15 sec , annealing at 54 c for 30 sec , and extension at 72 c for 30 sec . in addition , the no - template negative control ( h2o ) was routinely run in every pcr . the melting curve was examined at the end of amplification process to ensure the specificity of pcr products . expression level of eps receptors were normalized against gapdh expression ( internal calibrator for equal rna template loading and normalization ) . to determine the relative quantification of gene expression , comparative cycle of threshold ( ct ) method with arithmetic formula ( 2 ) data are shown as meansem statistical analysis was performed by one - way anova and followed by post hoc tukey s test . male wistar rats ( 150 - 200 g ) were supplied from the animal house of ahvaz jundishapur university of medical sciences . they were maintained under standard conditions of humidity , temperature ( 22 2 c ) and light / dark cycle ( 12 hr : 12 hr ) . all experiments were carried out in accordance with ethics committee of ahvaz jundishapur university of medical sciences ( ir.ajums.rec.1394.661 ) . twenty - four rats were randomly assigned into 4 groups ( 6 per group ) . fasted rats were anesthetized with a mixture of ketamine and xylasine ( 60 + 15 mg / kg , ip ) . depth of anesthesia was monitored throughout the experiment by the pedal withdrawal ( toe pinch ) reflex every 30 - 45 min . if the reflex was observed , a supplemental dose of anesthetics ( 1/3 of initial dose ) was administered to maintain adequate anesthesia . animal body temperature was controlled with a rectal thermometer and maintained at 370.5 c by using a homoeothermic blanket control system ( harvard , edenbridge , uk ) . anesthetized rats underwent a midline laparotomy , and then both the stomach and duodenum were exposed . a polyethylene catheter [ 3 mm , od ( outer diameter ) ] was inserted into the stomach through the duodenum and held in place by a ligature around the pylorus . at the beginning of each experiment , the lumen of the stomach was gently rinsed with isotonic saline ( ph=7 , 37 c ) until gastric wash out was clear . in the first set of experiments , to evaluate the effect of nahs on mucosal mrna expression of eps receptors in response to distention - induced gastric acid secretion , 12 rats were assigned into control , and nahs - treated rats . thirty min after surgical operation , isotonic saline ( 1.5 ml/100 g of body weight ; ph=7 , 37 c ) ( 11 ) was instilled into the stomach to stimulate the gastric acid output . nahs - treated rats received a single ip injection of nahs ( a h2s donor ) at 80 g / kg ( 7 ) along with gastric distention by isotonic saline . in the second set of experiments , to evaluate the effect of nahs on mucosal mrna expression of eps receptors in response to mucosal acidification 30 min after surgical operation , one ml of isotonic acidic solution ( ph=1 ) ( 12 ) was instilled into the stomach of experimental groups . nahs - treated rats were received a single ip injection of nahs at 80 g / kg ( 7 ) along with the instillation of acidic solution ( hcl ; ph=1 ) . ninety min after the instillation of isotonic saline or acidic solution , animals were sacrificed by an overdose of anesthetics , their stomachs were removed and gastric contents collected . the ph of each sample was measured with a digital ph meter ( istek ; inc , south korea ) . after that , the stomachs were opened along the greater curvature , rinsed with physiological saline and pinned out in ice - cold saline . to determine the gastric mucus wall , thirty mg of gastric mucosal tissues were quickly snap - frozen and stored in liquid nitrogen for mrna analysis . the total rna was extracted from the frozen tissue samples using rneasy plus min kit ( qiagen , qiagengmbh , hilden , germany ) . the purity and concentration of the extracted rna was determined spectrophotometerically at 260 and 280 nm wave - length ( nanodrop thermoscientific s.n : d015 ) . the cdna was synthesized from 1 g of the total rna using quantitect reverse transcription kit ( qiagen , qiagengmbh , hilden , germany ) according to the manufacturers instruction . the mrna levels of prostaglandin e2 receptors [ ep1 , ep3 , and ep4 ] , and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) were measured by quantitative real - time pcr ( qrt - pcr ) using a lightcycler 96 system ( roche diagnostics ) . the specific primers ( bioneer , daejeon , south korea ) for measurement of target genes and gapdh were used and the lengths for amplified products were as follows : gapdh ( forward : 5-tgctggtgctgagtatgtcgtg-3 and 5-ggagatgatgacccttttgg-3 , 101 bp ) ; ep1(forward : 5-tggcacagatacaggggatg-3and reverse : 5-gtgggacgtgaatccagaact-3 , 145 bp ) ; ep3 ( forward : 5-gtgtgtactgtccgtctgct-3and reverse : 5-tcaggttgttcatcatctggca-3,230 bp ) ; and ep4 ( forward : 5-gttctggcagagacggttca-3 and reverse : 5-aagttctcagcgaggtggtg-3 , 234 bp ) . all pcr amplifications were performed in duplicate reactions and in final volume of 20 l containing 2l cdna , 0.8 l of specific primers , 10 l of master mix sybr green(takara sybr _ premix ex taqtm ii , tli rnaseh plus , bulk[takara , bio inc , shiga , japan ] ) , 6.4 l ddh2o using the following protocol : pre - incubation at 95 c for 1 min to activate dna taq polymerase and 40 two - step cycles with denaturation at 95 c for 15 sec , annealing at 54 c for 30 sec , and extension at 72 c for 30 sec . in addition , the no - template negative control ( h2o ) was routinely run in every pcr . the melting curve was examined at the end of amplification process to ensure the specificity of pcr products . expression level of eps receptors were normalized against gapdh expression ( internal calibrator for equal rna template loading and normalization ) . to determine the relative quantification of gene expression , comparative cycle of threshold ( ct ) method with arithmetic formula ( 2 ) data are shown as meansem statistical analysis was performed by one - way anova and followed by post hoc tukey s test . as shown in figures 1a and 3a , analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep1 receptors while decreased mrna expression of ep4 receptors in response to mucosal acidification . nahs did not change the mrna expression of ep3 receptor in response to mucosal acidification ( figure 2a ) . figures 2b and 3b showed that the mucosal mrna expression of ep3 , and ep4 was significantly increased in response to distention - induced gastric acid secretion while mrna expression of ep1 was not affected in nahs - treated rats ( figure 1b ) . effect of sodium hydrosulfide ( nahs ) on mucosal mrna expression of ep1 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs significantly increased the mucosal mrna expression of ep1 receptors in response to mucosal acidification ( figure 1a ) while mrna expression of ep1 was not affected in response to distention - induced gastric acid secretion in nahs - treated rats ( figure 1b ) . ca : control acid ; and nahs80a : animals received a single intraperitoneally injection of nahs at 80 g / kg along with mucosal instilattion of acid solution ( ph=1 ) ; cd : control distention , and nahs80d : animals received a single intraperitoneally injection of nahs at 80 g / kg along with stimulating the gastric acid output by gastric distention * * * p<0.001 significant increase compared with the corresponding s control rats . data are expressed as meansem effect of sodium hydrosulfide on mucosal mrna expression of ep3 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep3 receptors in response to distention - induced gastric acid secretion ( fig . 2b ) while mrna expression of ep3 was not affected in response to mucosal acidification in nahs - treated rats ( fig . data are expressed as meansem effect of sodium hydrosulfide on mucosal mrna expression of ep4 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep4 receptors in response to distention - induced gastric acid secretion ( figure 3b ) while mrna expression of ep4 was significantly decreased in response to mucosal acidification in nahs - treated rats ( figure 3a).**p<0.01 significant versus corresponding s control groups . data are expressed as meansem as shown in figure 4b , sodium hydrosulfide increased the gastric mucus production in response to distention - induced gastric acid output . nahs did not affect the basal production of gastric mucus in response to mucosal acidification ( figure 4a ) . effect of sodium hydrosulfide on gastric wall mucus production in response to distention - induced gastric acid output and mucosal acidification . sodium hydrosulfide significantly increased the gastric mucus production in response to distention - induced gastric acid output ( figure 4b ) while did not affect the basal production of gastric mucus in response to mucosal acidification ( figure 4a).**p<0.01significant versus correspon - ding s control group . data are expressed as meansem as demonstrated in figures 5a and 5b , sodium hydrosulfide significantly increased the ph of gastric contents both in response to mucosal acidification and distention - induced gastric acid output . effect of sodium hydrosulfide on ph of gastric contents in response to distention - induced gastric acid output and mucosal acidification . sodium hydrosulfide significantly increased the ph of gastric contents in response to mucosal acidification ( figure 5a ) and distention - induced gastric acid output ( figure 5b ) . * as shown in figures 1a and 3a , analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep1 receptors while decreased mrna expression of ep4 receptors in response to mucosal acidification . nahs did not change the mrna expression of ep3 receptor in response to mucosal acidification ( figure 2a ) . figures 2b and 3b showed that the mucosal mrna expression of ep3 , and ep4 was significantly increased in response to distention - induced gastric acid secretion while mrna expression of ep1 was not affected in nahs - treated rats ( figure 1b ) . effect of sodium hydrosulfide ( nahs ) on mucosal mrna expression of ep1 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs significantly increased the mucosal mrna expression of ep1 receptors in response to mucosal acidification ( figure 1a ) while mrna expression of ep1 was not affected in response to distention - induced gastric acid secretion in nahs - treated rats ( figure 1b ) . ca : control acid ; and nahs80a : animals received a single intraperitoneally injection of nahs at 80 g / kg along with mucosal instilattion of acid solution ( ph=1 ) ; cd : control distention , and nahs80d : animals received a single intraperitoneally injection of nahs at 80 g / kg along with stimulating the gastric acid output by gastric distention * * * p<0.001 significant increase compared with the corresponding s control rats . data are expressed as meansem effect of sodium hydrosulfide on mucosal mrna expression of ep3 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep3 receptors in response to distention - induced gastric acid secretion ( fig . 2b ) while mrna expression of ep3 was not affected in response to mucosal acidification in nahs - treated rats ( fig . data are expressed as meansem effect of sodium hydrosulfide on mucosal mrna expression of ep4 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep4 receptors in response to distention - induced gastric acid secretion ( figure 3b ) while mrna expression of ep4 was significantly decreased in response to mucosal acidification in nahs - treated rats ( figure 3a).**p<0.01 significant versus corresponding s control groups . as shown in figure 4b , sodium hydrosulfide increased the gastric mucus production in response to distention - induced gastric acid output . nahs did not affect the basal production of gastric mucus in response to mucosal acidification ( figure 4a ) . effect of sodium hydrosulfide on gastric wall mucus production in response to distention - induced gastric acid output and mucosal acidification . sodium hydrosulfide significantly increased the gastric mucus production in response to distention - induced gastric acid output ( figure 4b ) while did not affect the basal production of gastric mucus in response to mucosal acidification ( figure 4a).**p<0.01significant versus correspon - ding s control group . as demonstrated in figures 5a and 5b , sodium hydrosulfide significantly increased the ph of gastric contents both in response to mucosal acidification and distention - induced gastric acid output . effect of sodium hydrosulfide on ph of gastric contents in response to distention - induced gastric acid output and mucosal acidification . sodium hydrosulfide significantly increased the ph of gastric contents in response to mucosal acidification ( figure 5a ) and distention - induced gastric acid output ( figure 5b ) . * * p<0.01 , and * * * p<0.001 significant versus corresponding s control groups . the present study showed that a single administration of sodium hydrosulfide : 1 . increased the mucus secretion , and mrna expression of ep3 as well as ep4 receptors of pge2 in response to distention - induced expression ; 2 . upregulated the mrna expre - ssion of ep1 receptors of pge2 while downregulated ep4 mrna expression in response to mucosal acidification ; and 3 . increased the ph of gastric contents both in response to distention - induced gastric acid secretion and mucosal acidification . the current results showed that nahs significantly increased the ph of gastric contents in response to mucosal application of acidic solution ( ph=1 ) . according to the real - time pcr results , this response can be resulted from dowregulation of ep4 receptors and at the same time upregulation of ep1 receptors in gastric mucosa . it is a general fact any increase in the ph of gastric content can be produce via decreasing the gastric acid output or increasing the gastric bicarbonate secretion and also simultaneous increase of hco3 and decrease of hcl . therefore , downregulating mrna expression of ep4 receptors can be lead to decreasing ep4 protein receptors in parietal cell membrane which in turn inhibits the gastric acid secretion . additionally , upregulating mrna expression of ep1 receptors by nahs could be the second reason for increasing the ph of gastric contents because of the activation of ep1 receptors results in hco3secretion by epithelial mucus cell ( 1 , 5 ) . therefore , nahs through increment the mucosal production of ligand [ prostaglandin e2 ] as shown by magierowski et al ( 9 ) and at the same time through modulating the receptor density of cell membrane ( decrement ep4 , while increment ep1 mrna receptors ) enhanced the ph of gastric contents . the current findings showed that nahs did not change the mucus content in response to mucosal acidification . it has been shown that activation of ep4 receptors on epithelial mucus cells by pge2 stimulate the mucus secretion ( 5 ) . our qrt - pcr results showed that the mrna expression of ep4 decreased in nahs - treated rats . the present results also showed that the mucus content was slightly but not significantly decreased while the ph of gastric contents effectively and significantly increased in nahs - treated rats . it was 2.520.23 in control rats while administration of nahs at 80 g / kg increased the ph to 4.330.38 . therefore , these results implied that when the gastric mucosa suddenly exposes to a highly acidic solution ( ph=1 ) , the preferred mechanism by which nahs protected the gastric mucosa is neutralizing the entered acid . as results showed nahs decreased the gastric acid output by downregulating mrna expression of ep4and induced the bicarbonate secretion by upregulating mrna expression of ep1 . the results of the present study indicated that nahs increased the rate of mucus secretion and enhanced the ph of gastric contents in response to distention - induced gastric acid secretion . as shown in figures 2b and 3b , the mrna expression of ep3 and ep4 increased in nahs - pretreated groups . ep3 receptors are expressed in the parietal cells of rat s stomach ( 2 ) . it has been shown that the activation of ep3 receptor inhibits the gastric acid output ( 4 ) . ep4 receptors are expressed by parietal ( 13 ) as well as mucus cells ( 2 ) in gastric mucosa . it has been shown that activation of ep4 receptors has two effects , stimulating the gastric acid output and inducing mucus secretion . in spite of parietal cells , in contrast to mucosal acidification experiment , the rate of acid increase is slower in rats underwent gastric distention and provides much more time for nahs to protect the gastric mucosa by two mechanisms : 1 . mucus production by upregulating ep4 mrna receptors and inhibiting the gastric acid by upregulating ep3 mrna receptors as evidenced by the present results . it also seems that the rate of mrna expression of ep4 receptors was higher in epithelial mucus cell than in parietal cells because the results did not show the acid excitatory effect of ep4 receptors . on the other hand , the qrt - pcr and the ph findings together suggest that when ep3 receptors on parietal cell membranes activate due to the simultaneous increase of mrna expression of ep3 as shown by current study and pge2 as shown by a previous research ( 9 ) , the membrane ep4 receptors inactivate or has lower affinity to pge2 . the present ph result was also support the activation of ep3 receptors rather than ep4 receptors in parietal cells . however , this study aimed to evaluate the effect of nahs on mucosal mrna expression of ep1 , ep3 , and ep4 of pge2 receptors but not their activation by nahs . thus , more studies need to exactly define the effect of nahs on eps activation . whether the affinity and activation of eps change by nahs is also remained to be defined by further future experiments . the results suggest nahs increased the ph of the gastric contents in response to mucosal acidification through dowregulating mucosal mrna expression of ep4 , and upregulating ep1 . the increased mucus and ph of gastric contents in nahs - treated rats in response to distention - induced gastric acid output is mainly mediated through upregulating mucosal mrna expression of ep3 and ep4 receptors .

objective(s):prostaglandins have been shown to mediate the gastro - protective effect of sodium hydrosulfide ( nahs ) but effect of nahs on mrna expression of prostaglandin e2 receptors ( ep1 , 3 - 4 ; eps ) has not been investigated . therefore , this study designed to evaluate the effect of nahs on mrna expression of eps receptors in response to mucosal acidification and distention - induced gastric acid secretion in rats.materials and methods : fasted rats were randomly assigned into 4 groups ( n=6/group ) . they were control , and nahs - treated groups . to evaluate the effect of nahs on mucosal mrna expression of eps receptors , the gastric mucosa exposed to stimulated gastric acid output and mucosal acidification . the pylorus sphincter catheterized for instillation of isotonic neutral saline or acidic solution . ninety min after beginning the experiments , animals sacrificed and the gastric mucosa collected to determine the ph , mucus secretion and to quantify the mrna expression of eps receptors by quantitative real - time pcr.results:present results showed that a ) nahs increased the mucus secretion , mrna expression of ep3 and ep4 receptors in response to distention - induced expression ; b ) the mrna expression of ep1 receptors increased while ep4 mrna receptors decreased in response to mucosal acidification in nahs - pretreated rats ; and c ) nahs increased ph of gastric contents both in response to distention - induced gastric acid secretion and mucosal acidification.conclusion:nahs behaves in a different manner . it effectively only increased the ph of gastric contents to reinforce the gastric mucosa against a highly acidic solution but modulated both acid and mucus secretion when the rate of acid increase in the stomach was slower .

Introduction Materials and Methods Animals Animal grouping and surgical procedures RNA extraction and cDNA synthesis Quantitative real-time PCR Statistical analysis Results Effect of NaHS on mucosal mRNA expression of EPs receptors in response to distention-induced gastric acid output and mucosal acidification Effects of NaHS on gastric wall mucus production in response to distention-induced gastric acid output and mucosal acidification Effects of NaHS on pH of gastric contents in response to distention-induced gastric acid output and mucosal acidification Discussion Conclusion Conflict of interest

it is well established that prostaglandins ( pgs ) protect the gastroduodenal mucosa against physiologic [ gastric acid and pepsin ] and non - physiologic irritants ( 1 ) . the involved mechanisms by which pgs maintain the integrity of gastric mucosa are decreasing the gastric acid output and increasing the mucus and bicarbonate secretion as well as improving the gastric mucosal blood flow ( 2 ) . pge2 has dual effect on gastric acid secretion in rats ( 2 ) . at low concentrations , inhibits acid secretion through activation of ep3 receptors while at high concentration , stimulates the gastric acid output by activating ep4 receptors . the gastro - protective effect of the third gasotran- smitter , hydrogen sulfide ( h2s ) , is well established ( 6 ) . h2s and its donor [ nahs ] have been shown to protect the gastric mucosa through different pathways such as potentiating the gastric mucosal barrier [ increasing mucosal blood flow , mucus , bicarbonate secretion , etc ] and inhibiting the gastric acid output ( 6 - 8 ) . it has been shown that nahs through increasing the mucosal production of pge2 protect the rat s gastric mucosa against water immersion stress - induced gastric lesions ( 9 ) . another research has been demonstrated that endogenous and exogenous h2s increased the ph of gastric contents by upregulating the mucosal mrna expression of sodium bicarbonate cotransporter1 [ nbc1 ] in rats ( 10 ) . therefore , previous reports have shown the effects of nahs on mucosal production of pge2 and bicarbonate as well as mucus secretion , but on the best of our knowledge its effect on mucosal mrna expression of pge2 receptors has not been investigated . therefore , the present study designed to evaluate the effect of nahs on mrna expression of pge2 receptors [ ep1 , ep3 and ep4 ) in gastric mucosa in rats . to determine this effect of nahs , the gastric mucosa exposed to distention - induced gastric acid output and mucosal application of hydrochloric acid ( ph=1 ) . twenty - four rats were randomly assigned into 4 groups ( 6 per group ) . at the beginning of each experiment , the lumen of the stomach was gently rinsed with isotonic saline ( ph=7 , 37 c ) until gastric wash out was clear . in the first set of experiments , to evaluate the effect of nahs on mucosal mrna expression of eps receptors in response to distention - induced gastric acid secretion , 12 rats were assigned into control , and nahs - treated rats . thirty min after surgical operation , isotonic saline ( 1.5 ml/100 g of body weight ; ph=7 , 37 c ) ( 11 ) was instilled into the stomach to stimulate the gastric acid output . in the second set of experiments , to evaluate the effect of nahs on mucosal mrna expression of eps receptors in response to mucosal acidification 30 min after surgical operation , one ml of isotonic acidic solution ( ph=1 ) ( 12 ) was instilled into the stomach of experimental groups . nahs - treated rats were received a single ip injection of nahs at 80 g / kg ( 7 ) along with the instillation of acidic solution ( hcl ; ph=1 ) . ninety min after the instillation of isotonic saline or acidic solution , animals were sacrificed by an overdose of anesthetics , their stomachs were removed and gastric contents collected . to determine the gastric mucus wall , thirty mg of gastric mucosal tissues were quickly snap - frozen and stored in liquid nitrogen for mrna analysis . the mrna levels of prostaglandin e2 receptors [ ep1 , ep3 , and ep4 ] , and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) were measured by quantitative real - time pcr ( qrt - pcr ) using a lightcycler 96 system ( roche diagnostics ) . twenty - four rats were randomly assigned into 4 groups ( 6 per group ) . at the beginning of each experiment , the lumen of the stomach was gently rinsed with isotonic saline ( ph=7 , 37 c ) until gastric wash out was clear . in the first set of experiments , to evaluate the effect of nahs on mucosal mrna expression of eps receptors in response to distention - induced gastric acid secretion , 12 rats were assigned into control , and nahs - treated rats . thirty min after surgical operation , isotonic saline ( 1.5 ml/100 g of body weight ; ph=7 , 37 c ) ( 11 ) was instilled into the stomach to stimulate the gastric acid output . in the second set of experiments , to evaluate the effect of nahs on mucosal mrna expression of eps receptors in response to mucosal acidification 30 min after surgical operation , one ml of isotonic acidic solution ( ph=1 ) ( 12 ) was instilled into the stomach of experimental groups . nahs - treated rats were received a single ip injection of nahs at 80 g / kg ( 7 ) along with the instillation of acidic solution ( hcl ; ph=1 ) . ninety min after the instillation of isotonic saline or acidic solution , animals were sacrificed by an overdose of anesthetics , their stomachs were removed and gastric contents collected . to determine the gastric mucus wall , thirty mg of gastric mucosal tissues were quickly snap - frozen and stored in liquid nitrogen for mrna analysis . the mrna levels of prostaglandin e2 receptors [ ep1 , ep3 , and ep4 ] , and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) were measured by quantitative real - time pcr ( qrt - pcr ) using a lightcycler 96 system ( roche diagnostics ) . the specific primers ( bioneer , daejeon , south korea ) for measurement of target genes and gapdh were used and the lengths for amplified products were as follows : gapdh ( forward : 5-tgctggtgctgagtatgtcgtg-3 and 5-ggagatgatgacccttttgg-3 , 101 bp ) ; ep1(forward : 5-tggcacagatacaggggatg-3and reverse : 5-gtgggacgtgaatccagaact-3 , 145 bp ) ; ep3 ( forward : 5-gtgtgtactgtccgtctgct-3and reverse : 5-tcaggttgttcatcatctggca-3,230 bp ) ; and ep4 ( forward : 5-gttctggcagagacggttca-3 and reverse : 5-aagttctcagcgaggtggtg-3 , 234 bp ) . as shown in figures 1a and 3a , analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep1 receptors while decreased mrna expression of ep4 receptors in response to mucosal acidification . nahs did not change the mrna expression of ep3 receptor in response to mucosal acidification ( figure 2a ) . figures 2b and 3b showed that the mucosal mrna expression of ep3 , and ep4 was significantly increased in response to distention - induced gastric acid secretion while mrna expression of ep1 was not affected in nahs - treated rats ( figure 1b ) . effect of sodium hydrosulfide ( nahs ) on mucosal mrna expression of ep1 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs significantly increased the mucosal mrna expression of ep1 receptors in response to mucosal acidification ( figure 1a ) while mrna expression of ep1 was not affected in response to distention - induced gastric acid secretion in nahs - treated rats ( figure 1b ) . ca : control acid ; and nahs80a : animals received a single intraperitoneally injection of nahs at 80 g / kg along with mucosal instilattion of acid solution ( ph=1 ) ; cd : control distention , and nahs80d : animals received a single intraperitoneally injection of nahs at 80 g / kg along with stimulating the gastric acid output by gastric distention * * * p<0.001 significant increase compared with the corresponding s control rats . data are expressed as meansem effect of sodium hydrosulfide on mucosal mrna expression of ep3 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep3 receptors in response to distention - induced gastric acid secretion ( fig . 2b ) while mrna expression of ep3 was not affected in response to mucosal acidification in nahs - treated rats ( fig . data are expressed as meansem effect of sodium hydrosulfide on mucosal mrna expression of ep4 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep4 receptors in response to distention - induced gastric acid secretion ( figure 3b ) while mrna expression of ep4 was significantly decreased in response to mucosal acidification in nahs - treated rats ( figure 3a). data are expressed as meansem as shown in figure 4b , sodium hydrosulfide increased the gastric mucus production in response to distention - induced gastric acid output . nahs did not affect the basal production of gastric mucus in response to mucosal acidification ( figure 4a ) . effect of sodium hydrosulfide on gastric wall mucus production in response to distention - induced gastric acid output and mucosal acidification . sodium hydrosulfide significantly increased the gastric mucus production in response to distention - induced gastric acid output ( figure 4b ) while did not affect the basal production of gastric mucus in response to mucosal acidification ( figure 4a). data are expressed as meansem as demonstrated in figures 5a and 5b , sodium hydrosulfide significantly increased the ph of gastric contents both in response to mucosal acidification and distention - induced gastric acid output . effect of sodium hydrosulfide on ph of gastric contents in response to distention - induced gastric acid output and mucosal acidification . sodium hydrosulfide significantly increased the ph of gastric contents in response to mucosal acidification ( figure 5a ) and distention - induced gastric acid output ( figure 5b ) . * as shown in figures 1a and 3a , analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep1 receptors while decreased mrna expression of ep4 receptors in response to mucosal acidification . nahs did not change the mrna expression of ep3 receptor in response to mucosal acidification ( figure 2a ) . figures 2b and 3b showed that the mucosal mrna expression of ep3 , and ep4 was significantly increased in response to distention - induced gastric acid secretion while mrna expression of ep1 was not affected in nahs - treated rats ( figure 1b ) . effect of sodium hydrosulfide ( nahs ) on mucosal mrna expression of ep1 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs significantly increased the mucosal mrna expression of ep1 receptors in response to mucosal acidification ( figure 1a ) while mrna expression of ep1 was not affected in response to distention - induced gastric acid secretion in nahs - treated rats ( figure 1b ) . ca : control acid ; and nahs80a : animals received a single intraperitoneally injection of nahs at 80 g / kg along with mucosal instilattion of acid solution ( ph=1 ) ; cd : control distention , and nahs80d : animals received a single intraperitoneally injection of nahs at 80 g / kg along with stimulating the gastric acid output by gastric distention * * * p<0.001 significant increase compared with the corresponding s control rats . data are expressed as meansem effect of sodium hydrosulfide on mucosal mrna expression of ep3 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep3 receptors in response to distention - induced gastric acid secretion ( fig . 2b ) while mrna expression of ep3 was not affected in response to mucosal acidification in nahs - treated rats ( fig . data are expressed as meansem effect of sodium hydrosulfide on mucosal mrna expression of ep4 receptor of pge2 in response to distention - induced gastric acid output and mucosal acidification . analysis of qrt - pcr results showed that nahs treatment significantly increased the mucosal mrna expression of ep4 receptors in response to distention - induced gastric acid secretion ( figure 3b ) while mrna expression of ep4 was significantly decreased in response to mucosal acidification in nahs - treated rats ( figure 3a). as shown in figure 4b , sodium hydrosulfide increased the gastric mucus production in response to distention - induced gastric acid output . nahs did not affect the basal production of gastric mucus in response to mucosal acidification ( figure 4a ) . effect of sodium hydrosulfide on gastric wall mucus production in response to distention - induced gastric acid output and mucosal acidification . sodium hydrosulfide significantly increased the gastric mucus production in response to distention - induced gastric acid output ( figure 4b ) while did not affect the basal production of gastric mucus in response to mucosal acidification ( figure 4a). as demonstrated in figures 5a and 5b , sodium hydrosulfide significantly increased the ph of gastric contents both in response to mucosal acidification and distention - induced gastric acid output . effect of sodium hydrosulfide on ph of gastric contents in response to distention - induced gastric acid output and mucosal acidification . sodium hydrosulfide significantly increased the ph of gastric contents in response to mucosal acidification ( figure 5a ) and distention - induced gastric acid output ( figure 5b ) . the present study showed that a single administration of sodium hydrosulfide : 1 . increased the mucus secretion , and mrna expression of ep3 as well as ep4 receptors of pge2 in response to distention - induced expression ; 2 . upregulated the mrna expre - ssion of ep1 receptors of pge2 while downregulated ep4 mrna expression in response to mucosal acidification ; and 3 . increased the ph of gastric contents both in response to distention - induced gastric acid secretion and mucosal acidification . the current results showed that nahs significantly increased the ph of gastric contents in response to mucosal application of acidic solution ( ph=1 ) . according to the real - time pcr results , this response can be resulted from dowregulation of ep4 receptors and at the same time upregulation of ep1 receptors in gastric mucosa . it is a general fact any increase in the ph of gastric content can be produce via decreasing the gastric acid output or increasing the gastric bicarbonate secretion and also simultaneous increase of hco3 and decrease of hcl . therefore , downregulating mrna expression of ep4 receptors can be lead to decreasing ep4 protein receptors in parietal cell membrane which in turn inhibits the gastric acid secretion . additionally , upregulating mrna expression of ep1 receptors by nahs could be the second reason for increasing the ph of gastric contents because of the activation of ep1 receptors results in hco3secretion by epithelial mucus cell ( 1 , 5 ) . therefore , nahs through increment the mucosal production of ligand [ prostaglandin e2 ] as shown by magierowski et al ( 9 ) and at the same time through modulating the receptor density of cell membrane ( decrement ep4 , while increment ep1 mrna receptors ) enhanced the ph of gastric contents . the current findings showed that nahs did not change the mucus content in response to mucosal acidification . it has been shown that activation of ep4 receptors on epithelial mucus cells by pge2 stimulate the mucus secretion ( 5 ) . our qrt - pcr results showed that the mrna expression of ep4 decreased in nahs - treated rats . the present results also showed that the mucus content was slightly but not significantly decreased while the ph of gastric contents effectively and significantly increased in nahs - treated rats . it was 2.520.23 in control rats while administration of nahs at 80 g / kg increased the ph to 4.330.38 . therefore , these results implied that when the gastric mucosa suddenly exposes to a highly acidic solution ( ph=1 ) , the preferred mechanism by which nahs protected the gastric mucosa is neutralizing the entered acid . as results showed nahs decreased the gastric acid output by downregulating mrna expression of ep4and induced the bicarbonate secretion by upregulating mrna expression of ep1 . the results of the present study indicated that nahs increased the rate of mucus secretion and enhanced the ph of gastric contents in response to distention - induced gastric acid secretion . as shown in figures 2b and 3b , the mrna expression of ep3 and ep4 increased in nahs - pretreated groups . it has been shown that the activation of ep3 receptor inhibits the gastric acid output ( 4 ) . it has been shown that activation of ep4 receptors has two effects , stimulating the gastric acid output and inducing mucus secretion . in spite of parietal cells , in contrast to mucosal acidification experiment , the rate of acid increase is slower in rats underwent gastric distention and provides much more time for nahs to protect the gastric mucosa by two mechanisms : 1 . mucus production by upregulating ep4 mrna receptors and inhibiting the gastric acid by upregulating ep3 mrna receptors as evidenced by the present results . it also seems that the rate of mrna expression of ep4 receptors was higher in epithelial mucus cell than in parietal cells because the results did not show the acid excitatory effect of ep4 receptors . on the other hand , the qrt - pcr and the ph findings together suggest that when ep3 receptors on parietal cell membranes activate due to the simultaneous increase of mrna expression of ep3 as shown by current study and pge2 as shown by a previous research ( 9 ) , the membrane ep4 receptors inactivate or has lower affinity to pge2 . however , this study aimed to evaluate the effect of nahs on mucosal mrna expression of ep1 , ep3 , and ep4 of pge2 receptors but not their activation by nahs . thus , more studies need to exactly define the effect of nahs on eps activation . the results suggest nahs increased the ph of the gastric contents in response to mucosal acidification through dowregulating mucosal mrna expression of ep4 , and upregulating ep1 . the increased mucus and ph of gastric contents in nahs - treated rats in response to distention - induced gastric acid output is mainly mediated through upregulating mucosal mrna expression of ep3 and ep4 receptors .

despite being regarded as highly preventable , injuries account for an increasing share of childhood mortality in the world [ 1 , 2 ] . the most common causes of child mortality and morbidity by injury are road traffic crash , drowning , poisoning , burns , and falls . these injuries are unevenly distributed between countries [ 1 , 3 ] and , within countries , between socioeconomic groups , to the detriment of the more disadvantaged families and communities . reviews published throughout the years , be they focused on a specific childhood injury cause or setting [ 46 ] or covering several of them [ 711 ] strongly substantiate this notion . injuries are in fact acknowledged as one of the causes of childhood mortality with the steepest socioeconomic gradient [ 115 ] . despite this knowledge , in the medical and public health literature alone , a great deal of research is regularly published on socioeconomic disparities in childhood injury that specifically aims at measuring the magnitude of those differences . some injury causes like traffic - related ones are frequently studied [ 2 , 711 ] , whereas others , like burns and drownings , receive far less attention . as this literature is highly descriptive in nature ( as is the case for studies on socioeconomic differences in injuries in general ) , there is a paucity of studies on the mechanisms susceptible to generate those differences , which poses challenges to prevention work . likewise , the reasons why differences exist in socioeconomic disparities across studies are seldom thoroughly addressed . some of the reviews mentioned above that , inspired by lead authors on social inequalities in health , put forward key mechanisms that help understanding why socioeconomic disparities may arise [ 4 , 911 ] but they deal with the why differences exist and how to combat differences questions in generic terms and they provide little insight regarding why socioeconomic differences vary both in magnitude and direction , either with increasing age or across settings . it considers the five leading causes of child mortality and morbidity mentioned above , thus allowing a more accurate coverage of the whole age spectrum and helping to highlight whether and how socioeconomic disparities in injuries vary over age category and causes . as a basis for the paper , we revisited and updated two recent reviews commissioned by the world health organisation and conducted in sequence by our research team ( see below ) . here , we examine and reflect on two decades of studies ( 1990 to 2009 ) on socioeconomic disparities in injuries among children aged up to 18 years . the findings are discussed in light of various conceptual approaches to the understanding of the socioeconomic differences in health and safety . the source reviews [ 10 , 11 ] encompassed empirical studies on socioeconomic differences in injuries published in the medical and public health literature during the period 19902006 and , in preparing this paper , an additional three years were added ( 20072009 ) . the articles initially sought for were original research articles that examined socioeconomic disparities in injury risk across socioeconomic groups all ages and all injuries in the first review and unintentional injuries in children in the second one . the articles were obtained through a literature search in the databases of safetylit and the national library of medicine 's medline . for the former database , all studies included under social disparities swedish , and danish language studies published between january 1990 and december 2009 were identified using the keywords injury or injuries or accident or accidents in conjunction with educational status or education or social class or socioeconomic status or occupation or income or social position or socioeconomic position or socioeconomic context or social context or deprivation or socioeconomic factors or socioeconomic characteristics or residence characteristics or neighbourhood and infant or infants or child or children or childhood or adolescent or adolescents or adolescence or youth . additional studies were also identified from the reference lists in selected articles and in those of the reviews listed above . it is important to note that , in the injury field , the safetylit database has both breadth ( number of journals included ) and depth ( coverage from each journal 's backfiles ) . in fact , it has been found that of five commonly used databases , embase , psycinfo , pubmed / medline , safetylit , and web of science ( including the science citation index and the social science citation index ) , the database with the greatest breadth and depth of coverage for journals that publish articles in the injury prevention and safety promotion ( ipsp ) field is safetylit . the safetylit database coverage includes all ipsp - relevant journals from each of the listed databases and , for the journals that are found in the source lists of the other databases , a greater depth of coverage of backfile years . further , the safetylit database contains articles from journals that are not included in any of the other listed databases . because of the wider scope of the original review , and the change in focus in the subsequent updates ( second review and the current one ) , it is unfortunately not possible to specify the total number of articles originally identified from the literature searches that deal with socioeconomic differences in childhood injuries . as a consequence , we also can not specify the number ( or proportion ) of them that meet the selection criteria presented below . from the original literature searches , titles and abstracts were scanned for relevance independently by at least two of the authors . full papers were then obtained to check for further relevance and procede with data extraction ( see below ) . to be included in the current review , they examined the relationship between socioeconomic status ( ses ) and injury at an individual- or area - level as the primary research question , 2 . they considered one or several of the five major cause of mortality and morbidity among children , 3 . they included denominators ( i.e. , population data rather than only injury data ) and assessments of significance between groups or areas ( e.g. , testing for significance or providing confidence intervals ) . these latter criteria guaranteed a minimum level of strength for any single study and no further assessment of the quality of evidence was applied . the age upper limit was relaxed for traffic - related injuries as motor - vehicle driver ( up to 24 years old ) . ( over 20 in a former review ) or specific body parts [ 10 , 11 ] that lack both insight into the understanding of the phenomenon and useful information on which to design intervention strategies and influence policy . a data extraction form was devised and used to record details from each study included . the details retained for the current paper can be found in the results tables 2to 7 . in case of disagreement between the reviewers , table 1 presents an overview of the studies reviewed by injury cause ( traffic being split into four categories ) , severity level ( mortality versus morbidity ) , country , and type of relationship between ses and injury . the majority of the studies reviewed were conducted in high income countries and focused on nonfatal outcomes . as the bulk of those studies considered boys and girls simultaneously road traffic injuries are by far the most studied cause of health disparities in the child injury field . the vast majority of these studies are from european countries ( 26 out of 37 studies ) , and from sweden and the united kingdom in particular . both area- and individual - based studies are represented as well as two multilevel ones . six studies investigated road traffic injuries combined for all kinds of road users . all but one study showed a positive relationship between level of deprivation and road traffic injury [ 1823 ] . a multilevel study from south korea on young children up to 5 years showed that deprivation has a clear positive relationship with mortality by transport - related causes . another study from south korea found that transport - related mortality among boys , 1014 and 1519 years , in families with low income were more than twice as high as the mortality among their peers in families with higher income . this is partly supported by a study from australia that showed an increased mortality inequality for motor vehicle accidents for boys , 014 and 1524 years , but for females only in the age group 1524 years . swedish studies , on the other hand , found a similar social patterning for both sexes [ 22 , 23 ] . all studies show a positive relationship between individual socioeconomic disadvantage or deprivation of the living area and pedestrian injuries [ 2445 ] . one of the studies observed that the association between deprivation and increased pedestrian casualties in england is stronger among children than among older age groups . children in the most deprived areas have up to a four times higher risk for pedestrian injuries than children in the least deprived . [ 31 , 32 ] additional studies from canada and the us support this finding [ 27 , 28 , 30 ] . similar findings were also reported in a study from greece where less wealthy towns have twice as many pedestrian injuries compared to wealthier ones , and in sweden ( stockholm ) where poor areas have approximately 90% higher risk than the most affluent areas for pedestrian injuries . the study from greece indicates that boys are disproportionately disadvantaged regarding pedestrian injuries when they reside in less wealthy towns . individual - based studies from sweden and the united kingdom show that children in families with low socioeconomic position ( measured in terms of parental social class , education or disposable income ) are at greater risk for bicycle injuries [ 18 , 24 , 33 , 34 , 45 ] . these findings are in line with area - based studies from the united kingdom , [ 26 , 29 , 32 ] , ireland and canada [ 27 , 30 ] showing that children from the most deprived areas have significantly higher risk for bicycle injuries than their peers from less deprived areas . in contrast with this , swedish area - based studies show that contextual socioeconomic attributes of the living area are not significant for injuries sustained as bicyclists [ 46 , 47 ] . motorcycle injuries were considered only in three studies , two from sweden and one from australia . the swedish studies were based on individual data and showed a positive relationship between socioeconomic diadvantage and road traffic injuries as motorcyclists [ 24 , 48 ] . these findings are supported by an area - based study from australia showing that children in the most disadvantaged quintile were more likely to be hospitalized for motorcycle injuries than children in the least disadvantaged quintile . one individual - based study showed that children of unskilled workers have significantly higher odds for injuries as a moped user as compared to children of intermediate and high - level salaried employees on the other hand , two area - based studies found that living in areas with higher levels of deprivation reduced the risk for moped injuries [ 39 , 46 ] . area - based studies from canada , australia and the united kingdom found that children from the most deprived areas have significantly higher risks for injuries as car occupants than their peers from the least deprived areas [ 29 , 30 , 38 ] . these results are in line with a multilevel study from sweden that showed that , after adjusting for compositional factors , there was still unexplained area variability for injuries among motorvehicle riders . individual - based studies from sweden and the united kingdom showed that young people in the most disadvantaged families have an increased risk for injuries as a car driver compared to children in the most advantaged families [ 24 , 34 , 49 , 50 ] . tables 5 ( individual level studies ) , 6 ( area level studies ) and 7 ( multilevel studies ) describe the studies that have dealt with causes of injuries other than road traffic - related ones . as the third column of each table indicates , most studies considered several causes at a time ( sometimes including rtis ) . in the text below , the cause specific results are presented . drowning was considered in only three studies . a study from bangladesh , based on an individual level household survey , found greater socioeconomic disadvantage was associated with greater drowning mortality and morbidity among the under five . in south korea , one , a multilevel study conducted among small children ( 05 years ) , found a positive relationship between area - level deprivation and risk of drowning , after adjustment for sex and individual level ses variables . considering older children ( 1014 and 1519 years ) and parental income as an individual level indicator , the other korean study found no evidence of mortality differences by income level for either sex or age group . of the eleven studies that examined socioeconomic disparities in poisoning injury , the majority are ecological ( n = 7 ) and all but one ( england and wales ) examined nonfatal injuries . except for one from peru , all only the peruvian study , among 018 year olds in lima , found no association between household poverty or parental education and child poisoning . the other three studies , from high - income countries , observed large socioeconomic disparities . a danish national study on unintentional home injuries , adjusting for sex , age , distance from hospital and several family , and household factors , observed a gradient of increasing risk of nonfatal poisoning with decreasing parental education and income . similarly , canadian children ( < 18 years ) from low - income families had odds of poisoning injuries which were 60% higher than those children from well - off families . poisoning deaths were higher among children from low social classes in england and wales . studies on injury hospitalisations in children aged 014 years conducted in new south wales ( australia ) and in qubec ( canada ) showed that children in the most deprived quintile had a 52% and 68% higher risk , respectively , than children in the least deprived quintile . in england , there was a clear gradient of increasing risk of poisoning with increasing deprivation among 04 years olds in the east midlands and among 014 years olds in trent . in the former study , it was also observed that gradients were particularly steep for benzodiazepines , antidepressants , cough and cold remedies , and organic solvents . similarly , the two most deprived quintiles had significantly higher hospital admission rates for poisoning than the three least deprived quintiles in wales . in stockholm , the results depended on the measure of ses but not low material deprivation was associated with poisoning among children aged 015 years . in ireland , although 012 year olds living the most deprived areas of north and west belfast had 3.65 the risk of being treated in the emergency department for poisoning compared to those in the least deprived areas , the association failed to reach significance . in the studies reviewed , the definition of what constitutes a burn differed , some studies only dealt with injuries related to fire and flames , other included scalds from hot liquids . in several studies unlike other causes , an almost equal number of individual and area levels studies have been conducted . three individual level studies were conducted in low and middle income settings and they were based on survey or questionnaire data gathered from caregivers . in the ashanti region ( ghana ) , one study found that maternal education was not significantly associated with burn injuries ( with evidence of a physical scar ) among children aged 05 years . the other two studies , both from lima ( peru ) , found evidence of disparities . among 017 year olds , low income and crowding were strongly associated with increased risk , and better maternal education had a protective effect . in the other study , household poverty was associated with an increased odds of burn injuries but low education of the household head was not . individual level studies from high income countries showed strong positive associations between socioeconomic disadvantage and burn injuries . in england and wales , deaths rates from burn injuries were 16 and 38 times higher among children from families with the least favourable occupational status compared to those from the most favourable ones . burn incidence rates were also higher among danish children from families with low income and low education . when scalds by hot liquids and burns on cookers were analysed as separate categories of burn injuries , the socioeconomic differences increased . in alberta ( canada ) , children ( < 18 years ) from low - income families ( defined as those receiving subsidies for healthcare insurance premiums ) had considerably higher odds of burn injuries compared with children whose families required no financial assistance . in contrast , income was not related to fatal fire events among children less than five in tennessee , after adjustment for several maternal and child characteristics . although low maternal education was associated with a more than threefold increase in fatal fire events , confidence intervals were wide ( see table 5 ) . low income of census tracts was associated with higher rates of burns ( nonfatal and fatal cases combined ) among 019 year olds in dallas , among 016 years olds in northern manhattan , and among 014 year olds in philadelphia . in trent ( 19921997 ) socioeconomic gradients for burn and scald hospital admissions were marked , children living in the most deprived areas had a 3.5-fold higher risk than those in the least deprived areas . a similar gradient was also observed in admission rates for 014 year olds in wales . studies on hospitalisation for burns in children aged 014 years conducted in new south wales and in quebec also revealed strong positive associations between area deprivation and the risk of burn injury . and a study in cape town ( south africa ) found that poor housing conditions , socioeconomic barriers , and child dependency were associated with children 's ( 012 years ) burns in a graded fashion . although a study in stockholm found that a higher concentration of people with low ses increased the risk of burns / scalds among children aged 015 years , moderate compared to low material deprivation was associated with reduced risk of burn injuries . the association between economic deprivation of the living area of children aged 012 years , and burns / scalds did not reach significance in north and west belfast . almost all of the eighteen fall studies reviewed were area - based and examined nonfatal outcomes . most were from europe , from sweden and the united kingdom in particular . with the exception of some swedish studies , [ 30 , 40 , 47 , 61 ] low paternal social class was associated with an increased risk of fatal falls in england and wales . similarly , low education and low income were independently associated with nonfatal falls among danish children . when examined as separate categories , the risk of falls from playground equipment increased with greater socioeconomic disadvantage but falls from bunk beds did not . low parental ses , adjusted for parental country of birth , single parent home , and receipt of welfare benefits were associated with a slightly increased risk of falls only for the youngest group of children ( 04 years ) . in a peruvian study , fall injuries among 018 year olds were not associated with household poverty or with low parental education . however , the combination of household poverty and low parental education increased the odds of these injuries by 30% . this is particularly the case for studies that stratify by sex , age , or subdiagnoses of falls ( e.g. , falls from same level , falls from furniture ) . in ireland , fall injuries were considerably higher in the most , as compared to the least deprived areas , but the difference in rates only reached significance for low falls ( < 1 metre ) . results from three swedish studies reveal considerable variations in socioeconomic disparities when considering various types of falls and looking at age group and sex of the child separately ; [ 40 , 46 , 61 ] and even differences over time are reported . most strikingly , both aggravating and protective effects are reported . for instance , one of these studies , among young children ( 05 years ) , reported protective effects ( about 30% ) for falls at the same level ( the largest diagnosis ) and from heights , and aggravating effects for falls from items of furniture ( about 34% ) . no association was found with falls from playground equipment . among older children ( 615 years ) , protective effects were found for falls from playground equipment , falls from trees and sports - related falls , while aggravating effects for material deprivation ( moderate level only ) were reported for falls on the same level . the study including a time perspective on falls among boys and girls in the age groups 1014 and 1519 years observed that the association between area deprivation and fall injuries changed markedly over time among girls aged 1519 years , from being protective in the early nineties ( 199395 ) to being aggravating in the early 2000s ( 200305 ) . no such changes were observed among younger girls or among boys of both age groups , where associations were weak in both time periods . a recent canadian study ( quebec ) also observed that associations between area material deprivation and hospitalization for falls among 014 year old children were only significant for particular types of falls . deprivation was associated with an increased risk of falls from stairs and from a building , and with a decreased risk for falls on the same level . in contrast , an australian study separated falls that occurred in the playground and those that did not , and found children in the most disadvantaged areas had a reduced risk of both kinds of injury compared with those in the least deprived areas . children in the poorest quintile had a 42% higher risk of fall injuries compared to children in the richest quintile . in a study concerned with trends in socioeconomic disparities across urban areas of canada , the risk of death from falls increased by 29% for each unit change in income quintile , from highest to lowest . similar results were observed in the united kingdom and the united states . in wales and trent , there were clear gradients of increasing hospital admission rates among 014 year olds with increased deprivation . children from moderately and largely low - income census tracts in northern manhattan had a 50% and 90% higher risk of severe fall injuries ( including deaths ) than those in better - off areas . there was no association between area material deprivation or socioeconomic status and swedish children 's hospitalisation for fall - related injuries . and in england , the association between an index of multiple deprivation and serious child fall injuries disappeared following adjustment for ethnicity , lone parent families , and households without a car . in addition , a multilevel study in korea found that district level deprivation , adjusted for sex and individual level ses variables , was not associated with fall mortality among 05 year olds . children are vulnerable to injuries for various types of reasons and , as they develop , their injury pattern changes [ 1 , 2 , 62 ] . by considering several injury causes , a broader spectrum of potential hazards it is also possible to examine whether socioeconomic differences tend to remain constant across causes and settings . unfortunately , not all injury causes have been studied for their socioeconomic patterning to the same extent and an across - causes examination is rendered more difficult . in particular , drowning has rarely been investigated , despite the importance of these injuries in child morbidity and mortality . an additional drawback is the strong bias of the knowledge at hand to the conditions prevailing in a few high income countries , the evidence therefore being mainly representative of some types of governments , economies , and forms of social stratification ( see also below ) . from a methodological point of view , despite 20 years of empirical contributions , studies remain predominantly descriptive , [ 4 , 7 , 911 ] and , for most injury causes , rather than being conducted at the individual level ( using measures such as maternal or paternal occupation , education , class or income , and household economy ) , many studies are area - based ( using measures such as neighbourhood deprivation , percentage of low income households , and percentage of unemployed ) . the choice of observation unit is also very much country - dependant , with many studies from the uk for instance being area - based while others from sweden are individual - based . in the case of area - based studies , it can be argued that , while designs of that kind are regarded as much weaker than individual level ones , contextual and environmental modification ( reducing exposure by e.g. , eliminating , modifying , or separating sources of danger ) is regarded as a very powerful and effective measure for primary injury prevention . an earlier review and subsequent studies also show that the living environment may play a role in childhood injury causation , independent of individual attributes . an additional observation is the paucity of studies that present data for boys and girls separately or for different age strata . this is further discussed below . although differences in studies ' characteristics ( e.g. , method , sample size , setting , and age range ) and methodology ( e.g. , data collection tools , relationship characterization , and injury severity criteria ) make comparisons complicated , a number of observations nonetheless arise . as expected from previous reviews , [ 411 ] there tend to be substantial socioeconomic disparities in injuries throughout childhood and in all major causes of unintentional injuries . interestingly also , the magnitude of the differences varies between causes and , within causes , between contexts as well as , when considered , between age groups . to explain the very existence of socioeconomic differences in injuries , the notion of fundamental causes is useful . the fundamental causes theory suggests that a range of resources susceptible to protect one 's health and safety are socially distributed , implying that people of higher ses hold an advantage in warding off threats to their and their offspring's wellbeing . the theory predicts that ses is more strongly associated with health outcomes for which prevention and treatment measures are known , which largely applies to unintentional injuries [ 1 , 2 ] . poverty and material deprivation are acknowledged risk factors of child injuries , [ 1 , 2 , 711 , 62 ] with two broad mechanisms contributing to this . one obvious one is that children from poor families and from more deprived areas are more exposed to a wider range of hazards , what towner and colleagues call proximate tier and others , differential an additional mechanism is that their care provider or themselves lack the means to protect themselves in their home or in their community ( e.g. , the means to afford safe equipments or devices ) , differential vulnerability [ 911 ] . in a recent review , schwebel and gaines have summarized the situation in the following manner : ( ) in homes where financial and temporal resources are limited , both tangible ( e.g. , smoke alarms , bicycle helments , and outlet covers ) and intangible ( e.g. parental supervision , parental teaching of rules about safety ) mechanisms to prevent child injury are lacking or inadequate . ( [ 62 , pages 246 - 247 ] ) . the theory of fundamental causes also predicts that the strength of the association between socioeconomic status and health would vary across countries ( or settings ) depending on how any given country ( or setting ) compensates for socioeconomic disparities . compensation can occur in many different ways that are meaningful to combat socioeconomic inequality in health and safety . one is through social welfare policies that contribute to enhance either individual social mobility or individual living circumstances ( e.g. , housing and commuting conditions ) ; thereby minimizing exposure in amount and in kind . one can also imagine that welfare policies render possible better equity in care , which in turn can reduce injury lethality and minimize differential consequences of those injuries sustained . child pedestrian injuries for instance are associated with very steep social gradients in the uk , where only area - based studies have been conducted , and with negligible ones in sweden , where studies are predominantly individual - based . it is unclear whether area - based differences would also apply at the individual level in the uk ( i.e. , the ecological fallacy coming into play ) . to explain the situation in sweden , one can mention the existence of safety - for - all measures [ 65 , 66 ] that have long been on the agenda of the swedish transport sector ( see a discussion in [ 10 , 11 ] ) . but it is of note that equity - oriented measures from outside the transport sector are also very likely to have contributed to buffer the negative effect of lower ses , minimizing exposure disparities between socioeconomic groups . measures with that potential are for instance employment policies ( for both men and women ) combined with child care services , child access to recreational environments ( other than the street ) , and limited distance to and from school for all . the latter reduces both child exposure to traffic ( in duration ) and variability of exposure across living areas . it is a considerable knowledge gap that so few studies were conducted in low / middle - income countries where the burden of injuries is far greater . the bulk of the evidence at hand stems from high - income countries and , very often , countries from northern europe . this , in combination with the use of different indicators of ses between studies , impedes our ability to further explore how the current empirical evidence supports the theory of fundamental causes . processes like globalisation , urbanisation , motorisation , and environmental change could negatively impact on child safety and increase differences both between and within countries . additionally , in times of economic difficulties , as is the case when working on this paper , some factors are likely to exacerbate socioeconomic disparities . of note is disturbed parental supervision , previously proposed as an explanation to increased infant mortality due to unintentional injuries in metropolitan california in time of economic recession . also troubling is a reduction in individual insurance , such as that of insured motorists or house owners and tenants . during a recession , less basic societal investment in safety in the form of for example , built - in safety , routine maintenance and reparation , and non universal / restrictive access to trauma care will put additional responsibility on individuals who are already under pressure . whereas the above helps clarifying differences between settings or countries , it does not bring much light on age - based differences in disparities within similar settings . it is possible that in an environment like the home , where small children spend a great share of their time , the vulnerabilities inherent to poverty are more detrimental to them ( given their development process and their dependency on their caregivers ) [ 2 , 9 ] . as few studies have investigated the matter , it is difficult to draw conclusions . nonetheless , studies indicate that , for an injury cause as common as falls , disparities are stonger in the younger children than in older ones or disparities exist only in the young . because poverty exacerbates home injury risks in the young , it is possible that home safety interventions based on the uptake of safe practices may not be fully adapted to the situation of children from lower ses . but there are examples of home safety education programs that have reached normative improvements and some significant injury reductions even in resource poor households and neighborhoods that can inspire policy and practive [ 6971 ] . west for his part has suggested that a process of equalisation of socioeconomic differences in injury risks may arise at school ages and be more pronounced for a number of health outcomes , among which are unintentional injuries ( accidents ) . west poses that during that period of life , factors other than family , home background , and neighbourhood ( i.e. , class - related factors ) come into play and counteract the impact of class - related ones ; a process of class - patterning equalisation occurs [ 72 , 73 ] . among other mechanisms , the school , peer group , and youth culture may strongly impact on children 's life - style choices and behaviours ) [ 7476 ] . the process would run from childhood through to young adulthood but its impact is expected to be at a maximum during early youth ( in secondary school ) . still , according to west , in the case of unintentional injuries , equalisation would be effected by means of similarity in the activities pursued by young people from different social backgrounds in the school and peer - group contexts . to date should further studies of the like be conducted , gender differences would definitely need to be taken into account . it is indeed unfortunate that so few studies investigated whether socioeconomic disparities are more detrimental to boys than to girls or even , whether sex differences appear hand in hand with the increasing age of the child . whereas we have known for a long time that boys have greater injury mortality and morbidity than girls , we do not know how the factors contributing to those differences are socially distributed biology , cognition , socialization , and exposure opportunity . poorer chances of survival and poor health , when generated by social processes to the detriment of the less well - off , impede basic human rights [ 65 , 66 , 77 ] . sectoral examples of passive safety dealing with physical exposures show that tackling material deprivation in the home through better housing conditions , [ 64 , 78 ] or modifying the traffic environment [ 64 , 7881 ] can do much to level up safety differentials between members of different social groups . given that injuries are the leading cause of death and disability among children worldwide , abatement strategies of the like that reduce injury risks for all children can only be welcomed . one could posit that the more the injury cause varies across socioeconomic groups ( the theory of fundamental causes applying ) , the less likely it is that behavioural interventions will make a significant contribution to either disparity or global risk reduction [ 10 , 11 , 71 ] . one eloquent example of the problem arising with behavioural or safe practice campaigns is the case of burn injuries and fires in the uk where socioeconomic disparities are huge and where interventions aiming at the installation of burn detectors in resources - poor areas , even if subsidised , all failed to achieve their injury reduction goals . the same reasoning could apply to pedestrian injuries when the preventive measures put forward rely on enhanced parental supervision [ 10 , 70 ] . although numerous interventions have been evaluated and promoted as effective , few have been conducted that assess whether those interventions are equally effective in all socioeconomic groups ( or areas ) or if they help reduce differences between those groups . in other words , very few interventions have been evaluated for their potential in childhood injury inequality reduction . below is a summary of interventions discussed that have the potential to reduce both injury rates overall and socioeconomic disparities in injuries . several of them have been discussed in earlier policy documents and reviews [ 1 , 2 , 911 , 70 , 71 ] safety - oriented legislation or regulation that determines minimum standards and conditions under which a number of activities can not be performed or imposes safe behaviours and practices that would not be largely adopted on a voluntary basis . level up the safety of the physical environment through passive safety measures can be achieved through engineering and product development . it is a matter of modifying , isolating , separating or eliminating the sources of danger . community - based prevention programmes that intend to tackle the safety level of communities by combining strategies like behavioural and environmental changes , in some instances together with enforcing legislation and subsidies . home safety education and home visit programmes aiming at promoting safe practices in the home and also for the prevention of both unintentional and intentional injuries . the creation of attractive places for recreation as the fewer off - street play areas that are offered , the more the street environment becomes not only an area for traffic but also one for recreation . the literature on socioeconomic disparities in child injuries is abundant but reviews are few . by reviewing the literature on several childhood injury causes , a broader spectrum of potential hazards the findings at hand are biased to some causes ( especially traffic - related injuries , burns and falls ) and some high income countries . in the main , they offer support to the notion that low ses is often greatly detrimental to child safety . injuries are highly preventable and socioeconomic differences in wealth need not be reflected in differences in safety . variations between causes and , within causes , between settings and countries suggest that the prevention of inequities in child safety needs not only that direct mechanisms of injuries be tackled but also remote and fundamental ones inherent to poverty .

injuries are one of the major causes of both death and social inequalities in health in children . this paper reviews and reflects on two decades of empirical studies ( 1990 to 2009 ) published in the peer - reviewed medical and public health literature on socioeconomic disparities as regards the five main causes of childhood unintentional injuries ( i.e. , traffic , drowning , poisoning , burns , falls ) . studies have been conducted at both area and individual levels , the bulk of which deal with road traffic , burn , and fall injuries . as a whole and for each injury cause separately , their results support the notion that low socioeconomic status is greatly detrimental to child safety but not in all instances and settings . in light of variations between causes and , within causes , between settings and countries , it is emphasized that the prevention of inequities in child safety requires not only that proximal risk factors of injuries be tackled but also remote and fundamental ones inherent to poverty .

1. Introduction 2. Review Methods 3. Results 4. Discussion 5. Conclusions

despite being regarded as highly preventable , injuries account for an increasing share of childhood mortality in the world [ 1 , 2 ] . the most common causes of child mortality and morbidity by injury are road traffic crash , drowning , poisoning , burns , and falls . these injuries are unevenly distributed between countries [ 1 , 3 ] and , within countries , between socioeconomic groups , to the detriment of the more disadvantaged families and communities . injuries are in fact acknowledged as one of the causes of childhood mortality with the steepest socioeconomic gradient [ 115 ] . despite this knowledge , in the medical and public health literature alone , a great deal of research is regularly published on socioeconomic disparities in childhood injury that specifically aims at measuring the magnitude of those differences . as this literature is highly descriptive in nature ( as is the case for studies on socioeconomic differences in injuries in general ) , there is a paucity of studies on the mechanisms susceptible to generate those differences , which poses challenges to prevention work . likewise , the reasons why differences exist in socioeconomic disparities across studies are seldom thoroughly addressed . some of the reviews mentioned above that , inspired by lead authors on social inequalities in health , put forward key mechanisms that help understanding why socioeconomic disparities may arise [ 4 , 911 ] but they deal with the why differences exist and how to combat differences questions in generic terms and they provide little insight regarding why socioeconomic differences vary both in magnitude and direction , either with increasing age or across settings . it considers the five leading causes of child mortality and morbidity mentioned above , thus allowing a more accurate coverage of the whole age spectrum and helping to highlight whether and how socioeconomic disparities in injuries vary over age category and causes . here , we examine and reflect on two decades of studies ( 1990 to 2009 ) on socioeconomic disparities in injuries among children aged up to 18 years . the findings are discussed in light of various conceptual approaches to the understanding of the socioeconomic differences in health and safety . the source reviews [ 10 , 11 ] encompassed empirical studies on socioeconomic differences in injuries published in the medical and public health literature during the period 19902006 and , in preparing this paper , an additional three years were added ( 20072009 ) . the articles initially sought for were original research articles that examined socioeconomic disparities in injury risk across socioeconomic groups all ages and all injuries in the first review and unintentional injuries in children in the second one . the articles were obtained through a literature search in the databases of safetylit and the national library of medicine 's medline . for the former database , all studies included under social disparities swedish , and danish language studies published between january 1990 and december 2009 were identified using the keywords injury or injuries or accident or accidents in conjunction with educational status or education or social class or socioeconomic status or occupation or income or social position or socioeconomic position or socioeconomic context or social context or deprivation or socioeconomic factors or socioeconomic characteristics or residence characteristics or neighbourhood and infant or infants or child or children or childhood or adolescent or adolescents or adolescence or youth . additional studies were also identified from the reference lists in selected articles and in those of the reviews listed above . it is important to note that , in the injury field , the safetylit database has both breadth ( number of journals included ) and depth ( coverage from each journal 's backfiles ) . in fact , it has been found that of five commonly used databases , embase , psycinfo , pubmed / medline , safetylit , and web of science ( including the science citation index and the social science citation index ) , the database with the greatest breadth and depth of coverage for journals that publish articles in the injury prevention and safety promotion ( ipsp ) field is safetylit . the safetylit database coverage includes all ipsp - relevant journals from each of the listed databases and , for the journals that are found in the source lists of the other databases , a greater depth of coverage of backfile years . further , the safetylit database contains articles from journals that are not included in any of the other listed databases . because of the wider scope of the original review , and the change in focus in the subsequent updates ( second review and the current one ) , it is unfortunately not possible to specify the total number of articles originally identified from the literature searches that deal with socioeconomic differences in childhood injuries . as a consequence , we also can not specify the number ( or proportion ) of them that meet the selection criteria presented below . to be included in the current review , they examined the relationship between socioeconomic status ( ses ) and injury at an individual- or area - level as the primary research question , 2 . they considered one or several of the five major cause of mortality and morbidity among children , 3 . they included denominators ( i.e. the details retained for the current paper can be found in the results tables 2to 7 . in case of disagreement between the reviewers , table 1 presents an overview of the studies reviewed by injury cause ( traffic being split into four categories ) , severity level ( mortality versus morbidity ) , country , and type of relationship between ses and injury . as the bulk of those studies considered boys and girls simultaneously road traffic injuries are by far the most studied cause of health disparities in the child injury field . all studies show a positive relationship between individual socioeconomic disadvantage or deprivation of the living area and pedestrian injuries [ 2445 ] . one of the studies observed that the association between deprivation and increased pedestrian casualties in england is stronger among children than among older age groups . individual - based studies from sweden and the united kingdom show that children in families with low socioeconomic position ( measured in terms of parental social class , education or disposable income ) are at greater risk for bicycle injuries [ 18 , 24 , 33 , 34 , 45 ] . the swedish studies were based on individual data and showed a positive relationship between socioeconomic diadvantage and road traffic injuries as motorcyclists [ 24 , 48 ] . one individual - based study showed that children of unskilled workers have significantly higher odds for injuries as a moped user as compared to children of intermediate and high - level salaried employees on the other hand , two area - based studies found that living in areas with higher levels of deprivation reduced the risk for moped injuries [ 39 , 46 ] . individual - based studies from sweden and the united kingdom showed that young people in the most disadvantaged families have an increased risk for injuries as a car driver compared to children in the most advantaged families [ 24 , 34 , 49 , 50 ] . tables 5 ( individual level studies ) , 6 ( area level studies ) and 7 ( multilevel studies ) describe the studies that have dealt with causes of injuries other than road traffic - related ones . in the text below , the cause specific results are presented . in south korea , one , a multilevel study conducted among small children ( 05 years ) , found a positive relationship between area - level deprivation and risk of drowning , after adjustment for sex and individual level ses variables . of the eleven studies that examined socioeconomic disparities in poisoning injury , the majority are ecological ( n = 7 ) and all but one ( england and wales ) examined nonfatal injuries . the other three studies , from high - income countries , observed large socioeconomic disparities . studies on injury hospitalisations in children aged 014 years conducted in new south wales ( australia ) and in qubec ( canada ) showed that children in the most deprived quintile had a 52% and 68% higher risk , respectively , than children in the least deprived quintile . in the former study , it was also observed that gradients were particularly steep for benzodiazepines , antidepressants , cough and cold remedies , and organic solvents . in stockholm , the results depended on the measure of ses but not low material deprivation was associated with poisoning among children aged 015 years . in ireland , although 012 year olds living the most deprived areas of north and west belfast had 3.65 the risk of being treated in the emergency department for poisoning compared to those in the least deprived areas , the association failed to reach significance . in the studies reviewed , the definition of what constitutes a burn differed , some studies only dealt with injuries related to fire and flames , other included scalds from hot liquids . in several studies unlike other causes , an almost equal number of individual and area levels studies have been conducted . in the ashanti region ( ghana ) , one study found that maternal education was not significantly associated with burn injuries ( with evidence of a physical scar ) among children aged 05 years . in the other study , household poverty was associated with an increased odds of burn injuries but low education of the household head was not . low income of census tracts was associated with higher rates of burns ( nonfatal and fatal cases combined ) among 019 year olds in dallas , among 016 years olds in northern manhattan , and among 014 year olds in philadelphia . the association between economic deprivation of the living area of children aged 012 years , and burns / scalds did not reach significance in north and west belfast . low parental ses , adjusted for parental country of birth , single parent home , and receipt of welfare benefits were associated with a slightly increased risk of falls only for the youngest group of children ( 04 years ) . in a peruvian study , fall injuries among 018 year olds were not associated with household poverty or with low parental education . in ireland , fall injuries were considerably higher in the most , as compared to the least deprived areas , but the difference in rates only reached significance for low falls ( < 1 metre ) . results from three swedish studies reveal considerable variations in socioeconomic disparities when considering various types of falls and looking at age group and sex of the child separately ; [ 40 , 46 , 61 ] and even differences over time are reported . for instance , one of these studies , among young children ( 05 years ) , reported protective effects ( about 30% ) for falls at the same level ( the largest diagnosis ) and from heights , and aggravating effects for falls from items of furniture ( about 34% ) . the study including a time perspective on falls among boys and girls in the age groups 1014 and 1519 years observed that the association between area deprivation and fall injuries changed markedly over time among girls aged 1519 years , from being protective in the early nineties ( 199395 ) to being aggravating in the early 2000s ( 200305 ) . in contrast , an australian study separated falls that occurred in the playground and those that did not , and found children in the most disadvantaged areas had a reduced risk of both kinds of injury compared with those in the least deprived areas . children in the poorest quintile had a 42% higher risk of fall injuries compared to children in the richest quintile . in a study concerned with trends in socioeconomic disparities across urban areas of canada , the risk of death from falls increased by 29% for each unit change in income quintile , from highest to lowest . children from moderately and largely low - income census tracts in northern manhattan had a 50% and 90% higher risk of severe fall injuries ( including deaths ) than those in better - off areas . there was no association between area material deprivation or socioeconomic status and swedish children 's hospitalisation for fall - related injuries . and in england , the association between an index of multiple deprivation and serious child fall injuries disappeared following adjustment for ethnicity , lone parent families , and households without a car . children are vulnerable to injuries for various types of reasons and , as they develop , their injury pattern changes [ 1 , 2 , 62 ] . by considering several injury causes , a broader spectrum of potential hazards it is also possible to examine whether socioeconomic differences tend to remain constant across causes and settings . in particular , drowning has rarely been investigated , despite the importance of these injuries in child morbidity and mortality . an additional drawback is the strong bias of the knowledge at hand to the conditions prevailing in a few high income countries , the evidence therefore being mainly representative of some types of governments , economies , and forms of social stratification ( see also below ) . from a methodological point of view , despite 20 years of empirical contributions , studies remain predominantly descriptive , [ 4 , 7 , 911 ] and , for most injury causes , rather than being conducted at the individual level ( using measures such as maternal or paternal occupation , education , class or income , and household economy ) , many studies are area - based ( using measures such as neighbourhood deprivation , percentage of low income households , and percentage of unemployed ) . in the case of area - based studies , it can be argued that , while designs of that kind are regarded as much weaker than individual level ones , contextual and environmental modification ( reducing exposure by e.g. , eliminating , modifying , or separating sources of danger ) is regarded as a very powerful and effective measure for primary injury prevention . , data collection tools , relationship characterization , and injury severity criteria ) make comparisons complicated , a number of observations nonetheless arise . as expected from previous reviews , [ 411 ] there tend to be substantial socioeconomic disparities in injuries throughout childhood and in all major causes of unintentional injuries . interestingly also , the magnitude of the differences varies between causes and , within causes , between contexts as well as , when considered , between age groups . to explain the very existence of socioeconomic differences in injuries , the notion of fundamental causes is useful . poverty and material deprivation are acknowledged risk factors of child injuries , [ 1 , 2 , 711 , 62 ] with two broad mechanisms contributing to this . , the means to afford safe equipments or devices ) , differential vulnerability [ 911 ] . , smoke alarms , bicycle helments , and outlet covers ) and intangible ( e.g. the theory of fundamental causes also predicts that the strength of the association between socioeconomic status and health would vary across countries ( or settings ) depending on how any given country ( or setting ) compensates for socioeconomic disparities . compensation can occur in many different ways that are meaningful to combat socioeconomic inequality in health and safety . child pedestrian injuries for instance are associated with very steep social gradients in the uk , where only area - based studies have been conducted , and with negligible ones in sweden , where studies are predominantly individual - based . it is unclear whether area - based differences would also apply at the individual level in the uk ( i.e. to explain the situation in sweden , one can mention the existence of safety - for - all measures [ 65 , 66 ] that have long been on the agenda of the swedish transport sector ( see a discussion in [ 10 , 11 ] ) . but it is of note that equity - oriented measures from outside the transport sector are also very likely to have contributed to buffer the negative effect of lower ses , minimizing exposure disparities between socioeconomic groups . it is a considerable knowledge gap that so few studies were conducted in low / middle - income countries where the burden of injuries is far greater . the bulk of the evidence at hand stems from high - income countries and , very often , countries from northern europe . processes like globalisation , urbanisation , motorisation , and environmental change could negatively impact on child safety and increase differences both between and within countries . additionally , in times of economic difficulties , as is the case when working on this paper , some factors are likely to exacerbate socioeconomic disparities . during a recession , less basic societal investment in safety in the form of for example , built - in safety , routine maintenance and reparation , and non universal / restrictive access to trauma care will put additional responsibility on individuals who are already under pressure . whereas the above helps clarifying differences between settings or countries , it does not bring much light on age - based differences in disparities within similar settings . it is possible that in an environment like the home , where small children spend a great share of their time , the vulnerabilities inherent to poverty are more detrimental to them ( given their development process and their dependency on their caregivers ) [ 2 , 9 ] . as few studies have investigated the matter , it is difficult to draw conclusions . nonetheless , studies indicate that , for an injury cause as common as falls , disparities are stonger in the younger children than in older ones or disparities exist only in the young . because poverty exacerbates home injury risks in the young , it is possible that home safety interventions based on the uptake of safe practices may not be fully adapted to the situation of children from lower ses . west for his part has suggested that a process of equalisation of socioeconomic differences in injury risks may arise at school ages and be more pronounced for a number of health outcomes , among which are unintentional injuries ( accidents ) . west poses that during that period of life , factors other than family , home background , and neighbourhood ( i.e. among other mechanisms , the school , peer group , and youth culture may strongly impact on children 's life - style choices and behaviours ) [ 7476 ] . still , according to west , in the case of unintentional injuries , equalisation would be effected by means of similarity in the activities pursued by young people from different social backgrounds in the school and peer - group contexts . it is indeed unfortunate that so few studies investigated whether socioeconomic disparities are more detrimental to boys than to girls or even , whether sex differences appear hand in hand with the increasing age of the child . poorer chances of survival and poor health , when generated by social processes to the detriment of the less well - off , impede basic human rights [ 65 , 66 , 77 ] . given that injuries are the leading cause of death and disability among children worldwide , abatement strategies of the like that reduce injury risks for all children can only be welcomed . one could posit that the more the injury cause varies across socioeconomic groups ( the theory of fundamental causes applying ) , the less likely it is that behavioural interventions will make a significant contribution to either disparity or global risk reduction [ 10 , 11 , 71 ] . one eloquent example of the problem arising with behavioural or safe practice campaigns is the case of burn injuries and fires in the uk where socioeconomic disparities are huge and where interventions aiming at the installation of burn detectors in resources - poor areas , even if subsidised , all failed to achieve their injury reduction goals . although numerous interventions have been evaluated and promoted as effective , few have been conducted that assess whether those interventions are equally effective in all socioeconomic groups ( or areas ) or if they help reduce differences between those groups . below is a summary of interventions discussed that have the potential to reduce both injury rates overall and socioeconomic disparities in injuries . several of them have been discussed in earlier policy documents and reviews [ 1 , 2 , 911 , 70 , 71 ] safety - oriented legislation or regulation that determines minimum standards and conditions under which a number of activities can not be performed or imposes safe behaviours and practices that would not be largely adopted on a voluntary basis . it is a matter of modifying , isolating , separating or eliminating the sources of danger . home safety education and home visit programmes aiming at promoting safe practices in the home and also for the prevention of both unintentional and intentional injuries . the creation of attractive places for recreation as the fewer off - street play areas that are offered , the more the street environment becomes not only an area for traffic but also one for recreation . the literature on socioeconomic disparities in child injuries is abundant but reviews are few . by reviewing the literature on several childhood injury causes , a broader spectrum of potential hazards the findings at hand are biased to some causes ( especially traffic - related injuries , burns and falls ) and some high income countries . in the main , they offer support to the notion that low ses is often greatly detrimental to child safety . variations between causes and , within causes , between settings and countries suggest that the prevention of inequities in child safety needs not only that direct mechanisms of injuries be tackled but also remote and fundamental ones inherent to poverty .

the annealing helicase hepa - related protein ( harp ; also known as smarcal1 ) is an atp - dependent enzyme capable of rewinding dna that is bound by the single - stranded dna ( ssdna)-binding protein replication protein a ( rpa ) . such rpa - bound structures can arise during normal processes that involve unwinding of the dna duplex , such as dna replication , dna repair and transcription . harp has been shown to play an important role during both dna replication and repair , and cells lacking harp exhibit replication defects and persistent rpa binding . the importance of harp has been further highlighted by the finding that mutations in human harp cause a rare disorder known as schimke immuno - osseous dysplasia ( siod ) . while the molecular basis of siod is not fully understood , a potential link between an increase in chromosome breakage and mutations in harp is consistent with a role of harp in dna repair . harp binds preferentially and with high affinity to forked dna structures ( i.e. , split ends ) , which are formed at the ends of ssdna regions . harp interacts directly with rpa through a conserved motif near the n terminus of harp . while this interaction is dispensable for the annealing helicase activity of harp , it helps localize harp to sites of dna replication and repair . in addition to the annealing helicase activity of harp , which regulates the amount of rpa - bound ssdna in the cell , rpa can be regulated through phosphorylation . rpa , a heterotrimer consisting of rpa1 , rpa2 and rpa3 , is preferentially phosphorylated on the n terminus of its rpa2 subunit . several kinases phosphorylate rpa including cyclin - dependent kinase ( cdk ) , ataxia - telangiectasia mutated ( atm ) , atm and rad3-related ( atr ) , and the dna - dependent protein kinase ( dna - pk ) . changes in rpa phosphorylation have been observed during cell - cycle progression and in response to dna damage , which ultimately leads to hyperphoshorylation of rpa . the downstream effects of rpa phosphorylation depend on the residues that are phosphorylated and include checkpoint activation , the regulation of dna repair , and the modulation of rpa - dna interactions . like rpa , harp is also phosphorylated in response to replication stress and dna damage , although the significance of harp phosphorylation is less clear . atm , atr and dna - pk are all capable of phosphorylating harp . despite the knowledge that harp associates with rpa , and that both factors are substrates for multiple kinases , little is known about which factors form the preferred harp complex . in this study , we found that , after rpa , dna - pk is the most abundant factor in the harp complex . a stable interaction of dna - pk with harp is mediated by rpa and suggests a tendency for harp to associate with rpa bound by dna - pk . using dna - pk purified from hela cells by a rapid dna - affinity protocol , we found that while dna - pk is able to phosphorylate rpa in vitro , it is unable to phosphorylate rpa in the presence of harp . dna - pk also phosphorylates harp in vitro and the phosphorylation of harp by dna - pk does not significantly affect its annealing helicase activity . together , our data suggest that harp inhibits the function or activity of rpa by both eliminating ssdna via the annealing helicase activity of harp and by preventing rpa phosphorylation by dna - pk . as previously reported , we purified harp from hela cells that constitutively express a flag - tagged copy of harp by tandem - affinity purification . following the purification , we observed that in addition to rpa , three other factors stably co - purify with harp . these proteins were identified by mass spectrometry as the subunits of the dna - pk heterotrimer ( i.e. , dna - pkcs , ku80 , and ku70 ) . other reports have identified dna - pk subunits as part of a group of potential cofactors for harp , but our analysis reveals that rpa and dna - pk are the two most abundant factors that stably co - purify with harp ( fig . , we did not detect wrn helicase , a factor previously reported to associate with harp , suggesting that wrn may be part of a less abundant or less stable harp complex . ( a ) the harp - containing complex was purified from hela cells by tandem anti - flag / anti - ha purification and resolved by sds - page and silver staining . ( b ) harp , rpa , and dna - pk co - fractionate on glycerol gradients . the purified harp complex was resolved on a 1030% glycerol gradient and the migration of the proteins determined by sds - page and silver staining . to gain insight into the stability of the harp - rpa - dna - pk interactions we collected fractions off the top of the gradient and analyzed them by sds - page and silver staining . we found that the dna - pk heterotrimer is present in the same fractions that contain both harp and rpa ( fig . 1b , fractions 7 to 11 ) , suggesting that harp , rpa , and dna - pk associate in a single , stable complex . to examine how dna - pk affects the activity of harp , we purified the dna - pk heterotrimer from hela cells using a one - step dna - affinity protocol ( fig . we first crosslinked sonicated poly-[di : dc ] dna fragments to sepharose beads and then used the free dna ends to purify dna - pk from hela nuclear extracts . we initially prepared nuclear extracts from 4 l of hela cells and pre - incubated the extracts with plasmid dna ( which has no free dna ends ) to remove non - specific dna - binding proteins . we then incubated the pre - cleared extracts with the dna - resin that contains free dna ends . the beads were then washed to remove the unbound proteins , and the bound proteins were eluted using a step gradient of increasing nacl . under these conditions , the collected fractions consisted almost entirely of the dna - pk heterotrimer , which eluted from the resin in the fractions containing between 0.3 to 0.4 m of nacl ( fig . a scaled - down version of the protocol was used to extract dna - pk from hela cells from a single 15 cm dish , showing that small - scale preparations of dna - pk can be rapidly made ( fig . isolation of native dna - pk by a single - step dna - affinity purification method . ( a ) dna was crosslinked to sepharose beads and used to extract dna - pk , which preferentially binds to free dna ends . ( b ) nuclear extracts from 4 l of hela cells were pre - incubated with circular plasmid dna to bind non - specific dna binding proteins . the extract was then passed through the crosslinked beads , the unbound proteins were washed off , and the bound proteins were eluted by increasing salt step gradient . ( c ) a scaled - down purification was performed with nuclear extracts prepared from a single 15 cm dish . the peak fraction from the salt gradient was analyzed by sds - page followed by silver staining . using the native dna - pk purified from hela cell extracts , we first asked whether dna - pk interacts directly with harp or associates with the complex through an interaction with rpa . following several independent purifications of the harp complex , we noticed that the dna - pk is often less abundant than harp and rpa . we considered the possibility that dna - pk , which is known to associate with rpa , is present in the harp complex through a direct interaction with rpa . to determine whether rpa is required for dna - pk to stably associate with the harp complex , we tested whether a mutant version of harp , which contains a 10 amino acid substitution in the n - terminal rpa - interaction motif , can interact with dna - pk . we incubated purified rpa , dna - pk with either wild - type or mutant harp , immunoprecipitated the harp proteins using anti - flag antibodies , and analyzed the co - precipitating factors by western blotting ( fig . as previously reported , we found that wild - type but not mutant harp interacts with rpa . moreover , dna - pk readily co - precipitates with wild - type harp but not the mutant harp , suggesting that a direct interaction between harp and dna - pk is not stable . we can not rule out the possibility that rpa and dna - pk compete for the same binding site on harp and that the mutant harp fails to interact with both rpa and dna - pk . however , we think this is unlikely as our gradient analysis suggests that harp , rpa , and dna - pk all appear to stably interact in one single complex . our findings suggest that harp exhibits a preference , or tendency , to interact with rpa bound to dna - pk . ( a ) purified wild - type ( wt ) or mutant ( mut ) harp proteins were purified by a baculovirus system and incubated with bacterially expressed rpa and native dna - pk . ( b ) the harp proteins were then immunoprecipitated using anti - flag antibodies , and the immunoprecipitated material was analyzed by western blotting . ( c ) in vitro kinase assays were performed by incubating purified dna - pk with harp , rpa , [ p]-labeled atp and plasmid dna . ( d ) the in vitro kinase assays were performed in the presence of equal amounts of wt or mut harp proteins . we next used the purified dna - pk to investigate the phosphorylation of harp and rpa . in order to examine whether harp is a substrate for dna - pk and whether harp affects the phosphorylation of rpa by dna - pk , we performed in vitro kinase assays . we incubated dna - pk with harp , the rpa heterotrimer , or both in the presence of double - stranded plasmid dna and [ p]-atp . we then resolved the proteins by sds - page , dried and scanned the gel using a phosphorimager . we found that both harp and rpa can be independently phosphorylated in vitro by dna - pk ( fig . when both harp and rpa are present in the reactions , the phosphorylation of harp and rpa2 by dna - pk is substantially reduced ( fig . in fact , we were unable to detect any rpa phosphorylation in the presence of harp . one potential mechanism by which harp could block the phosphorylation of rpa is by binding and masking the phosphorylation residues of rpa2 . this idea is supported by the fact that the n terminus of harp contains an rpa2-interacting motif . on the other hand , it is also possible that harp interferes with rpa2 phosphorylation through a more subtle mechanism , such as by interfering with the kinase activity of dna - pk or by weakening dna - pk - rpa2 interactions . in order to address these potential models , we compared the levels of phosphorylation of rpa2 by dna - pk in the presence of wild - type and mutant harp proteins . the mutant harp does not stably interact with rpa and should be less likely to mask rpa2 phosphorylation sites . we found that both wild - type and mutant harp proteins are able to block rpa2 phosphorylation by dna - pk ( fig . in addition , while the levels of phosphorylation of wild - type harp are reduced in the presence of rpa , the levels of phosphorylation of mutant harp remain high in the presence of rpa . these findings suggest that harp does not simply mask the phosphorylation sites of rpa through the stable interaction of the two factors . it is likely that a more complex mechanism is behind the ability of harp to block the phosphorylation of rpa2 . for example , in addition to the stable interaction between harp and rpa that is mediated by the conserved n terminus of harp , multiple weaker interactions may exist between other regions of harp and rpa . these interactions may be sufficient for both wild - type and mutant harp to block the phosphorylation of rpa2 . furthermore , interactions between harp and dna - pk may exist that are not strong enough for the mutant harp to co - purify with dna - pk , but are strong enough for harp to affect the ability of dna - pk to phosphorylate rpa . in contrast , the observed reduction in the phosphorylation of harp by dna - pk and in the presence of rpa may be explained by the masking of phosphorylation sites on harp by rpa . this would explain why mutant harp , which fails to stably interact with rpa , does not show the reduced levels of phosphorylation seen with wild - type harp . the role that phosphorylation of harp plays in the cell is poorly understood although it has been suggested that phosphorylation of some residues may affect the activity of harp . to test whether dna - pk affects the atp - dependent activity of harp , we performed annealing helicase assays in the presence or absence of purified dna - pk . we generated rpa - bound dna substrates using purified rpa and plasmid dna ( fig . in contrast , if the rpa - bound substrate is rewound , the resulting double - stranded plasmid dna will be in a relaxed form following precipitation . supercoiled and relax dna are resolved by agarose gel electrophoresis . as previously reported , we observed that harp , in the absence of dna - pk , rewinds the unwound regions of the dna in an atp - dependent manner ( fig . 4b , lanes 3 and 4 ) . we also found that addition of dna - pk to the rpa - bound dna in the absence of harp resulted in a modest decrease in the stability of the rpa - ssdna complex in an atp - independent manner ( fig . 4b , lanes 5 and 6 ) . reactions in which both harp and dna - pk were added to the rpa - bound substrate did not show any change in the annealing helicase activity of harp ( fig . 4 , lanes 7 and 8) . under the conditions we tested , harp is able to fully rewind the rpa - bound dna in the presence of dna - pk and atp . it should be noted that in the reactions that contained both harp and dna - pk , we pre - incubated harp with dna - pk in the presence or absence of atp to allow for harp phosphorylation before the addition of the rpa - bound dna . based on this finding , and the fact that dna - pk is present in the harp complex through its binding to rpa , it appears likely that rpa is the main target of the kinase activity of dna - pk . the presence of dna - pk does not significantly affect the annealing helicase activity of harp . annealing helicase assays were performed in the presence or absence of dna - pk using a plasmid partially loaded with rpa . for the reactions that contain both dna - pk and harp , the two factors were preincubated in the presence or absence of atp prior to addition of the rpa - bound dna substrate . following the reactions , the purified dna was analyzed by agarose gel electrophoresis and visualized by ethidium bromide ( etbr ) staining . the role of phosphorylation in the regulation of the activities of rpa is not entirely understood . some studies have reported that dna - pk preferentially phosphorylates rpa that is bound to ssdna near junctions with dsdna , such as at fork dna , which are also the preferred binding sites for harp . this suggests that harp , rpa , and dna - pk may function together at these unique dna sites . our in vitro pull - down experiments were performed in the absence of dna and demonstrate that harp , rpa , and dna - pk do not require dna for their interaction ( fig . whether significant amounts of harp , rpa , and dna - pk are interacting without dna in the nucleus is unclear . the purification of the harp complex may result in extraction of the three factors off of the dna . our study also found that harp interferes with rpa phosphorylation both by its annealing helicase activity , which limits the amount of ssdna in the cell , and by preventing phosphorylation of rpa by protein : protein interactions . thus , it appears that harp plays a multifaceted role in regulating the function of rpa . the connection between mutations in harp and siod , a disease with a pleiotropic phenotype , is not fully understood . the diverse roles of harp and rpa in modulating the levels of ssdna in the cell likely impact many processes such as dna replication , repair , and transcription , ultimately contributing to the complex nature of siod . the native harp - containing complex was purified from hela cell nuclear extracts by tandem - affinity purification as previously reported . the glycerol gradients were performed by loading the purified harp complex onto a 4 ml , 10 to 30% linear gradient in harp buffer ( 20 mm hepes , ph 7.6 , 1.5 mm mgcl2 , 10% glycerol , 0.05% np40 , 1 mm dtt , 0.2 mm pmsf ) + 0.1 m kcl . the gradients were centrifuged at 170 000 g for 3 h at 4 c . the native dna - pk heterotrimer was purified by a single step dna - affinity chromatography method . poly-[di : dc ] ( amersham ) was resuspended in h2o and briefly sonicated ( three times for 2 s each ) to fragment the polymers into smaller fragments . the fragments were then crosslinked to cnbr - activated sepharose ( ge ) according to the manufacturer protocol . nuclear extracts were prepared from hela cells using 0.42 m kcl extraction as previously described and dialyzed to 0.1 m kcl . the extracts were then incubated with plasmid dna ( at a concentration of 130 g / ml ) on ice for 10 min . the extract was then centrifuged at 16 000 g for 10 min to remove precipitations . the clarified extract was then incubated with the crosslinked sepharose beads for 10 min at 4 c on a rotator . the mix was poured into a 10 ml disposable column and the resin was allowed to settle . the column was drained and the resin washed four times with 5 column volumes of harp buffer + 0.1 m nacl . the human rpa used in all of the assays was expressed and purified from bacteria as previously described . harp ( 0.5 g ) , rpa ( 0.5 g ) , dna - pk ( 0.1 g ) were incubated in 20 l of binding buffer ( 20 mm hepes [ k+ ] , ph 7.6 , 0.1 m kcl , 5 mm mgcl2 , 3% [ v / v ] glycerol , 0.25 mg / ml bsa , 0.05 mm edta , 0.5 mm dtt , 0.01% ( v / v ) np-40 ) for 20 min at 30 c . the reactions were then diluted to 200 l with binding buffer and incubated with 10 l of anti - flag resin for 3 h at 4 c . the resin was washed three times with binding buffer and the proteins eluted with sds - sample buffer . for the in vitro kinase assays , harp ( 0.4 g ) , rpa ( 0.5 g ) , dna - pk ( 0.3 g ) were incubated in 20 l of binding buffer with plasmid dna ( 60 ng ) and [ p]-atp for 15 min at 30 c . the reactions were stopped by the addition of sds sample buffer and resolved by sds - page . plasmid dna was incubated with rpa in 1 te in the presence of topoisomerase i. this results in the loading of rpa onto the plasmid due to transient opening of the dna . the rpa bound substrate is then incubated with harp in annealing helicase buffer ( 10 mm tris - hcl , ph 7.9 , 20 mm kcl , 2 mm mgcl2 , 0.5 mm dtt , 0.1 mm edta ) , and in the presence or absence of atp ( utp was included in the minus atp reactions ) . the dna is then precipitated and resolved by agarose gel electrophoresis . where indicated , 0.5 g of dna - pk was included and in reactions that contained both dna - pk and harp , the dna - pk and harp were pre - incubated with or without atp for 15 min before combining with the rpa - bound dna substrate . the native harp - containing complex was purified from hela cell nuclear extracts by tandem - affinity purification as previously reported . the glycerol gradients were performed by loading the purified harp complex onto a 4 ml , 10 to 30% linear gradient in harp buffer ( 20 mm hepes , ph 7.6 , 1.5 mm mgcl2 , 10% glycerol , 0.05% np40 , 1 mm dtt , 0.2 mm pmsf ) + 0.1 m kcl . the gradients were centrifuged at 170 000 g for 3 h at 4 c . the native dna - pk heterotrimer was purified by a single step dna - affinity chromatography method . poly-[di : dc ] ( amersham ) was resuspended in h2o and briefly sonicated ( three times for 2 s each ) to fragment the polymers into smaller fragments . the fragments were then crosslinked to cnbr - activated sepharose ( ge ) according to the manufacturer protocol . nuclear extracts were prepared from hela cells using 0.42 m kcl extraction as previously described and dialyzed to 0.1 m kcl . the extracts were then incubated with plasmid dna ( at a concentration of 130 g / ml ) on ice for 10 min . the extract was then centrifuged at 16 000 g for 10 min to remove precipitations . the clarified extract was then incubated with the crosslinked sepharose beads for 10 min at 4 c on a rotator . the mix was poured into a 10 ml disposable column and the resin was allowed to settle . the column was drained and the resin washed four times with 5 column volumes of harp buffer + 0.1 m nacl . the human rpa used in all of the assays was expressed and purified from bacteria as previously described . harp ( 0.5 g ) , rpa ( 0.5 g ) , dna - pk ( 0.1 g ) were incubated in 20 l of binding buffer ( 20 mm hepes [ k+ ] , ph 7.6 , 0.1 m kcl , 5 mm mgcl2 , 3% [ v / v ] glycerol , 0.25 mg / ml bsa , 0.05 mm edta , 0.5 mm dtt , 0.01% ( v / v ) np-40 ) for 20 min at 30 c . the reactions were then diluted to 200 l with binding buffer and incubated with 10 l of anti - flag resin for 3 h at 4 c . the resin was washed three times with binding buffer and the proteins eluted with sds - sample buffer . for the in vitro kinase assays , harp ( 0.4 g ) , rpa ( 0.5 g ) , dna - pk ( 0.3 g ) were incubated in 20 l of binding buffer with plasmid dna ( 60 ng ) and [ p]-atp for 15 min at 30 c . the reactions were stopped by the addition of sds sample buffer and resolved by sds - page . plasmid dna was incubated with rpa in 1 te in the presence of topoisomerase i. this results in the loading of rpa onto the plasmid due to transient opening of the dna . the rpa bound substrate is then incubated with harp in annealing helicase buffer ( 10 mm tris - hcl , ph 7.9 , 20 mm kcl , 2 mm mgcl2 , 0.5 mm dtt , 0.1 mm edta ) , and in the presence or absence of atp ( utp was included in the minus atp reactions ) . the dna is then precipitated and resolved by agarose gel electrophoresis . where indicated , 0.5 g of dna - pk was included and in reactions that contained both dna - pk and harp , the dna - pk and harp were pre - incubated with or without atp for 15 min before combining with the rpa - bound dna substrate .

the hepa - related protein ( harp / smarcal1 ) is an atp - dependent annealing helicase that is capable of rewinding dna structures that are stably unwound due to binding of the single - stranded dna ( ssdna)-binding protein replication protein a ( rpa ) . harp has been implicated in maintaining genome integrity through its role in dna replication and repair , two processes that generate rpa - coated ssdna . in addition , mutations in harp cause a rare disease known as schimke immuno - osseous dysplasia . in this study , we purified harp containing complexes with the goal of identifying the predominant factors that stably associate with harp . we found that harp preferentially interacts with rpa molecules that are bound to the dna - dependent protein kinase ( dna - pk ) . we also found that rpa is phosphorylated by dna - pk in vitro , while the rpa - harp complexes are not . our results suggest that , in addition to its annealing helicase activity , which eliminates the natural binding substrate for rpa , harp blocks the phosphorylation of rpa by dna - pk .

Introduction Results and Discussion Materials and Methods Native HARP complex Single-step purification of DNA-PK Pull-down and in vitro kinase assays Annealing helicase assays

the annealing helicase hepa - related protein ( harp ; also known as smarcal1 ) is an atp - dependent enzyme capable of rewinding dna that is bound by the single - stranded dna ( ssdna)-binding protein replication protein a ( rpa ) . such rpa - bound structures can arise during normal processes that involve unwinding of the dna duplex , such as dna replication , dna repair and transcription . harp has been shown to play an important role during both dna replication and repair , and cells lacking harp exhibit replication defects and persistent rpa binding . the importance of harp has been further highlighted by the finding that mutations in human harp cause a rare disorder known as schimke immuno - osseous dysplasia ( siod ) . while the molecular basis of siod is not fully understood , a potential link between an increase in chromosome breakage and mutations in harp is consistent with a role of harp in dna repair . while this interaction is dispensable for the annealing helicase activity of harp , it helps localize harp to sites of dna replication and repair . in addition to the annealing helicase activity of harp , which regulates the amount of rpa - bound ssdna in the cell , rpa can be regulated through phosphorylation . several kinases phosphorylate rpa including cyclin - dependent kinase ( cdk ) , ataxia - telangiectasia mutated ( atm ) , atm and rad3-related ( atr ) , and the dna - dependent protein kinase ( dna - pk ) . changes in rpa phosphorylation have been observed during cell - cycle progression and in response to dna damage , which ultimately leads to hyperphoshorylation of rpa . the downstream effects of rpa phosphorylation depend on the residues that are phosphorylated and include checkpoint activation , the regulation of dna repair , and the modulation of rpa - dna interactions . like rpa , harp is also phosphorylated in response to replication stress and dna damage , although the significance of harp phosphorylation is less clear . atm , atr and dna - pk are all capable of phosphorylating harp . despite the knowledge that harp associates with rpa , and that both factors are substrates for multiple kinases , little is known about which factors form the preferred harp complex . in this study , we found that , after rpa , dna - pk is the most abundant factor in the harp complex . a stable interaction of dna - pk with harp is mediated by rpa and suggests a tendency for harp to associate with rpa bound by dna - pk . using dna - pk purified from hela cells by a rapid dna - affinity protocol , we found that while dna - pk is able to phosphorylate rpa in vitro , it is unable to phosphorylate rpa in the presence of harp . dna - pk also phosphorylates harp in vitro and the phosphorylation of harp by dna - pk does not significantly affect its annealing helicase activity . together , our data suggest that harp inhibits the function or activity of rpa by both eliminating ssdna via the annealing helicase activity of harp and by preventing rpa phosphorylation by dna - pk . as previously reported , we purified harp from hela cells that constitutively express a flag - tagged copy of harp by tandem - affinity purification . following the purification , we observed that in addition to rpa , three other factors stably co - purify with harp . these proteins were identified by mass spectrometry as the subunits of the dna - pk heterotrimer ( i.e. other reports have identified dna - pk subunits as part of a group of potential cofactors for harp , but our analysis reveals that rpa and dna - pk are the two most abundant factors that stably co - purify with harp ( fig . , we did not detect wrn helicase , a factor previously reported to associate with harp , suggesting that wrn may be part of a less abundant or less stable harp complex . ( b ) harp , rpa , and dna - pk co - fractionate on glycerol gradients . the purified harp complex was resolved on a 1030% glycerol gradient and the migration of the proteins determined by sds - page and silver staining . to gain insight into the stability of the harp - rpa - dna - pk interactions we collected fractions off the top of the gradient and analyzed them by sds - page and silver staining . we found that the dna - pk heterotrimer is present in the same fractions that contain both harp and rpa ( fig . 1b , fractions 7 to 11 ) , suggesting that harp , rpa , and dna - pk associate in a single , stable complex . to examine how dna - pk affects the activity of harp , we purified the dna - pk heterotrimer from hela cells using a one - step dna - affinity protocol ( fig . we then incubated the pre - cleared extracts with the dna - resin that contains free dna ends . under these conditions , the collected fractions consisted almost entirely of the dna - pk heterotrimer , which eluted from the resin in the fractions containing between 0.3 to 0.4 m of nacl ( fig . a scaled - down version of the protocol was used to extract dna - pk from hela cells from a single 15 cm dish , showing that small - scale preparations of dna - pk can be rapidly made ( fig . isolation of native dna - pk by a single - step dna - affinity purification method . ( a ) dna was crosslinked to sepharose beads and used to extract dna - pk , which preferentially binds to free dna ends . using the native dna - pk purified from hela cell extracts , we first asked whether dna - pk interacts directly with harp or associates with the complex through an interaction with rpa . following several independent purifications of the harp complex , we noticed that the dna - pk is often less abundant than harp and rpa . we considered the possibility that dna - pk , which is known to associate with rpa , is present in the harp complex through a direct interaction with rpa . to determine whether rpa is required for dna - pk to stably associate with the harp complex , we tested whether a mutant version of harp , which contains a 10 amino acid substitution in the n - terminal rpa - interaction motif , can interact with dna - pk . we incubated purified rpa , dna - pk with either wild - type or mutant harp , immunoprecipitated the harp proteins using anti - flag antibodies , and analyzed the co - precipitating factors by western blotting ( fig . as previously reported , we found that wild - type but not mutant harp interacts with rpa . moreover , dna - pk readily co - precipitates with wild - type harp but not the mutant harp , suggesting that a direct interaction between harp and dna - pk is not stable . we can not rule out the possibility that rpa and dna - pk compete for the same binding site on harp and that the mutant harp fails to interact with both rpa and dna - pk . however , we think this is unlikely as our gradient analysis suggests that harp , rpa , and dna - pk all appear to stably interact in one single complex . our findings suggest that harp exhibits a preference , or tendency , to interact with rpa bound to dna - pk . ( a ) purified wild - type ( wt ) or mutant ( mut ) harp proteins were purified by a baculovirus system and incubated with bacterially expressed rpa and native dna - pk . ( c ) in vitro kinase assays were performed by incubating purified dna - pk with harp , rpa , [ p]-labeled atp and plasmid dna . we next used the purified dna - pk to investigate the phosphorylation of harp and rpa . in order to examine whether harp is a substrate for dna - pk and whether harp affects the phosphorylation of rpa by dna - pk , we performed in vitro kinase assays . we incubated dna - pk with harp , the rpa heterotrimer , or both in the presence of double - stranded plasmid dna and [ p]-atp . we found that both harp and rpa can be independently phosphorylated in vitro by dna - pk ( fig . when both harp and rpa are present in the reactions , the phosphorylation of harp and rpa2 by dna - pk is substantially reduced ( fig . one potential mechanism by which harp could block the phosphorylation of rpa is by binding and masking the phosphorylation residues of rpa2 . on the other hand , it is also possible that harp interferes with rpa2 phosphorylation through a more subtle mechanism , such as by interfering with the kinase activity of dna - pk or by weakening dna - pk - rpa2 interactions . in order to address these potential models , we compared the levels of phosphorylation of rpa2 by dna - pk in the presence of wild - type and mutant harp proteins . we found that both wild - type and mutant harp proteins are able to block rpa2 phosphorylation by dna - pk ( fig . in addition , while the levels of phosphorylation of wild - type harp are reduced in the presence of rpa , the levels of phosphorylation of mutant harp remain high in the presence of rpa . these findings suggest that harp does not simply mask the phosphorylation sites of rpa through the stable interaction of the two factors . it is likely that a more complex mechanism is behind the ability of harp to block the phosphorylation of rpa2 . for example , in addition to the stable interaction between harp and rpa that is mediated by the conserved n terminus of harp , multiple weaker interactions may exist between other regions of harp and rpa . these interactions may be sufficient for both wild - type and mutant harp to block the phosphorylation of rpa2 . furthermore , interactions between harp and dna - pk may exist that are not strong enough for the mutant harp to co - purify with dna - pk , but are strong enough for harp to affect the ability of dna - pk to phosphorylate rpa . in contrast , the observed reduction in the phosphorylation of harp by dna - pk and in the presence of rpa may be explained by the masking of phosphorylation sites on harp by rpa . this would explain why mutant harp , which fails to stably interact with rpa , does not show the reduced levels of phosphorylation seen with wild - type harp . the role that phosphorylation of harp plays in the cell is poorly understood although it has been suggested that phosphorylation of some residues may affect the activity of harp . to test whether dna - pk affects the atp - dependent activity of harp , we performed annealing helicase assays in the presence or absence of purified dna - pk . we generated rpa - bound dna substrates using purified rpa and plasmid dna ( fig . in contrast , if the rpa - bound substrate is rewound , the resulting double - stranded plasmid dna will be in a relaxed form following precipitation . as previously reported , we observed that harp , in the absence of dna - pk , rewinds the unwound regions of the dna in an atp - dependent manner ( fig . we also found that addition of dna - pk to the rpa - bound dna in the absence of harp resulted in a modest decrease in the stability of the rpa - ssdna complex in an atp - independent manner ( fig . reactions in which both harp and dna - pk were added to the rpa - bound substrate did not show any change in the annealing helicase activity of harp ( fig . under the conditions we tested , harp is able to fully rewind the rpa - bound dna in the presence of dna - pk and atp . it should be noted that in the reactions that contained both harp and dna - pk , we pre - incubated harp with dna - pk in the presence or absence of atp to allow for harp phosphorylation before the addition of the rpa - bound dna . based on this finding , and the fact that dna - pk is present in the harp complex through its binding to rpa , it appears likely that rpa is the main target of the kinase activity of dna - pk . the presence of dna - pk does not significantly affect the annealing helicase activity of harp . annealing helicase assays were performed in the presence or absence of dna - pk using a plasmid partially loaded with rpa . for the reactions that contain both dna - pk and harp , the two factors were preincubated in the presence or absence of atp prior to addition of the rpa - bound dna substrate . the role of phosphorylation in the regulation of the activities of rpa is not entirely understood . some studies have reported that dna - pk preferentially phosphorylates rpa that is bound to ssdna near junctions with dsdna , such as at fork dna , which are also the preferred binding sites for harp . this suggests that harp , rpa , and dna - pk may function together at these unique dna sites . our in vitro pull - down experiments were performed in the absence of dna and demonstrate that harp , rpa , and dna - pk do not require dna for their interaction ( fig . whether significant amounts of harp , rpa , and dna - pk are interacting without dna in the nucleus is unclear . the purification of the harp complex may result in extraction of the three factors off of the dna . our study also found that harp interferes with rpa phosphorylation both by its annealing helicase activity , which limits the amount of ssdna in the cell , and by preventing phosphorylation of rpa by protein : protein interactions . thus , it appears that harp plays a multifaceted role in regulating the function of rpa . the connection between mutations in harp and siod , a disease with a pleiotropic phenotype , is not fully understood . the diverse roles of harp and rpa in modulating the levels of ssdna in the cell likely impact many processes such as dna replication , repair , and transcription , ultimately contributing to the complex nature of siod . the glycerol gradients were performed by loading the purified harp complex onto a 4 ml , 10 to 30% linear gradient in harp buffer ( 20 mm hepes , ph 7.6 , 1.5 mm mgcl2 , 10% glycerol , 0.05% np40 , 1 mm dtt , 0.2 mm pmsf ) + 0.1 m kcl . harp ( 0.5 g ) , rpa ( 0.5 g ) , dna - pk ( 0.1 g ) were incubated in 20 l of binding buffer ( 20 mm hepes [ k+ ] , ph 7.6 , 0.1 m kcl , 5 mm mgcl2 , 3% [ v / v ] glycerol , 0.25 mg / ml bsa , 0.05 mm edta , 0.5 mm dtt , 0.01% ( v / v ) np-40 ) for 20 min at 30 c . for the in vitro kinase assays , harp ( 0.4 g ) , rpa ( 0.5 g ) , dna - pk ( 0.3 g ) were incubated in 20 l of binding buffer with plasmid dna ( 60 ng ) and [ p]-atp for 15 min at 30 c . plasmid dna was incubated with rpa in 1 te in the presence of topoisomerase i. this results in the loading of rpa onto the plasmid due to transient opening of the dna . the rpa bound substrate is then incubated with harp in annealing helicase buffer ( 10 mm tris - hcl , ph 7.9 , 20 mm kcl , 2 mm mgcl2 , 0.5 mm dtt , 0.1 mm edta ) , and in the presence or absence of atp ( utp was included in the minus atp reactions ) . where indicated , 0.5 g of dna - pk was included and in reactions that contained both dna - pk and harp , the dna - pk and harp were pre - incubated with or without atp for 15 min before combining with the rpa - bound dna substrate . the native dna - pk heterotrimer was purified by a single step dna - affinity chromatography method . harp ( 0.5 g ) , rpa ( 0.5 g ) , dna - pk ( 0.1 g ) were incubated in 20 l of binding buffer ( 20 mm hepes [ k+ ] , ph 7.6 , 0.1 m kcl , 5 mm mgcl2 , 3% [ v / v ] glycerol , 0.25 mg / ml bsa , 0.05 mm edta , 0.5 mm dtt , 0.01% ( v / v ) np-40 ) for 20 min at 30 c . for the in vitro kinase assays , harp ( 0.4 g ) , rpa ( 0.5 g ) , dna - pk ( 0.3 g ) were incubated in 20 l of binding buffer with plasmid dna ( 60 ng ) and [ p]-atp for 15 min at 30 c . plasmid dna was incubated with rpa in 1 te in the presence of topoisomerase i. this results in the loading of rpa onto the plasmid due to transient opening of the dna . the rpa bound substrate is then incubated with harp in annealing helicase buffer ( 10 mm tris - hcl , ph 7.9 , 20 mm kcl , 2 mm mgcl2 , 0.5 mm dtt , 0.1 mm edta ) , and in the presence or absence of atp ( utp was included in the minus atp reactions ) . where indicated , 0.5 g of dna - pk was included and in reactions that contained both dna - pk and harp , the dna - pk and harp were pre - incubated with or without atp for 15 min before combining with the rpa - bound dna substrate .

degenerative mitral valve disease represents the main cause of mitral regurgitation ( mr ) in the western countries . mitral valve repair has become the treatment of choice of degenerative mr because of its well known advantages compared to mitral valve replacement . the repair techniques introduced and popularized by carpentier and coworkers have provided predictable and durable long - term results . the most favourable outcomes have been described in patients with isolated prolapse of the posterior leaflet treated by a simple quadrangular resection and annuloplasty . on the other hand , less gratifying results have been associated with correction of mitral regurgitation due to anterior , bileaflet or commissural prolapse . all of carpentier s techniques for mitral valve repair have been developed to realize an anatomical reconstruction of the diseased mitral valve . however , to reach this goal , they can be technically demanding and time consuming , particularly in the setting of anterior , bileaflet and commissural lesions . an alternative approach to the regurgitant degenerative mitral valve was introduced by our group in 1991 and named edge - to - edge technique . revolutionary idea standing behind this new surgical approach is that the competence of a regurgitant mitral valve can be effectively restored with a the key point of this surgical method is to identify with accuracy where the regurgitant jet is located . exactly at this level the free edge of the diseased leaflet is sutured to the corresponding edge of the opposing leaflet thereby eliminating the mitral incompetence . when the regurgitant jet is in the central part of the mitral valve , the application of the edge - to - edge technique produces a mitral valve with a double orifice configuration ( double orifice repair ) ( figure 1 ) . the edge - to - edge technique used as a double orifice repair . depending on the extension and location of the suture performed , the two orifices can have similar or significantly different sizes . in case of commissural prolapse or flail , on the other hand , the application of the edge - to - edge technique is usually called paracommissural edge - to - edge repair and leads to a single orifice mitral valve with a relatively smaller area ( figure 2 ) . the edge - to - edge technique used as a paracommissural repair in many situations , the regurgitant jet of the mitral valve is not a single one . particularly in the setting of barlow s disease with severe bileaflet prolapse , for example , multiple regurgitant jets are usually found along the entire line of coaptation of the anterior and posterior leaflet . in this case , the edge - to - edge approximation of the middle scallop of the anterior and posterior leaflet ( a2 and p2 ) allows the elimination of most of the mitral insufficiency while the residual smaller regurgitant jets are effectively corrected by a ring annuloplasty . the first edge - to - edge repair case was performed in 1991 and more than 1500 published cases have been accumulated worldwide so far with the longest follow - up being now longer than 15 years . the technique is attractive because of its simplicity , reproducibility and effectiveness in different settings . indeed , due to its versatility , the edge - to - edge technique has been used for correction of mitral regurgitation due to different etiologies and mechanisms including bileaflet prolapse ( barlow s disease ) , anterior leaflet prolapse , commissural prolapse , functional mitral regurgitation secondary to ischemic or idiopathic dilated cardiomyopathy . moreover it has been adopted as a rescue procedure in case of suboptimal result of conventional mitral repair operation and to prevent or treat systolic anterior motion of the anterior leaflet . the edge - to - edge technique for bileaflet prolapse in barlow s disease in patients with barlow s disease , all the components of the mitral valve apparatus are affected by a severe myxomatous degeneration which leads to generalized bileaflet prolapse , severe annular dilatation and severe mitral insufficiency . echocardiography usually shows diffuse prolapse of most of the anterior and posterior leaflets with multiple jets of regurgitation along the entire coaptation line . intraoperative inspection demonstrates large , redundant leaflets with irregular free edges and an increased number of clefts ( figure 3 ) . intraoperative view og a mitral valve affected by barlow 's disease with important redundancy of all the valve tissue , multiple scallops , chordal elongation and severe bileaflet prolapse . such a generalized disease can certainly be effectively treated by performing an anatomical reconstruction of the mitral valve at annular , valvular and subvalvular level . however a rather complex operation requiring a long aortic cross - clamp time is usually required . the advantage of using the edge - to - edge approach in this challenging setting is that , with a single and standardized surgical act , it is possible to correct the multiple anatomical defects typically present in barlow s disease . indeed , just by suturing the middle scallop of the anterior and posterior leaflet ( a2 to p2 ) followed by ring annuloplasty , the edge - to - edge technique restores valve competence . the edge - to - edge suture is able to correct leaflets redundancy and prolapse without riscking post - operative sam . the edge - to - edge repair is carried out through a conventional median sternotomy or , in selected patients , through a small right anterior thoracotomy . following aortic and bicaval cannulation , in normothermic cardiopulmonary by - pass , the aorta is clamped and intermittent cold - blood cardioplegia is delivered into the ascending aorta to obtain cardioplegic arrest . the mitral valve is usually approached through the left atrium after careful dissection of the interatrial groove . in barlow s disease the mitral annulus is severely dilated and the leaflets are affected by severe myxomatous degeneration . in the segments with the most extensive pathologic involvement , however , are the middle scallops of the anterior and posterior leaflets ( a2 and p2 ) . chordal elongation and rupture are very common and may be responsible for multiple prolapses or flailing lesions . the first step is to identify exactly the middle of the anterior and posterior leaflets by inspecting the subvalvular apparatus . the first stitch of the edge - to - edge suture has to be positioned exactly the anatomical middle of the two leaflets . this step is very important to avoid valve distortion and residual valve regurgitation . to be sure of this technical point if the correct position of the stitch is confirmed , a continuous mattress suture of 4 - 0 polypropylene is passed along the whole length of the middle scallop of the anterior and posterior leaflets ( a2 and p2 ) . a second over - and - over continuous suture is then added to stabilize the repair . in barlow s disease the bites have to be rather deep , usually around 1 cm , to reduce as much as possible the height of the redundant leaflets . usually a wide suture , connecting the whole p2 free edge to the opposing a2 , is necessary . when a flail segment is present , this has to be included in the edge - to - edge suture . therefore the position of the stitch may be somewhat asymmetric , corresponding to the center of the flail portion of the leaflet . in this case the size of the two orifices will be different , one being larger than the other one . such an event should not raise any concern as far as both of them are competent and the sum of their areas is above 2.5 cm2 for a normal size patient . a flexible or semirigid ring annuloplasty is always added to complete and stabilize the repair ( figure 4 ) . once ring implantation has been completed , the final competence of the two mitral orifices is evaluated by forceful saline filling of the left ventricle . the residual mitral area is assessed by direct inspection and , in case of doubts , hegar valve dilators can be introduced into the orifices to be sure that a global valve area of at least 2.5 cm in normal size patients has been left . transesophageal echo reassessment of the valve is routinely performed after weaning from cardiopulmonary by - pass . typically no residual mitral regurgitation is present and two diastolic flows can be visualized through the double orifice mitral valve . the valve area is commonly assessed by a planimetric method using the transgastric , short - axis view . the simplicity of the edge - to - edge technique makes it possible to repair complex mitral valves with a cross - clamp time which , in our experience , has been 333.9 minutes . our experience with the edge - to - edge technique for the surgical treatment of bileaflet prolapse in the context of barlow s disease includes 648 consecutive patients submitted to mitral valve repair from 1991 to march 2008 . actuarial survival at 5 years was 92%4.5% and freedom from reoperation 91%4.2% with no patients requiring late reoperation for mitral valve stenosis . follow - ups echocardiography show good results of the repair , with stable competence and no progression of valve stenosis : the mean mitral valve area , assessed in a subgroup of 75 patients , was 10.22.1 cm preoperatively , decreased to 3.70.8 cm after repair and did not significantly change at a mean follow - up of 1.3 years ( 113 patient - years , range 1 month-5 years ) remaining 3.60.97 cm . considering the unfavorable anatomical features and the complexity of the mitral valve lesions of patients affected by barlow s disease , we consider these results very satisfactory . the edge - to - edge technique for anterior leaflet prolapse we have analyzed and reported the late outcome of a series of 133 patients with segmental prolapse of the anterior leaflet submitted to isolated edge - to - edge repair and mitral annuloplasty over a period of 10 years . no hospital deaths occurred and long - term ( up to 13 years ) clinical and echocardiographic follow - up was 100% complete . overall 10 year survival was 914.06% , freedom from cardiac death 95.82.83% and freedom from reoperation 962.3% with 93.2% of the patients being in nyha functional class i or ii at a mean follow - up of 4.53.12 years ( range 1 month-13.2 years ) . the mean mitral valve area after repair was 2.60.6 cm ( range 2 - 4 cm ) and the mean mitral gradient 3.11.5 mmhg ( range 1.8 - 5.8 mmhg ) . mitral stenosis was never detected immediately after surgery or later and , at the last follow - up , mitral regurgitation was absent or mild in 120 patients ( 90.1% ) , moderate in 10 patients ( 7.5% ) and severe in 3 patients ( 2.2% ) . these results were comparable to those obtained in our institution with patients submitted to standard quadrangular resection for prolapse of the posterior leaflet . thus in our experience with the edge - to - edge repair anterior mitral leaflet prolapse is no longer a risk factor for suboptimal results . these results were obtained in patients with segmental prolapse of the anterior leaflet , involving only one scallop . in presence of an extended anterior leaflet prolapse , involving more than one scallop , the edge - to - edge technique alone may not be sufficient to obtain a perfectly competent valve since a long suture would be required with higher risk of inducing mitral stenosis . under the above circumstances , the implantation of artificial chordae may be required in order to eliminate incompetence without excessively reducing the mitral valve area . the edge - to - edge technique for commissural prolapse mitral valve repair in patients with commissural lesions can be very challenging with conventional techniques . the edge - to - edge technique with annuloplasty is a rapid and reliable mitral reconstruction and may be applied to correct either one or both leaflet prolapses / flails at the commissure . in the cleveland clinic experience , more than 100 patients with commissural prolapse / flail have been treated with suture closure of the commissure , with no instances of mitral stenosis , suture dehiscence or recurrent prolapse in the follow - up . in our opinion , commissural edge - to - edge repair with annuloplasty is probably the simplest and most reproducible method to repair commissural lesions . we have employed this technique for approximation of the mitral valve leaflets at the commissural area in 115 patients for managing isolated commissural prolapse or flail . the incidence of reoperation has been very low ( 2 out of 114 hospital survivors , 1.7% ) . an echocardiographic follow - up was performed in 108 patients ( all with severe preoperative mr ) at a mean postoperative time of 2.31.9 years ( median 2.0 years , range 1 - 8.3 years ) . mitral regurgitation was absent in 60 patients ( 55.6% ) , mild in 43 ( 39.8% ) , moderate in three ( 2.8% ) and severe in two ( 1.9% , both reoperated on ) . no instance of mitral stenosis was ever documented as indicated by the low transvalvular gradients recorded echocardiographically . the longest follow - up is now approaching nine years and the satisfactory echocardiographic data obtained so far support a strategy of early mitral repair in the difficult setting of commissural mitral valve incompetence . because of its excellent clinical and echocardiographic result , it remains the method of choice to correct isolated commissural prolapse or flail at our institution . the edge - to - edge technique for functional mitral regurgitation in dilated cardiomyopathy mitral regurgitation is regarded as secondary ( or functional ) when the valve leaflets and chordae are structurally normal and the valve dysfunction is due to altered ventricular shape and dimension ( remodelling ) with displacement of one or both papillary muscles . annular dilatation is often concomitantly present , particularly when the valve insufficiency is severe and long - standing and the left ventricle is remarkably dilated . undersized annuloplasty ( at least two sizes under ) using a complete rigid ring is the standard operation , effective in most cases . to enhance the likelihood of a successful and durable repair , the addition of other procedures has been proposed including the edge - to - edge repair . in regard to the latter technique , the cleveland clinic group reported a disappointing 24% recurrence rate of moderately - severe ( 3 + ) mitral regurgitation 2 years after edge - to - edge repair of functional mitral regurgitation . these results , however , can mainly be explained by the fact that , in that series , the edge - to - edge technique was always employed in association with a posterior flexible band , which may not prevent the recurrence of annular dilatation in the setting of advanced dilated cardiomyopathy . indeed , the patients requiring reoperation almost invariably presented re - dilatation of the mitral annulus . in our institution the edge - to - edge was performed with an undersized annuloplasty whenever substantial apical tenting was present ( coaptation depth > 1 cm ) . however , from a technical point of view , the concomitant annuloplasty was always performed with a complete rigid or semirigid ring and the site of leaflet approximation was literally guided by the echocardiographic findings . this approach significantly increased the durability of mitral repair in our series with a freedom from failure at 1.5 years of 95%3.4% compared to 7712.1% registered in those patients submitted to isolated undersized annuloplasty in our department in the same time frame ( p=0.04 ) . moreover , in this same study , the use of the edge - to - edge technique was identified as the only significant predictor of durability of the repair , which confirms the role of this technique in this challenging setting . the edge - to - edge technique has also been used as a rescue procedure in patients with significant residual mitral regurgitant after conventional mitral repair . gatti and coworkers reported the adoption of this strategy in 11 patients . at intraoperative ecocardiography , residual mitral regurgitation jet area improved from 3.00.8 cm , after conventional repair , to 0.70.9 cm after rescue edge - to - edge the original reconstruction was not taken down and the additional cardiopulmonary bypass time was only 14.92.8 min . the edge - to - edge suture was just added where indicated by the echo findings . other authors , however , have reported suboptimal results with the edge - to - edge used to treat residual regurgitation after complex mitral repair . indeed , in this context , the efficacy of the edge - to - edge repair is very difficult to predict considering that it should be able to restore the mitral valve competence after a failed conventional repair . moreover , particularly if the first surgery included a resection procedure , the shortest edge - to - edge approximation should be applied to avoid the risk of mitral stenosis . the edge - to - edge technique to prevent or treat systolic anterior motion ( sam ) systolic anterior motion ( sam ) of the anterior mitral leaflet causing left ventricular outflow tract obstruction remains one of the most common complications after mitral valve repair . it has been postulated that use of the edge - to - edge technique might be effective to prevent or treat sam occurring after mitral repair . mascagni and colleagues reported the use of the edge - to - edge technique to prevent post - repair sam in four patients . more recently brinster and coworkers adopted the edge - to - edge repair for this purpose in twenty patients undergoing mitral valve reconstruction . in all cases sam was completely eliminated and the mean mr grade in the immediate postoperative period was 0.50.7 . no patient developed mitral stenosis and none developed sam or progressive mr at follow - up . the freedom from reoperation at 4 years was 100% . also the cleveland clinic reported on the use of the edge - to - edge technique for the treatment of sam . in particular bhudia and coworkers described the application of the edge - to - edge to lessen the likelihood of sam in patients with hypertrophic obstructive cardiomyopathy ( hocm ) submitted to myectomy . elimination of sam was achieved in the 14 patients treated although systolic anterior motion could still be demonstrated occasionally due to displacement of the lateral portion of the anterior leaflet which had not been fixed to the posterior leaflet by the edge - to - edge suture which had been placed in the central part of the mitral valve . although further studies are required , the edge - to - edge seems to have a role in eliminating sam post - repair or in preventing the occurrence of systolic anterior motion in patients with pre - repair echocardiographic predictors of sam . the minimally invasive approach option in patients with severe degenerative mitral regurgitation an early surgery policy is recommended whenever a very high probability of repair can be ensured . those patients are usually young , asymptomatic , with good lv function and no significant comorbidities . therefore they are ideal candidates for a minimally invasive mitral repair operation performed through a small right thoracotomy . the edge - to - edge technique , because of its technical simplicity and reproducibility , can be easily applied through such an approach . from a technical point of view patients are intubated with a double - lumen endotracheal tube and a 14 f cannula is placed percutaneously through the right jugular vein into the superior vena cava . a 6 to 8-cm minithoracotomy is then performed through the fourth intercostal space and a soft tissue retractor is used for spreading the ribs . one port is created laterally to the incision to introduce both a 5 mm video scope and a co2 line to flush the operative field . following cannulation of the femoral vessels , cardiopulmonary bypass ( cpb ) is established between femoral artery and femoral and jugular veins , at 28 - 30c . aortic cross - clamp is performed by using the chitwood transthoracic clamp inserted through the second or third intercostal space and intermittent antegrade cardioplegia is delivered through an aortic root catheter . the mitral valve is exposed in all cases through a left atriotomy using a transthoracic atrial retractor positioned in the fourth intercostal space . whenever the view is suboptimal , the inserted camera is used to improve valve assessment and reconstruction . even complex degenerative mitral disease can be effectively corrected through such a right minithoracotomy by using the edge - to - edge technique . in our experience this minimally invasive strategy did not compromise the durability of repair providing a very high patient satisfaction in terms of cosmetic results , postoperative pain and time to full recovery after the operation . edge - to - edge repair and annuloplasty a concomitant annuloplasty represents a key factor for the long - term durability of the edge - to - edge repair . indeed , in absence of annuloplasty , further dilatation of the mitral annulus can take place after surgery leading to recurrent mr . although at the beginning of our experience the edge - to - edge technique has been adopted in some cases without an associated annuloplasty , today we recommend to add always an annuloplasty to the edge - to - edge repair , if technically feasible , to increase the coaptation surface of the leaflets , enhance the competence of the valve , reduce the stress on the edge - to - edge suture and prevent the possibility of subsequent annular dilatation . we have been able to demonstrate that the lack of annuloplasty is one of the most important factors of edge - to - edge repair failure with a freedom from reoperation at 5 years which decreases from 92% in patients with annuloplasty to 79% in those without . double orifice edge - to - edge repair and hemodynamics when the edge - to - edge technique is adopted as a double orifice repair , the morphology of the mitral valve becomes that of a valve with two orifices . which are the possible hemodynamic effects of such an unusual configuration ? this question was formulated since the introduction of this new technique and addressed by performing computational model studies which demonstrated that the hemodynamic performance of a double - orifice mitral valve depends exclusively on the total valve area and on the cardiac output and not on the double orifice shape . in double - orifice valve configuration , the velocity of the flow through each orifice is very similar to the one observed through a single orifice valve of area equal to the sum of the areas of the two orifices . moreover the flow velocities through the two orifices are exactly the same , even when the orifice sizes are significantly different , which means that the doppler sampling of any of the two orifices is sufficient to assess the hemodynamic of the mitral valve . indeed , in our clinical practice , patients previously submitted to double orifice repair demonstrated at doppler examination blood flow velocities comparable in the two orifices . edge - to - edge repair and the risk of functional mitral stenosis one of the main concerns which have been raised regarding the edge - to - edge technique has been its potential restrictive effect at rest and during exercise . over the years , however , it has been demonstrated that functional mitral stenosis does not develop after edge - to - edge repair either at baseline or under exercise . frapier and coworkers compared patients operated on either by carpentier s techniques or by the edge - to - edge repair . rest and exercise echocardiogram along with cardiorespiratory testing with maximal oxygen uptake were performed . at baseline , the mean mitral valve area was 2.5 cm after the edge - to - edge and 2.9 cm following classic mitral repair techniques ( p=0.0018 ) . however , despite the higher mitral valve area reduction , the edge - to - edge technique did not induce more transvalvular gradients than classical carpentier s repair . indeed , mean mitral gradients at rest were not significantly different between two groups being 3.8 mmhg in the edge - to - edge and 3.3 mmhg in the classic techniques group , respectively . moreover , at peak exercise , increase of the mitral gradient and maximum oxygen uptake ( vo2 max ) was comparable between two groups . this shows that the edge - to - edge repair is no more restrictive at peak exercise than classic repairs and provides the same efficiency on mitral regurgitation reduction and the same exercise tolerance than carpentier s techniques . in our institution we measured the gradients at rest across the mitral valve after edge - to - edge repair since the beginning of our experience . both immediately after surgery and at follow - up they have been very low with no evidence of mitral stenosis . moreover , an exercise echocardiographic study was specifically designed and performed to assess if the edge - to - edge mitral repair could be a limiting factor for exercise tolerance . thirty patients previously submitted to double orifice mitral valve repair , underwent exercise echocardiography ( 10 watt per minute ) . a ring annuloplasty was present in 28 of them ( 93% ) . at peak of the stress heart rate , systolic blood pressure and stroke volume significantly increased showing a physiologic behaviour of the mitral valve . the mean mitral valve gradient ( 2.81.3 mmhg vs 4.61.9 mmhg , p<0.00001 ) , maximum mitral valve gradient ( 6.42.8 mmhg vs 10.54.6 mmhg , p<0.00002 ) and systolic pulmonary artery pressure ( 22.86.1 mmhg vs 28.29.9 mmhg , p<0.001 ) increased but not to pathologic levels . planimetric valve area increased significantly ( 3.20.6 cm vs 4.30.7 cm2 , p<0.00001 ) ( figures 5 - 7 ) . response of mean ( a ) and maximun ( b ) mitral gradient to exercise after edge - to - edge repair . exercise - induced change in systolic pulmonary artery pressure after edge - to - edge repair . exercise - induced change in planimetric mitral valve area edge - to - edge repair . these data clearly demonstrate that artificially created double orifice valves follow a physiologic behavior during exercise with a good valvular reserve in response to the increased cardiac output , a mean transmitral gradient which remains below 5 mmhg and a pulmonary pressure which does not increase up to pathologic levels . percutaneous edge - to - edge mitral valve repair catheter adaptations of the edge - to - edge technique have recently enabled mitral valve repair to be performed using endovascular techniques and thus opening the possibility of nonsurgical treatment of mitral regurgitation . the devices simulating surgical edge - to - edge using a percutaneous approach with a transeptal puncture include the mitraclip ( evalve , menlo park , ca , usa ) and the mobius ii leaflet repair system ( edwards lifesciences ) . the mitraclip ( figure 8) is a polyester - covered device with two arms which are able to engage the mitral leaflets . mitraclip ( evalve , menlo park , ca , usa ) . after feasibility studies , the phase i clinical trial ( everest i ) has been completed and has demonstrated the efficacy of the device . the mobius ii is also a percutaneous edge - to - edge device which uses a surgical suture to realize anterior and posterior leaflet approximation . in an acute animal model the edge - to - edge repair was performed with this device but further development of the system has been halted . at present selected cases of functional and degenerative mitral regurgitation can be treated percutaneously with the mitraclip although a wider application of this therapeutic option is likely to take place when reliable systems to perform concomitant mitral annuloplasty will become available . the introduction of the edge - to - edge technique has provided an additional contribution to the treatment of mitral regurgitation . simplicity , reliability and effectiveness are the main advantages of the edge - to - edge technique and have led to its increasing widespread application in the surgical community and to its adoption by a percutaneous approach .

the edge - to - edge technique was introduced in the surgical armamentarium of mitral valve repair in 1991 and has progressively been used to restore mitral competence in the setting of degenerative , post - endocarditis and functional mitral regurgitation . appropriate indications and awareness of the important technical aspects of the procedure are prerequisites for a good outcome . the free edges of the mitral leaflets have to be approximated in correspondence of the site of the regurgitant jet in such a way that mitral regurgitation is corrected without producing stenosis . a prosthetic ring is usually implanted to stabilize the repair . middle and long - term results are now available for degenerative mitral regurgitation ( bileaflet prolapse , anterior leaflet prolapse and commissural prolapse ) . of particular interest is the finding that the edge - to - edge technique for correction of anterior leaflet prolapse is providing a freedom from reoperation similar to that obtained in patients with posterior leaflet prolapse treated with quadrangular resection . degenerative or post - endocarditis commissural prolapse / flail of the mitral valve can be effectively corrected by this technique . in patients with functional mitral regurgitation , the use of the edge - to - edge repair , added to the undersized annuloplasty , has been associated with a significantly lower recurrence of mitral regurgitation in the follow - up compared to isolated undersized annuloplasty . almost 20 years after its introduction , the edge - to - edge technique remains an effective and versatile method to treat mitral regurgitation . its simplicity and reproducibility have led to its clinical application by percutaneous methods opening a new age in the fascinating field of reconstructive mitral valve surgery .

Introduction Results Conclusions

degenerative mitral valve disease represents the main cause of mitral regurgitation ( mr ) in the western countries . mitral valve repair has become the treatment of choice of degenerative mr because of its well known advantages compared to mitral valve replacement . the repair techniques introduced and popularized by carpentier and coworkers have provided predictable and durable long - term results . the most favourable outcomes have been described in patients with isolated prolapse of the posterior leaflet treated by a simple quadrangular resection and annuloplasty . on the other hand , less gratifying results have been associated with correction of mitral regurgitation due to anterior , bileaflet or commissural prolapse . however , to reach this goal , they can be technically demanding and time consuming , particularly in the setting of anterior , bileaflet and commissural lesions . an alternative approach to the regurgitant degenerative mitral valve was introduced by our group in 1991 and named edge - to - edge technique . revolutionary idea standing behind this new surgical approach is that the competence of a regurgitant mitral valve can be effectively restored with a the key point of this surgical method is to identify with accuracy where the regurgitant jet is located . exactly at this level the free edge of the diseased leaflet is sutured to the corresponding edge of the opposing leaflet thereby eliminating the mitral incompetence . when the regurgitant jet is in the central part of the mitral valve , the application of the edge - to - edge technique produces a mitral valve with a double orifice configuration ( double orifice repair ) ( figure 1 ) . the edge - to - edge technique used as a double orifice repair . in case of commissural prolapse or flail , on the other hand , the application of the edge - to - edge technique is usually called paracommissural edge - to - edge repair and leads to a single orifice mitral valve with a relatively smaller area ( figure 2 ) . the edge - to - edge technique used as a paracommissural repair in many situations , the regurgitant jet of the mitral valve is not a single one . particularly in the setting of barlow s disease with severe bileaflet prolapse , for example , multiple regurgitant jets are usually found along the entire line of coaptation of the anterior and posterior leaflet . in this case , the edge - to - edge approximation of the middle scallop of the anterior and posterior leaflet ( a2 and p2 ) allows the elimination of most of the mitral insufficiency while the residual smaller regurgitant jets are effectively corrected by a ring annuloplasty . the first edge - to - edge repair case was performed in 1991 and more than 1500 published cases have been accumulated worldwide so far with the longest follow - up being now longer than 15 years . indeed , due to its versatility , the edge - to - edge technique has been used for correction of mitral regurgitation due to different etiologies and mechanisms including bileaflet prolapse ( barlow s disease ) , anterior leaflet prolapse , commissural prolapse , functional mitral regurgitation secondary to ischemic or idiopathic dilated cardiomyopathy . the edge - to - edge technique for bileaflet prolapse in barlow s disease in patients with barlow s disease , all the components of the mitral valve apparatus are affected by a severe myxomatous degeneration which leads to generalized bileaflet prolapse , severe annular dilatation and severe mitral insufficiency . such a generalized disease can certainly be effectively treated by performing an anatomical reconstruction of the mitral valve at annular , valvular and subvalvular level . the advantage of using the edge - to - edge approach in this challenging setting is that , with a single and standardized surgical act , it is possible to correct the multiple anatomical defects typically present in barlow s disease . indeed , just by suturing the middle scallop of the anterior and posterior leaflet ( a2 to p2 ) followed by ring annuloplasty , the edge - to - edge technique restores valve competence . the edge - to - edge suture is able to correct leaflets redundancy and prolapse without riscking post - operative sam . the edge - to - edge repair is carried out through a conventional median sternotomy or , in selected patients , through a small right anterior thoracotomy . the mitral valve is usually approached through the left atrium after careful dissection of the interatrial groove . the first stitch of the edge - to - edge suture has to be positioned exactly the anatomical middle of the two leaflets . a second over - and - over continuous suture is then added to stabilize the repair . in barlow s disease the bites have to be rather deep , usually around 1 cm , to reduce as much as possible the height of the redundant leaflets . when a flail segment is present , this has to be included in the edge - to - edge suture . once ring implantation has been completed , the final competence of the two mitral orifices is evaluated by forceful saline filling of the left ventricle . typically no residual mitral regurgitation is present and two diastolic flows can be visualized through the double orifice mitral valve . the simplicity of the edge - to - edge technique makes it possible to repair complex mitral valves with a cross - clamp time which , in our experience , has been 333.9 minutes . our experience with the edge - to - edge technique for the surgical treatment of bileaflet prolapse in the context of barlow s disease includes 648 consecutive patients submitted to mitral valve repair from 1991 to march 2008 . actuarial survival at 5 years was 92%4.5% and freedom from reoperation 91%4.2% with no patients requiring late reoperation for mitral valve stenosis . follow - ups echocardiography show good results of the repair , with stable competence and no progression of valve stenosis : the mean mitral valve area , assessed in a subgroup of 75 patients , was 10.22.1 cm preoperatively , decreased to 3.70.8 cm after repair and did not significantly change at a mean follow - up of 1.3 years ( 113 patient - years , range 1 month-5 years ) remaining 3.60.97 cm . the edge - to - edge technique for anterior leaflet prolapse we have analyzed and reported the late outcome of a series of 133 patients with segmental prolapse of the anterior leaflet submitted to isolated edge - to - edge repair and mitral annuloplasty over a period of 10 years . no hospital deaths occurred and long - term ( up to 13 years ) clinical and echocardiographic follow - up was 100% complete . overall 10 year survival was 914.06% , freedom from cardiac death 95.82.83% and freedom from reoperation 962.3% with 93.2% of the patients being in nyha functional class i or ii at a mean follow - up of 4.53.12 years ( range 1 month-13.2 years ) . mitral stenosis was never detected immediately after surgery or later and , at the last follow - up , mitral regurgitation was absent or mild in 120 patients ( 90.1% ) , moderate in 10 patients ( 7.5% ) and severe in 3 patients ( 2.2% ) . these results were comparable to those obtained in our institution with patients submitted to standard quadrangular resection for prolapse of the posterior leaflet . thus in our experience with the edge - to - edge repair anterior mitral leaflet prolapse is no longer a risk factor for suboptimal results . these results were obtained in patients with segmental prolapse of the anterior leaflet , involving only one scallop . in presence of an extended anterior leaflet prolapse , involving more than one scallop , the edge - to - edge technique alone may not be sufficient to obtain a perfectly competent valve since a long suture would be required with higher risk of inducing mitral stenosis . the edge - to - edge technique for commissural prolapse mitral valve repair in patients with commissural lesions can be very challenging with conventional techniques . the edge - to - edge technique with annuloplasty is a rapid and reliable mitral reconstruction and may be applied to correct either one or both leaflet prolapses / flails at the commissure . in the cleveland clinic experience , more than 100 patients with commissural prolapse / flail have been treated with suture closure of the commissure , with no instances of mitral stenosis , suture dehiscence or recurrent prolapse in the follow - up . in our opinion , commissural edge - to - edge repair with annuloplasty is probably the simplest and most reproducible method to repair commissural lesions . we have employed this technique for approximation of the mitral valve leaflets at the commissural area in 115 patients for managing isolated commissural prolapse or flail . the longest follow - up is now approaching nine years and the satisfactory echocardiographic data obtained so far support a strategy of early mitral repair in the difficult setting of commissural mitral valve incompetence . the edge - to - edge technique for functional mitral regurgitation in dilated cardiomyopathy mitral regurgitation is regarded as secondary ( or functional ) when the valve leaflets and chordae are structurally normal and the valve dysfunction is due to altered ventricular shape and dimension ( remodelling ) with displacement of one or both papillary muscles . to enhance the likelihood of a successful and durable repair , the addition of other procedures has been proposed including the edge - to - edge repair . in regard to the latter technique , the cleveland clinic group reported a disappointing 24% recurrence rate of moderately - severe ( 3 + ) mitral regurgitation 2 years after edge - to - edge repair of functional mitral regurgitation . these results , however , can mainly be explained by the fact that , in that series , the edge - to - edge technique was always employed in association with a posterior flexible band , which may not prevent the recurrence of annular dilatation in the setting of advanced dilated cardiomyopathy . indeed , the patients requiring reoperation almost invariably presented re - dilatation of the mitral annulus . in our institution the edge - to - edge was performed with an undersized annuloplasty whenever substantial apical tenting was present ( coaptation depth > 1 cm ) . however , from a technical point of view , the concomitant annuloplasty was always performed with a complete rigid or semirigid ring and the site of leaflet approximation was literally guided by the echocardiographic findings . this approach significantly increased the durability of mitral repair in our series with a freedom from failure at 1.5 years of 95%3.4% compared to 7712.1% registered in those patients submitted to isolated undersized annuloplasty in our department in the same time frame ( p=0.04 ) . moreover , in this same study , the use of the edge - to - edge technique was identified as the only significant predictor of durability of the repair , which confirms the role of this technique in this challenging setting . the edge - to - edge technique has also been used as a rescue procedure in patients with significant residual mitral regurgitant after conventional mitral repair . at intraoperative ecocardiography , residual mitral regurgitation jet area improved from 3.00.8 cm , after conventional repair , to 0.70.9 cm after rescue edge - to - edge the original reconstruction was not taken down and the additional cardiopulmonary bypass time was only 14.92.8 min . the edge - to - edge suture was just added where indicated by the echo findings . other authors , however , have reported suboptimal results with the edge - to - edge used to treat residual regurgitation after complex mitral repair . indeed , in this context , the efficacy of the edge - to - edge repair is very difficult to predict considering that it should be able to restore the mitral valve competence after a failed conventional repair . moreover , particularly if the first surgery included a resection procedure , the shortest edge - to - edge approximation should be applied to avoid the risk of mitral stenosis . the edge - to - edge technique to prevent or treat systolic anterior motion ( sam ) systolic anterior motion ( sam ) of the anterior mitral leaflet causing left ventricular outflow tract obstruction remains one of the most common complications after mitral valve repair . it has been postulated that use of the edge - to - edge technique might be effective to prevent or treat sam occurring after mitral repair . mascagni and colleagues reported the use of the edge - to - edge technique to prevent post - repair sam in four patients . more recently brinster and coworkers adopted the edge - to - edge repair for this purpose in twenty patients undergoing mitral valve reconstruction . also the cleveland clinic reported on the use of the edge - to - edge technique for the treatment of sam . in particular bhudia and coworkers described the application of the edge - to - edge to lessen the likelihood of sam in patients with hypertrophic obstructive cardiomyopathy ( hocm ) submitted to myectomy . elimination of sam was achieved in the 14 patients treated although systolic anterior motion could still be demonstrated occasionally due to displacement of the lateral portion of the anterior leaflet which had not been fixed to the posterior leaflet by the edge - to - edge suture which had been placed in the central part of the mitral valve . although further studies are required , the edge - to - edge seems to have a role in eliminating sam post - repair or in preventing the occurrence of systolic anterior motion in patients with pre - repair echocardiographic predictors of sam . the minimally invasive approach option in patients with severe degenerative mitral regurgitation an early surgery policy is recommended whenever a very high probability of repair can be ensured . the edge - to - edge technique , because of its technical simplicity and reproducibility , can be easily applied through such an approach . even complex degenerative mitral disease can be effectively corrected through such a right minithoracotomy by using the edge - to - edge technique . edge - to - edge repair and annuloplasty a concomitant annuloplasty represents a key factor for the long - term durability of the edge - to - edge repair . although at the beginning of our experience the edge - to - edge technique has been adopted in some cases without an associated annuloplasty , today we recommend to add always an annuloplasty to the edge - to - edge repair , if technically feasible , to increase the coaptation surface of the leaflets , enhance the competence of the valve , reduce the stress on the edge - to - edge suture and prevent the possibility of subsequent annular dilatation . we have been able to demonstrate that the lack of annuloplasty is one of the most important factors of edge - to - edge repair failure with a freedom from reoperation at 5 years which decreases from 92% in patients with annuloplasty to 79% in those without . double orifice edge - to - edge repair and hemodynamics when the edge - to - edge technique is adopted as a double orifice repair , the morphology of the mitral valve becomes that of a valve with two orifices . in double - orifice valve configuration , the velocity of the flow through each orifice is very similar to the one observed through a single orifice valve of area equal to the sum of the areas of the two orifices . moreover the flow velocities through the two orifices are exactly the same , even when the orifice sizes are significantly different , which means that the doppler sampling of any of the two orifices is sufficient to assess the hemodynamic of the mitral valve . edge - to - edge repair and the risk of functional mitral stenosis one of the main concerns which have been raised regarding the edge - to - edge technique has been its potential restrictive effect at rest and during exercise . over the years , however , it has been demonstrated that functional mitral stenosis does not develop after edge - to - edge repair either at baseline or under exercise . frapier and coworkers compared patients operated on either by carpentier s techniques or by the edge - to - edge repair . at baseline , the mean mitral valve area was 2.5 cm after the edge - to - edge and 2.9 cm following classic mitral repair techniques ( p=0.0018 ) . however , despite the higher mitral valve area reduction , the edge - to - edge technique did not induce more transvalvular gradients than classical carpentier s repair . indeed , mean mitral gradients at rest were not significantly different between two groups being 3.8 mmhg in the edge - to - edge and 3.3 mmhg in the classic techniques group , respectively . this shows that the edge - to - edge repair is no more restrictive at peak exercise than classic repairs and provides the same efficiency on mitral regurgitation reduction and the same exercise tolerance than carpentier s techniques . in our institution we measured the gradients at rest across the mitral valve after edge - to - edge repair since the beginning of our experience . moreover , an exercise echocardiographic study was specifically designed and performed to assess if the edge - to - edge mitral repair could be a limiting factor for exercise tolerance . response of mean ( a ) and maximun ( b ) mitral gradient to exercise after edge - to - edge repair . exercise - induced change in systolic pulmonary artery pressure after edge - to - edge repair . exercise - induced change in planimetric mitral valve area edge - to - edge repair . these data clearly demonstrate that artificially created double orifice valves follow a physiologic behavior during exercise with a good valvular reserve in response to the increased cardiac output , a mean transmitral gradient which remains below 5 mmhg and a pulmonary pressure which does not increase up to pathologic levels . percutaneous edge - to - edge mitral valve repair catheter adaptations of the edge - to - edge technique have recently enabled mitral valve repair to be performed using endovascular techniques and thus opening the possibility of nonsurgical treatment of mitral regurgitation . the devices simulating surgical edge - to - edge using a percutaneous approach with a transeptal puncture include the mitraclip ( evalve , menlo park , ca , usa ) and the mobius ii leaflet repair system ( edwards lifesciences ) . after feasibility studies , the phase i clinical trial ( everest i ) has been completed and has demonstrated the efficacy of the device . the mobius ii is also a percutaneous edge - to - edge device which uses a surgical suture to realize anterior and posterior leaflet approximation . in an acute animal model the edge - to - edge repair was performed with this device but further development of the system has been halted . the introduction of the edge - to - edge technique has provided an additional contribution to the treatment of mitral regurgitation . simplicity , reliability and effectiveness are the main advantages of the edge - to - edge technique and have led to its increasing widespread application in the surgical community and to its adoption by a percutaneous approach .

bariatric surgery is the most effective treatment for severe obesity often resulting in excess weight loss of 5080% [ 1 , 2 ] and improved psychological wellbeing and health - related quality of life ( hrqol ) [ 35 ] . in addition , particularly , roux en - y gastric bypass ( rygb ) has been shown to lead to remission of type 2 diabetes and consequently many severely obese individuals with type 2 diabetes seek out this surgical procedure . however , results vary and 7%50% of patients undergoing bariatric surgery do not achieve a successful weight loss ( % ewl < 50 ) or regain weight when the surgical effects begin to abate [ 79 ] . a growing body of research has suggested that emotional and psychological impairments after surgery influence or may even be the cause of the suboptimal weight loss and weight regain in some bariatric patients [ 10 , 11 ] . before surgery , bariatric patients report elevated levels of mental distress and reduced hrqol compared with the general and nonsurgical obese population . also , in bariatric populations , the prevalence of current psychiatric disorders has been found to be as high as 56% and up to 73% report to have had at least one psychiatric disorder at some point in their life . in defining a successful treatment outcome after bariatric surgery , unfortunately , the importance of postoperative psychological factors has often been overlooked in the bariatric literature and further research is highly needed . type 2 diabetes and lack of physical activity in bariatric patients have previously been associated with decreased weight loss after surgery [ 1416 ] . however , the predictive value of preoperative medical comorbidities and behavioural factors including type 2 diabetes and physical activity , for mental health and hrqol , is currently unclear . it is well established that medical comorbidities such as type 2 diabetes are associated with impaired mental health and hrqol both in the general and in the nonsurgical obese population [ 1820 ] . for instance , a large meta - analysis of 42 studies with a combined total of 20.218 subjects concluded that individuals with diabetes have twice the risk of depression compared with nondiabetic individuals . however , in a recent study , we investigated mental health and hrqol in 129 rygb candidates and found indications that the relation between type 2 diabetes and mental health may differ in the bariatric population . more specifically , results from this study showed that bariatric patients with type 2 diabetes presented for rygb surgery with better mental health and hrqol compared with nondiabetic patients , especially with regard to physical aspects of hrqol . these results suggest that , in our study , patients with type 2 diabetes sought out the surgical option at an earlier stage than the nondiabetic patients , primarily to target their diabetes . in contrast , the nondiabetic patients may have experienced severe mental and physical impairments before considering surgical obesity treatment . despite the observed psychological differences between patients with and without type 2 diabetes , no studies have investigated the predictive value of baseline type 2 diabetes for the mental health and hrqol after bariatric surgery . in addition , lack of physical activity has been associated with poor mental health and reduced hrqol in the bariatric population . in a large cohort of severely obese adults , king et al . found an inverse association between physical activity and mental health prior to undergoing bariatric surgery . furthermore , a study with 131 bariatric patients reported that both frequency and intensity of physical activity one year after rygb were independently associated with better mental hrqol and fewer depressive symptoms . these results suggest that the lack of physical activity may negatively predict mental health and hrqol after bariatric surgery . however , to our knowledge , no studies have prospectively investigated the effect of physical activity on mental health among bariatric patients . furthermore , while these studies provide some evidence of an association between physical activity and psychological wellbeing among bariatric patients , the majority of the research relies on self - reported physical activity [ 2325 ] and no studies have investigated the relation using an objective measure of the patients ' actual physical fitness level . to address these shortcomings in the literature , the aim of this study was to prospectively investigate the effect of objectively measured baseline type 2 diabetes and physical fitness level on mental health , hrqol , and weight - related body image after rygb . the current study is part of the larger gasmito study ( the national committee on health research ethics , study protocol number hc-2009 - 050 ) that prospectively investigates psychological and physiological factors in severely obese rygb patients with and without type 2 diabetes from the time before weight loss is started until weight stability is achieved 1.5 years after rygb . all patients were tested at four follow - ups : ( a ) at baseline , prior to weight loss , ( b ) about two months later after a diet induced weight loss , ( c ) 4 months after rygb , and , finally , ( d ) 18 months after rygb . each test session consisted of three test days which included a number of physiological tests including oral and intravenous glucose tolerance tests , 2-hour hyperinsulinemic euglycemic clamp , dual x - ray absorptiometry scan , and incremental bicycle vo2max test . furthermore , the psychological profile of the patients was assessed using a battery of standardized questionnaires including measures of mental symptoms , hrqol , body image , and lifestyle , which were administered at each of the four follow - ups . in total , 40 gasmito participants completed the psychological and physiological tests at baseline , 32 participants at follow - up ( b ) , 31 participants at follow - up ( c ) , and finally 23 participants at follow - up ( d ) . the main reason for dropout was the time associated with the multiple tests days and some tests were furthermore associated with some pain or discomfort . participants were sampled consecutively at the bariatric clinic at hamlet hospital or at kge hospital , two hospitals located in copenhagen and suburban copenhagen area , respectively . participants were contacted at their first presurgical consultation at the hospital or contacted over the phone after their initial consultation and invited to an information meeting before inclusion . all gasmito participants were informed verbally and in writing about the procedures and were asked to sign a consent form before being included in the study . inclusion criteria were in agreement with the danish guidelines for gastric bypass surgery prior to january 2011 , which allowed individuals between 18 and 60 years of age with a bmi > 40 kg / m or bmi > 35 kg / m and obesity - related comorbidities ( e.g. , type 2 diabetes ) to be considered for surgery . exclusion criteria for the current study comprised heart disease , renal disease , or hyper- or hypothyreosis . to assess mental symptoms , a short version of the danish this 90-item checklist assesses the severity of a wide range of mental symptoms and is scored on a scale ranging from 0 to 4 with 4 reflecting the highest level of symptom load . only the three subscales ( 35 items ) measuring somatization , depression , and anxiety were used , as these symptoms are expected to be prevalent among the patients . furthermore , the general severity index ( gsi ) , which reflects both number and severity of symptoms , was calculated as the mean score of all 35 items . to assess quality of life and general health status of the patients ( sf-36 ) , which consists of 36 items evaluating eight aspects of hrqol including physical function , physical role , physical pain , general health , vitality , social function , mental role , and mental wellbeing . raw scores from the sf-36 were transformed to scores ranging from 0 to 100 with 100 indicating the highest level of hrqol . information about the patients ' background , lifestyle , and medical status was obtained using a short version of the questionnaire that was developed as part of the copenhagen aging and midlife biobank ( camb ) . this questionnaire comprises 76 questions providing information on social background , social situation , lifestyle , and physical health status . the body image questionnaire ( biq ) was used to assess the patients ' body image and comprises 50 items scored on a scale from 1 to 5 , with 5 indicating a more positive body image . a mean item score for weight - related items only was calculated as the sum of items 3 , 15 , 19 , 24 , 27 , 30 , 31 , 34 , 40 , 45 , 48 , and 50 divided by 12 . this score was expected to be especially sensitive to weight - related changes in body image . the plasma samples were cooled and centrifuged at 2000 g for 10 minutes before the plasma was stored at 80c until analysis was conducted . remission of type 2 diabetes was defined as hba1c < 42.1 mmol mol , fasting glucose < 5.6 mmol l , and no medication . the vo2max ( ml / min / kg ) is an objective measure of physical fitness level and a direct indicator of the extent of physical activity . a test was considered valid when leveling off was achieved , which was defined as unchanged oxygen uptake despite increasing work load , and when a respiratory exchange ratio greater than 1.15 was observed . maximal oxygen uptake was calculated as the highest average uptake during 20 seconds of exercise . finally , a dual x - ray absorptiometry scan ( dxa scan ) was performed to examine the body composition and body fat distribution of the patients . descriptive statistics were used to investigate patient characteristics at baseline . to avoid losing information due to missing follow - up data points , we analysed the repeated data using a random intercept multilevel model , allowing intercepts to vary between subjects , with within - subjects level 1 ( follow - up time ) and between - subjects level 2 with the subjects gasmito i d as grouping variable for the measures . all analyses of the repeated data were performed using the genlinmixed procedure in spss 20.0 with the robust standard error option to accommodate nonnormality in some of the outcome variables . to investigate the effect of baseline diabetes and physical fitness level on the psychological outcome variables , the multilevel model included follow - up time , sex , age , baseline bmi , baseline vo2max , baseline diabetes , and their interaction with follow - up time as fixed effects covariates . age , bmi , and vo2max at baseline were included as continuous variables in the interaction analyses with follow - up time . in total , 40 bariatric patients were included in the gasmito study , 30% ( n = 12 ) men and 70% ( n = 28 ) women . the participants ' mean age at baseline was 39.5 ( 8.9 ) years , their mean bmi was 42.5 ( 4.5 ) kg / m , and their mean vo2max was 21.2 ( 4.7 ) ml / kg / min , reflecting a very poor to poor level of physical fitness . finally , 35% ( n = 14 ) of the participants had type 2 diabetes at baseline and 65% ( n = 26 ) were classified as nondiabetic . patients with and without diabetes had similar age , weight , bmi , and vo2max prior to surgery . however , body fat % was lower in the diabetic than in the nondiabetic patients ( 45.1% ( 5.9 ) versus 49.6% ( 5.5 ) , p = 0.024 ) . table 1 shows the baseline results for weight , bmi , vo2max , mental symptoms ( scl-90 ) , hrqol ( sf-36 ) , and weight - related body image ( biq ) and the difference in each subscale score from one follow - up to another . these weight - related improvements were highly significant from baseline to follow - up ( b ) immediately before surgery and continued to be highly significant throughout the surgical course . at follow - up ( d ) , 18 months after rygb , the mean percent excess weight loss ( % ewl ) from baseline reached 65% ( 12 ) and percent weight loss ( % wl ) was 30% ( 6 ) , indicating a successful surgical weight loss outcome . patients with and without type 2 diabetes at baseline did not differ significantly with regard to % ewl . at 18-month follow - up furthermore , of the 14 patients with baseline type 2 diabetes , 50% ( n = 7 ) experienced total remission after surgery and only 21% ( n = 3 ) clearly met the criteria for diabetes ( hba1c > 48 ) postoperatively . also , vo2max increased significantly after surgery possibly explained by the decreased body mass of the patients . at 18-month follow - up , the bariatric patients reported a significant reduction of mental symptoms and a significant increase in hrqol and weight - related body image compared with baseline values , indicated by lower scores on the somatization , depression , anxiety , and gsi subscale of the scl-90 and higher scores on the eight sf-36 subscales and the weight - related biq subscale ( p < 0.05 for all subscales ) . as table 1 shows , the improvements were evident in most of the scl-90 and sf-36 subscales from the time immediately before surgery ( follow - up ( b ) ) to four months after surgery ( follow - up ( c ) ) with the most pronounced improvements observed on the physical hrqol subscales . of the scl-90 and sf-36 subscales , only somatization and social function did not improve during the first four postoperative months . furthermore , the somatization , depression , gsi , physical function , vitality , and social function subscales improved postoperatively until 18 months after surgery . finally , the weight - related body image score increased significantly throughout the surgical course and was , together with physical function , the only subscale demonstrating significant changes at each follow - up . in the applied multilevel model , the interaction between follow - up time and type 2 diabetes at baseline was found to be significant for six of the thirteen investigated subscales after adjusting for main effects of sex , age , baseline bmi , baseline vo2max , and their interaction with follow - up time . figure 1 shows the effect of baseline diabetes across follow - ups for the six subscales where this interaction was found to be significant . the only significant main effect of baseline diabetes was observed for the emotional role subscale of the sf-36 ( p = 0.011 ) . however , the interaction between follow - up time and diabetes was significant for the somatization ( p = 0.005 ) , the depression ( p = 0.004 ) , the anxiety ( p = 0.036 ) , and the gsi ( p = 0.001 ) subscales of the scl-90 . in addition , the interaction between follow - up time and diabetes was significant for the physical function ( p = 0.002 ) and the physical role ( p = 0.016 ) subscales of the sf-36 . as illustrated by figure 1 , these results indicate that differences in mental distress and physical aspects of hrqol between patients with and without baseline diabetes were only substantial at baseline assessment , but not at the later follow - ups . no significant main or interaction effects of diabetes were observed for the weight - related body image subscale . physical fitness ( vo2max ) made modest contributions to variations in mental symptoms and hrqol but not weight - related body image . main effects of vo2max were observed for the physical function ( p = 0.031 ) , the physical role ( p = 0.015 ) , and the vitality ( p = 0.018 ) subscales of the sf-36 with higher baseline vo2max associated with higher scores on these subscales . also , the interaction between follow - up time and baseline vo2max was significant for the somatization ( p = 0.000 ) subscale of the scl-90 and the physical role ( p = 0.016 ) and general health ( p = 0.024 ) subscales of the sf-36 suggesting that the effect of baseline physical fitness level on these subscales declines after surgery . to illustrate this , baseline vo2max was recoded into a binary low - high variable using the median ( vo2max = 21.7 ml / min / kg ) to distinguish the two groups . figure 2 shows the effect of baseline physical fitness level on the three subscales , somatization , physical role , and general health , across the four follow - ups . more specifically , the main effect of sex was significant for physical function ( p = 0.030 ) and weight - related body image ( p = 0.004 ) with women demonstrating lower scores than men on these subscales . the interaction between follow - up time and sex was significant for the depression ( p = 0.005 ) score of the scl-90 and the physical function ( p = 0.015 ) , the general health ( p = 0.024 ) , the vitality ( p = 0.032 ) , and the mental health ( p = 0.010 ) scores of the sf-36 . in general , these interactions reflected that sex differences tended to become smaller across follow - ups . however , for the vitality and the mental health subscale of the sf-36 , the direction of the association between sex and these subscale scores changed after surgery and sex differences remained at 18-month follow - up with women reporting higher scores than men . furthermore , a main effect of age was observed for the somatization ( p = 0.028 ) , the depression ( p = 0.007 ) , and the gsi ( p = 0.010 ) subscales with younger patients showing more mental distress than their older counterparts . the interaction between follow - up time and age was significant for the somatization ( p = 0.007 ) , the depression ( p = 0.002 ) , the gsi ( p = 0.015 ) , the vitality ( p = 0.001 ) , the mental health ( p = 0.016 ) , and the weight - related body image ( p = 0.048 ) scores indicating that in general the effect of age on these subscales declines across follow - ups . baseline bmi was of limited predictive value and only showed significant main effects for weight - related body image ( p = 0.040 ) where a lower bmi at baseline was associated with higher body image scores . finally , the interaction between follow - up time and baseline bmi was significant for the vitality ( p = 0.000 ) and the mental health ( p = 0.000 ) subscales suggesting that the effect of baseline bmi on these two subscales decreases from follow - up ( a ) to follow - up ( d ) . to our knowledge , this study was the first study to prospectively investigate the importance of type 2 diabetes and physical fitness at baseline for mental health , hrqol , and weight - related body image among bariatric patients undergoing rygb . in line with consistent findings from prior research [ 4 , 10 ] , mental health , hrqol , and weight - related body image improved significantly from preoperative levels . improvements were most pronounced four months after rygb particularly in physical aspects of hrqol and weight - related body image . the interaction between follow - up time and baseline diabetes was significant for mental health and physical hrqol outcomes indicating that the difference in mental symptoms and physical hrqol between diabetic and nondiabetic patients declines across follow - ups . diabetic patients endorsed fewer mental symptoms and had a higher hrqol and better weight - related body image preoperatively compared with nondiabetic patients . this indicates that diabetic patients seeking rygb may be motivated by their diabetes in contrast to nondiabetic patients that may seek out the surgical option due to substantial obesity - related distress . however , interestingly , the differences in mental symptoms and hrqol between patients with and without diabetes at baseline resolved around the time of surgery . this also suggests that the psychological impairments in the nondiabetic patients may be related to the obese state and therefore immediately influenced by weight loss . it could be speculated that positive expectations to the operation outcome generate an experience of optimism that positively affects the level of mental symptoms and hrqol in the nondiabetic patients around the time of surgery . in fact , improvements in mental health and hrqol were more pronounced initially after surgery in patients that did not have preoperative diabetes compared with patients with diabetes at baseline in spite of the fact that diabetes resolved in 50% of these patients after surgery . one possible explanation is that the differences in psychological wellbeing between patients with and without diabetes reflect preoperative differences in weight - related mental health and hrqol . prior research has suggested that type 2 diabetes is not associated with major impairments of mental and physical functioning among bariatric candidates . in contrast , nondiabetic patients report marked impairments in psychological and physical wellbeing prior to surgery indicating that nondiabetic patients seek out bariatric surgery at a more debilitating stage of the obese state . thus , as suggested , it is possible that bariatric candidates with diabetes primarily seek out surgery to target their diabetes , whereas some bariatric patients without diabetes at baseline seek surgery because of impaired mental health and hrqol related to obesity . it could therefore be speculated that nondiabetic patients experience a major relief when undergoing weight loss and surgery and this might intensify the improvements in mental health and hrqol among these patients initially after surgery . this explanation is supported by a study that investigated motivational factors among 208 gastric banding patients . interestingly , this study found that patients who sought bariatric surgery due to medical comorbidities , primarily hypertension and type 2 diabetes , had fewer depressive symptoms and higher hrqol scores compared with patients who sought surgery to improve other factors including appearance , physical fitness , and physical limitations . however , research investigating the influence of preoperative diabetes on mental health and hrqol after bariatric surgery is currently lacking and should therefore be investigated further . significant increases in physical activity have consistently been reported in prior research [ 23 , 25 ] . these studies have mainly used self - reported physical activity with the risk for patients to overreport their physical activity due to misperceptions or reporting bias . among the bariatric patients in our study , however , preoperative level of physical fitness did not contribute to the differences between diabetic and nondiabetic patients and vo2max was only a main predictor of improvements in the physical function , the physical role , and the vitality subscale of the sf-36 with higher vo2max associated with better physical hrqol . furthermore , the interaction between follow - up time and vo2max was only significant for the somatization subscale of the scl-90 and the physical role and general health subscales of the sf-36 . this may seem surprising since prior research has found that physical activity strongly predicts weight loss outcome [ 14 , 25 ] , fewer depressive symptoms , and higher mental hrqol one year after rygb . however , in contrast to the majority of this research , we used an objective measure as indicator of physical activity . vo2max depends on body mass that is why the observed increase in physical fitness level of our patients may reflect weight loss rather than increased physical activity . thus , with regard to the predictive value of physical activity , the use of different assessment methods may therefore explain the inconsistency between our results and prior findings . finally , numerous attempts have been made to identify demographic predictors of weight loss after bariatric surgery with conflicting results [ 33 , 34 ] . furthermore , the effect of demographic factors including sex and age on psychological outcome after bariatric surgery is understudied and currently unclear . in line with preoperative studies [ 3537 ] , our results showed that women presented for surgery with more mental distress , reduced hrqol , and poorer weight - related body image compared with men . also , younger patients tended to report worse psychological wellbeing compared with their older counterparts . a more positive weight - related body image was observed among patients with lower baseline bmi compared with the patients with a higher bmi . this effect of sex , age , and baseline bmi on mental distress , hrqol , and weight - related body image declined throughout the surgical course for most of the subscales . however , sex differences were observed on two aspects of hrqol 18 months after surgery with women reporting higher scores on the vitality and the mental health subscales of the sf-36 . thus , in terms of hrqol , it may seem that women benefit the most from surgery . in contrast , also using the sf-36 , masheb et al . investigated postoperative hrqol among 137 patients undergoing gastric bypass surgery and found that male sex predicted better mental hrqol 12 months after surgery . also , due to the relatively modest number of men participating in the current study , it is difficult to draw sound conclusions about the effect of sex on postoperative hrqol and further research is therefore highly needed . the current study is an important contribution to the very limited literature investigating type 2 diabetes and psychological outcome after bariatric surgery . the prospective study design and the use of objective methods to assess weight , bmi , type 2 diabetes , and physical fitness are important strengths of the study . in addition , the assessment of four psychological factors contributes to a fairly detailed psychological profile of the bariatric patients . the relatively small sample size , questioning the power of the study , and the lack of comparison with a nonsurgical obese control group are both limitations of the study . thus , significant findings may have been missed and , furthermore , it remains unclear whether the results are specific to the bariatric population or whether they also apply to the severely obese individuals in traditional weight loss programs . thus , it is not possible to infer causality and therefore not possible to examine whether baseline type 2 diabetes or baseline fitness level has causal effects on psychological outcome after rygb surgery . finally , patients were followed up to 18 months to two years after their operation . however , the american society for bariatric surgeons has recommended research to conduct at least 5-year follow - ups and the results of the current study should therefore be considered preliminary . we do acknowledge the importance of longer - term follow - ups and intend to pursue the possibility of extending the follow - up period . this study found significant improvements in bmi , mental health , hrqol , weight - related body image , and physical fitness among the gasmito patients undergoing rygb . the diabetic group had better mental health and hrqol on all psychological measures before surgery . however , these differences resolved around the time of surgery and no significant differences between the two groups were observed postoperatively . thus , this study found no evidence that baseline diabetes should be considered either a positive or a negative predictor of long - term mental health and hrqol after rygb surgery . furthermore , level of physical fitness ( vo2max ) as an indicator of physical activity was not a main predictor of mental health or hrqol outcome . future studies should prospectively investigate the long - term ( > 5-year follow - up ) effects of type 2 diabetes and physical activity on mental health and hrqol in a larger bariatric sample using objective methods to obtain information about weight , bmi , diabetes status , and physical activity . specifically , physical activity is of importance as this factor is clinically meaningful and can potentially be modified . research should therefore seek to clarify the most optimal type and intensity of physical activity that result in successful outcome with regard to both weight loss and psychological wellbeing after bariatric surgery .

objective . to investigate the predictive value of type 2 diabetes and lack of physical activity for mental health and health - related quality of life after roux - en - y gastric bypass . method . forty severely obese patients undergoing roux - en - y gastric bypass were included in the gasmito study . information about physiological and psychological factors was prospectively assessed at four time points , two times prior to surgery and two times after surgery . measures included oral and intravenous glucose tolerance tests , vo2max test , symptoms checklist ( scl-90 ) , short form health survey 36 ( sf-36 ) , body image questionnaire , and a questionnaire assessing sociodemographic factors and medical status . results . mean % excess weight loss was 65% ( 12 ) at 18-month follow - up and 50% of the participants with diabetes experienced total remission . also , significant improvements were observed with regard to physical fitness , mental distress , health - related quality of life , and weight - related body image ( p < 0.05 ) . the interaction between follow - up time and type 2 diabetes at baseline significantly predicted six of the thirteen psychological subscales ( p < 0.05 ) and , across the follow - ups , physical fitness level made modest contributions to variations in mental symptoms and hrqol but not weight - related body image . conclusion . the results suggest that baseline difference in mental symptoms and physical hrqol between diabetic and nondiabetic patients declines across follow - ups and resolves around the time of surgery .

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

bariatric surgery is the most effective treatment for severe obesity often resulting in excess weight loss of 5080% [ 1 , 2 ] and improved psychological wellbeing and health - related quality of life ( hrqol ) [ 35 ] . in addition , particularly , roux en - y gastric bypass ( rygb ) has been shown to lead to remission of type 2 diabetes and consequently many severely obese individuals with type 2 diabetes seek out this surgical procedure . a growing body of research has suggested that emotional and psychological impairments after surgery influence or may even be the cause of the suboptimal weight loss and weight regain in some bariatric patients [ 10 , 11 ] . type 2 diabetes and lack of physical activity in bariatric patients have previously been associated with decreased weight loss after surgery [ 1416 ] . however , the predictive value of preoperative medical comorbidities and behavioural factors including type 2 diabetes and physical activity , for mental health and hrqol , is currently unclear . it is well established that medical comorbidities such as type 2 diabetes are associated with impaired mental health and hrqol both in the general and in the nonsurgical obese population [ 1820 ] . however , in a recent study , we investigated mental health and hrqol in 129 rygb candidates and found indications that the relation between type 2 diabetes and mental health may differ in the bariatric population . more specifically , results from this study showed that bariatric patients with type 2 diabetes presented for rygb surgery with better mental health and hrqol compared with nondiabetic patients , especially with regard to physical aspects of hrqol . these results suggest that , in our study , patients with type 2 diabetes sought out the surgical option at an earlier stage than the nondiabetic patients , primarily to target their diabetes . despite the observed psychological differences between patients with and without type 2 diabetes , no studies have investigated the predictive value of baseline type 2 diabetes for the mental health and hrqol after bariatric surgery . in addition , lack of physical activity has been associated with poor mental health and reduced hrqol in the bariatric population . these results suggest that the lack of physical activity may negatively predict mental health and hrqol after bariatric surgery . furthermore , while these studies provide some evidence of an association between physical activity and psychological wellbeing among bariatric patients , the majority of the research relies on self - reported physical activity [ 2325 ] and no studies have investigated the relation using an objective measure of the patients ' actual physical fitness level . to address these shortcomings in the literature , the aim of this study was to prospectively investigate the effect of objectively measured baseline type 2 diabetes and physical fitness level on mental health , hrqol , and weight - related body image after rygb . the current study is part of the larger gasmito study ( the national committee on health research ethics , study protocol number hc-2009 - 050 ) that prospectively investigates psychological and physiological factors in severely obese rygb patients with and without type 2 diabetes from the time before weight loss is started until weight stability is achieved 1.5 years after rygb . all patients were tested at four follow - ups : ( a ) at baseline , prior to weight loss , ( b ) about two months later after a diet induced weight loss , ( c ) 4 months after rygb , and , finally , ( d ) 18 months after rygb . each test session consisted of three test days which included a number of physiological tests including oral and intravenous glucose tolerance tests , 2-hour hyperinsulinemic euglycemic clamp , dual x - ray absorptiometry scan , and incremental bicycle vo2max test . furthermore , the psychological profile of the patients was assessed using a battery of standardized questionnaires including measures of mental symptoms , hrqol , body image , and lifestyle , which were administered at each of the four follow - ups . in total , 40 gasmito participants completed the psychological and physiological tests at baseline , 32 participants at follow - up ( b ) , 31 participants at follow - up ( c ) , and finally 23 participants at follow - up ( d ) . inclusion criteria were in agreement with the danish guidelines for gastric bypass surgery prior to january 2011 , which allowed individuals between 18 and 60 years of age with a bmi > 40 kg / m or bmi > 35 kg / m and obesity - related comorbidities ( e.g. to assess quality of life and general health status of the patients ( sf-36 ) , which consists of 36 items evaluating eight aspects of hrqol including physical function , physical role , physical pain , general health , vitality , social function , mental role , and mental wellbeing . information about the patients ' background , lifestyle , and medical status was obtained using a short version of the questionnaire that was developed as part of the copenhagen aging and midlife biobank ( camb ) . remission of type 2 diabetes was defined as hba1c < 42.1 mmol mol , fasting glucose < 5.6 mmol l , and no medication . the vo2max ( ml / min / kg ) is an objective measure of physical fitness level and a direct indicator of the extent of physical activity . to avoid losing information due to missing follow - up data points , we analysed the repeated data using a random intercept multilevel model , allowing intercepts to vary between subjects , with within - subjects level 1 ( follow - up time ) and between - subjects level 2 with the subjects gasmito i d as grouping variable for the measures . to investigate the effect of baseline diabetes and physical fitness level on the psychological outcome variables , the multilevel model included follow - up time , sex , age , baseline bmi , baseline vo2max , baseline diabetes , and their interaction with follow - up time as fixed effects covariates . age , bmi , and vo2max at baseline were included as continuous variables in the interaction analyses with follow - up time . in total , 40 bariatric patients were included in the gasmito study , 30% ( n = 12 ) men and 70% ( n = 28 ) women . the participants ' mean age at baseline was 39.5 ( 8.9 ) years , their mean bmi was 42.5 ( 4.5 ) kg / m , and their mean vo2max was 21.2 ( 4.7 ) ml / kg / min , reflecting a very poor to poor level of physical fitness . finally , 35% ( n = 14 ) of the participants had type 2 diabetes at baseline and 65% ( n = 26 ) were classified as nondiabetic . however , body fat % was lower in the diabetic than in the nondiabetic patients ( 45.1% ( 5.9 ) versus 49.6% ( 5.5 ) , p = 0.024 ) . table 1 shows the baseline results for weight , bmi , vo2max , mental symptoms ( scl-90 ) , hrqol ( sf-36 ) , and weight - related body image ( biq ) and the difference in each subscale score from one follow - up to another . these weight - related improvements were highly significant from baseline to follow - up ( b ) immediately before surgery and continued to be highly significant throughout the surgical course . at follow - up ( d ) , 18 months after rygb , the mean percent excess weight loss ( % ewl ) from baseline reached 65% ( 12 ) and percent weight loss ( % wl ) was 30% ( 6 ) , indicating a successful surgical weight loss outcome . patients with and without type 2 diabetes at baseline did not differ significantly with regard to % ewl . at 18-month follow - up furthermore , of the 14 patients with baseline type 2 diabetes , 50% ( n = 7 ) experienced total remission after surgery and only 21% ( n = 3 ) clearly met the criteria for diabetes ( hba1c > 48 ) postoperatively . also , vo2max increased significantly after surgery possibly explained by the decreased body mass of the patients . at 18-month follow - up , the bariatric patients reported a significant reduction of mental symptoms and a significant increase in hrqol and weight - related body image compared with baseline values , indicated by lower scores on the somatization , depression , anxiety , and gsi subscale of the scl-90 and higher scores on the eight sf-36 subscales and the weight - related biq subscale ( p < 0.05 for all subscales ) . as table 1 shows , the improvements were evident in most of the scl-90 and sf-36 subscales from the time immediately before surgery ( follow - up ( b ) ) to four months after surgery ( follow - up ( c ) ) with the most pronounced improvements observed on the physical hrqol subscales . furthermore , the somatization , depression , gsi , physical function , vitality , and social function subscales improved postoperatively until 18 months after surgery . finally , the weight - related body image score increased significantly throughout the surgical course and was , together with physical function , the only subscale demonstrating significant changes at each follow - up . in the applied multilevel model , the interaction between follow - up time and type 2 diabetes at baseline was found to be significant for six of the thirteen investigated subscales after adjusting for main effects of sex , age , baseline bmi , baseline vo2max , and their interaction with follow - up time . however , the interaction between follow - up time and diabetes was significant for the somatization ( p = 0.005 ) , the depression ( p = 0.004 ) , the anxiety ( p = 0.036 ) , and the gsi ( p = 0.001 ) subscales of the scl-90 . in addition , the interaction between follow - up time and diabetes was significant for the physical function ( p = 0.002 ) and the physical role ( p = 0.016 ) subscales of the sf-36 . as illustrated by figure 1 , these results indicate that differences in mental distress and physical aspects of hrqol between patients with and without baseline diabetes were only substantial at baseline assessment , but not at the later follow - ups . no significant main or interaction effects of diabetes were observed for the weight - related body image subscale . physical fitness ( vo2max ) made modest contributions to variations in mental symptoms and hrqol but not weight - related body image . main effects of vo2max were observed for the physical function ( p = 0.031 ) , the physical role ( p = 0.015 ) , and the vitality ( p = 0.018 ) subscales of the sf-36 with higher baseline vo2max associated with higher scores on these subscales . also , the interaction between follow - up time and baseline vo2max was significant for the somatization ( p = 0.000 ) subscale of the scl-90 and the physical role ( p = 0.016 ) and general health ( p = 0.024 ) subscales of the sf-36 suggesting that the effect of baseline physical fitness level on these subscales declines after surgery . figure 2 shows the effect of baseline physical fitness level on the three subscales , somatization , physical role , and general health , across the four follow - ups . more specifically , the main effect of sex was significant for physical function ( p = 0.030 ) and weight - related body image ( p = 0.004 ) with women demonstrating lower scores than men on these subscales . the interaction between follow - up time and sex was significant for the depression ( p = 0.005 ) score of the scl-90 and the physical function ( p = 0.015 ) , the general health ( p = 0.024 ) , the vitality ( p = 0.032 ) , and the mental health ( p = 0.010 ) scores of the sf-36 . in general , these interactions reflected that sex differences tended to become smaller across follow - ups . however , for the vitality and the mental health subscale of the sf-36 , the direction of the association between sex and these subscale scores changed after surgery and sex differences remained at 18-month follow - up with women reporting higher scores than men . furthermore , a main effect of age was observed for the somatization ( p = 0.028 ) , the depression ( p = 0.007 ) , and the gsi ( p = 0.010 ) subscales with younger patients showing more mental distress than their older counterparts . the interaction between follow - up time and age was significant for the somatization ( p = 0.007 ) , the depression ( p = 0.002 ) , the gsi ( p = 0.015 ) , the vitality ( p = 0.001 ) , the mental health ( p = 0.016 ) , and the weight - related body image ( p = 0.048 ) scores indicating that in general the effect of age on these subscales declines across follow - ups . baseline bmi was of limited predictive value and only showed significant main effects for weight - related body image ( p = 0.040 ) where a lower bmi at baseline was associated with higher body image scores . finally , the interaction between follow - up time and baseline bmi was significant for the vitality ( p = 0.000 ) and the mental health ( p = 0.000 ) subscales suggesting that the effect of baseline bmi on these two subscales decreases from follow - up ( a ) to follow - up ( d ) . to our knowledge , this study was the first study to prospectively investigate the importance of type 2 diabetes and physical fitness at baseline for mental health , hrqol , and weight - related body image among bariatric patients undergoing rygb . in line with consistent findings from prior research [ 4 , 10 ] , mental health , hrqol , and weight - related body image improved significantly from preoperative levels . improvements were most pronounced four months after rygb particularly in physical aspects of hrqol and weight - related body image . the interaction between follow - up time and baseline diabetes was significant for mental health and physical hrqol outcomes indicating that the difference in mental symptoms and physical hrqol between diabetic and nondiabetic patients declines across follow - ups . diabetic patients endorsed fewer mental symptoms and had a higher hrqol and better weight - related body image preoperatively compared with nondiabetic patients . however , interestingly , the differences in mental symptoms and hrqol between patients with and without diabetes at baseline resolved around the time of surgery . it could be speculated that positive expectations to the operation outcome generate an experience of optimism that positively affects the level of mental symptoms and hrqol in the nondiabetic patients around the time of surgery . in fact , improvements in mental health and hrqol were more pronounced initially after surgery in patients that did not have preoperative diabetes compared with patients with diabetes at baseline in spite of the fact that diabetes resolved in 50% of these patients after surgery . one possible explanation is that the differences in psychological wellbeing between patients with and without diabetes reflect preoperative differences in weight - related mental health and hrqol . prior research has suggested that type 2 diabetes is not associated with major impairments of mental and physical functioning among bariatric candidates . in contrast , nondiabetic patients report marked impairments in psychological and physical wellbeing prior to surgery indicating that nondiabetic patients seek out bariatric surgery at a more debilitating stage of the obese state . thus , as suggested , it is possible that bariatric candidates with diabetes primarily seek out surgery to target their diabetes , whereas some bariatric patients without diabetes at baseline seek surgery because of impaired mental health and hrqol related to obesity . it could therefore be speculated that nondiabetic patients experience a major relief when undergoing weight loss and surgery and this might intensify the improvements in mental health and hrqol among these patients initially after surgery . interestingly , this study found that patients who sought bariatric surgery due to medical comorbidities , primarily hypertension and type 2 diabetes , had fewer depressive symptoms and higher hrqol scores compared with patients who sought surgery to improve other factors including appearance , physical fitness , and physical limitations . among the bariatric patients in our study , however , preoperative level of physical fitness did not contribute to the differences between diabetic and nondiabetic patients and vo2max was only a main predictor of improvements in the physical function , the physical role , and the vitality subscale of the sf-36 with higher vo2max associated with better physical hrqol . furthermore , the interaction between follow - up time and vo2max was only significant for the somatization subscale of the scl-90 and the physical role and general health subscales of the sf-36 . vo2max depends on body mass that is why the observed increase in physical fitness level of our patients may reflect weight loss rather than increased physical activity . thus , with regard to the predictive value of physical activity , the use of different assessment methods may therefore explain the inconsistency between our results and prior findings . in line with preoperative studies [ 3537 ] , our results showed that women presented for surgery with more mental distress , reduced hrqol , and poorer weight - related body image compared with men . a more positive weight - related body image was observed among patients with lower baseline bmi compared with the patients with a higher bmi . this effect of sex , age , and baseline bmi on mental distress , hrqol , and weight - related body image declined throughout the surgical course for most of the subscales . however , sex differences were observed on two aspects of hrqol 18 months after surgery with women reporting higher scores on the vitality and the mental health subscales of the sf-36 . investigated postoperative hrqol among 137 patients undergoing gastric bypass surgery and found that male sex predicted better mental hrqol 12 months after surgery . the current study is an important contribution to the very limited literature investigating type 2 diabetes and psychological outcome after bariatric surgery . the prospective study design and the use of objective methods to assess weight , bmi , type 2 diabetes , and physical fitness are important strengths of the study . thus , significant findings may have been missed and , furthermore , it remains unclear whether the results are specific to the bariatric population or whether they also apply to the severely obese individuals in traditional weight loss programs . however , the american society for bariatric surgeons has recommended research to conduct at least 5-year follow - ups and the results of the current study should therefore be considered preliminary . we do acknowledge the importance of longer - term follow - ups and intend to pursue the possibility of extending the follow - up period . this study found significant improvements in bmi , mental health , hrqol , weight - related body image , and physical fitness among the gasmito patients undergoing rygb . the diabetic group had better mental health and hrqol on all psychological measures before surgery . however , these differences resolved around the time of surgery and no significant differences between the two groups were observed postoperatively . thus , this study found no evidence that baseline diabetes should be considered either a positive or a negative predictor of long - term mental health and hrqol after rygb surgery . furthermore , level of physical fitness ( vo2max ) as an indicator of physical activity was not a main predictor of mental health or hrqol outcome . future studies should prospectively investigate the long - term ( > 5-year follow - up ) effects of type 2 diabetes and physical activity on mental health and hrqol in a larger bariatric sample using objective methods to obtain information about weight , bmi , diabetes status , and physical activity . research should therefore seek to clarify the most optimal type and intensity of physical activity that result in successful outcome with regard to both weight loss and psychological wellbeing after bariatric surgery .

heart and vascular system , including particularly the large elastic arteries , that is , the aorta , shows with advancing age a multitude of changes at different structural and functional levels [ 15 ] . as a result , vascular remodeling ( vr ) and medial degeneration ( md ) occur [ 2 , 4 ] . at the macroscopic level , these pathological entities induce weakening of aorta wall and a progressive stiffness [ 2 , 4 ] . endothelial dysfunction , increased oxidative stress , inflammatory reaction , inflammatory cell infiltration in aortic wall , apoptosis of vascular smooth muscle cells ( vsmc)s , degeneration of aortic media , and elastin fragmentation and degradation represent their microscopic alterations [ 2 , 4 ] . in turn , they can degenerate in aortic dilatation and aneurysm and increase the onset risk for complications , that is , aortic dissection and rupture . vr and md are the typical pathological entities of several aorta diseases , including the inherited syndromic and familial forms of thoracic aortic aneurysm ( taa ) and the sporadic forms . among these , sporadic taa is becoming a common and serious health risk because of growing enhance of old people in western populations [ 7 , 8 ] . aged population shows an increased incidence for sporadic taa with advancing of years , as recently reported by epidemiological studies executed in geographic regions with stable populations with little out- or in - migration , such as in minnesota and sweden [ 9 , 10 ] . another determining factor related to the population ageing is the increased number of hypertensive individuals . hypertension is , indeed , a widely prevalent and important risk factor for cardiovascular diseases , including sporadic taa , as established by recent guidelines . however , current research 's interest is enormously increasing , even if the literature data about its genetic , molecular , and cellular mechanisms are inconsistent . in addition , it is growing the opinion to consider thoracic aortic aneurysms , and particularly the sporadic forms , as immune / inflammatory diseases with a strong genetic component . he and colleagues observed an infiltration of inflammatory / immune cells in the media and adventitia from aorta samples of patients with sporadic taa [ 14 , 15 ] . accordingly , we detected a significant high number of cd3+cd4+cd8+cd68+cd20 + cells in tissue aorta samples from patients with stanford type a aortic dissection ( taad ) . a significant role of inflammatory variants in the taad risk was also identified in our study . this also leads to supposing that sporadic taa may be the result of a complex combination of factors , including a high genetic propensity , epidemiology factors , age - related vascular alterations , hemodynamic stress , chronic inflammation , and aortic injury . in this complex scenario , the identification of the pathways activated by these chronic stressors might be crucial , in order to translate experimental data in clinical new personalized measures of prevention , diagnosis , treatments , and management . in consistency with this suggestion , we propose that sporadic taa might be the consequence of an active stimulation of a particular inflammatory signaling pathway , the toll - like receptor ( tlr)-4-mediated signaling pathway , able to recognize both pathogens and endogenous ligands . an increasing number of studies underlines the weight of tlr4-mediated signaling pathway in several cardiovascular diseases ( cvd)s [ 23 , 3036 ] . in addition , its strong role in age - related aorta dysfunction , aneurysm 's onset , and its complications ( dissection or rupture ) recently is emerging . a histopathological study demonstrated the tlr4 expression 's profile in all cells of arterial wall , and particularly in endothelial cells ( ec)s and vsmcs . it also evidenced its functional importance in mediating physiological aorta homeostasis and maintaining of protection against pathogens and damaging cell molecules , as well as in inducting pathological aorta phenotypes . recent experimental investigations in animal and ex vivo models also emphasises its role in the vascular aorta alterations ( vr and md ) and their complications , that is , sporadic taa , by inducing or modulating increased expression and activation of endothelium dysfunction and remodeling aorta pathways ( i.e. , angiotensin converting enzyme ( ace ) , endothelial oxide nitric synthase ( enos ) , and metalloproteinase ( mmp ) pathways ) [ 3742 ] . in addition , genetic variants of tlr4-mediated signaling pathway have been associated with the susceptibility for several cvds [ 3036 ] . in particular , polymorphisms of tlr4 gene ( nm-138554.1 ) , and particularly the rs4986790 tlr4 polymorphism , have been associated with the risk for several cvds and other age - related diseases ( i.e. , alzheimer disease , prostate cancer , and diabetes ) , even if contrasting results have been reported [ 30 , 32 , 36 , 43 ] . in the light of this current evidence , we sought in the present study to investigate the potential role of tlr4-mediated signaling pathway ( promoter ) in sporadic taa . precisely , we analysed the weight of ten genetic variants related to tlr4-mediated signaling pathway in disease susceptibility , evocation of aorta abnormalities , systemic inflammation ( arterial - associated senescence secretor phenotype - aassp ) , and vascular biological ageing . thus , associations between their combined risk genotypes and typical pathological tissue phenotypes , systemic inflammatory mediator levels , inflammatory / immune infiltrate , apoptosis of vsmc cells , tissue mmp-9 amounts , and attrition of telomeres were also evaluated . our study included 161 individuals ( 127 men ( 78% ) and 34 ( 22% ) women ; mean age : 63 10.7 ) from western sicily enrolled precisely from january 2004 to july 2008 at time of their admission to cardiac surgery unit of palermo university hospital . they were affected by sporadic taa and diagnosed through imaging technologies ( i.e. , echo , ct and mri ) and with localization essentially in ascending aorta ( precisely in aortic sinus and tubular portion and sometimes only in tubular portion ) and in aortic bulb or both ( see table 1 ) . for the patient 's selection , histopathological analyses and exclusion criteria for syndromic and familial forms ( e.g. , marfan and ehler 's danlos syndromes ) and autoimmune connective tissue disorders were utilised . according to 2010 guidelines , elective or acute surgical treatment ( using wheat operation , bentall - de bono and tirone david surgical techniques , whenever possible ) and consequent resection were performed after evaluation of aortic transverse diameter sizes [ 44 , 45 ] . thus , demographic and clinical features , comorbidity conditions , and pharmacological treatments were collected ( see table 1 ) . the control group consisted of 128 subjects ( 61 ( 47% ) men and 67 ( 53% ) women ; mean age : 61.08 5.83 years ) , belonging to the same ethnic group of patients , in order to include in the study a very homogenous population . ethnicity was confirmed , since parents and grandparents of both patients and controls were born in western sicily . controls were in good health according to their clinical history and blood tests ( complete blood cell count , erythrocyte sedimentation rate , glucose , urea nitrogen , creatinine , electrolytes , c reactive protein , liver function tests , iron , and proteins ) ( see table 1 ) . their demographic and clinical features , comorbidity conditions , and pharmacological treatments were collected ( see table 1 ) . furthermore , echocardiography imaging examinations confirmed absence of any aorta wall histopatological abnormalities in all controls . in order to detect histopathological abnormalities , control ascending aortas were obtained from 30 individuals ( 20 men and 10 women , mean age : 63.9 10.3 ) who died for causes unrelated to aortic disease and having no sepsis at death time , as confirmed by autopsy ( see table 1 ) . our study received approval from local ethic committees and all participants gave their informed consent . histopathological investigations were performed only in 100 aorta samples obtained from aortic wall of patients who underwent surgical repair , since some patient 's aortas showed unsuitable histological conditions . the procedures used were previously reported in our recent studies [ 16 , 4649 ] and briefly described in online supplementary material . in addition , the following histological features were evaluated : ( 1 ) fibrosis ( defined as an increase in interstitial collagen ) ; ( 2 ) medionecrosis ( defined as a focal loss of smooth muscle cell nuclei in the media ) ; ( 3 ) cystic medial necrosis ( defined as mucoid material accumulation ) ; ( 4 ) focal or plurifocal medial apoptosis ; ( 5 ) elastic fragmentation ( defined as focal fragmentation of elastic lamellae in the media ) ; ( 6 ) amounts of mmp-9 ; ( 7 ) inflammatory / immune cell infiltration . histopathological abnormalities of aortic wall were graded and defined according to the definitions and grading systems used by bechtel and colleagues ( see details reported in the section of online supplementary materials and precisely in figures 1(s ) and 2(s ) . ) and previously described in our recent studies [ 16 , 4649 ] . in addition , typical phenotypes were detected as reported in online supplementary material and previously described in our recent study . specific monoclonal antibodies were used , and standard techniques were performed as described in online supplementary material . for detecting apoptosis , a tdt- ( terminal deoxynucleotidyl transferase- ) mediated x - dutp ( deoxyuridine triphosphate nucleotides ) nick end - labeling ( tunel ) reaction ( in situ cell death detection kit , roche diagnostics s.p.a , milano , italy ) was used and performed as reported in detail in online supplementary material . three apoptosis patterns were identified : absent , focal , and plurifocal medial apoptosis , as evidenced in figure 2(s ) in online supplementary material . staining was classified as low , moderate , or high amount , as reported in figure 2(s ) in online supplementary material . dna samples were obtained by blood samples from case and control individuals ( see online supplementary material ) . they were genotyped for ten snps located in promoter and coding regions of selected candidate genes codifying molecules related to tlr4-mediated signaling pathway and with biological effects able to modulate the susceptibility for several cvds , such as sporadic taa . information about these snps was acquired from dbsnp ncbi , the ensembl database ( http://www.ensembl.org/index.html ) , and the ucsc genome browser website ( http://genome.ucsc.edu ) and reported in table s1 of online supplementary material . plasma il-6 , tnf- , mmp-2 , mmp-9 , and crp levels were measured according to method reported in online supplementary material . a marker of telomere leukocyte length was determined using dna samples of 30 cases and 30 controls selected randomly , but having the same age and gender . significant differences among qualitative variables were calculated by using pearson test . to analyze significant relationships among quantitative variables , furthermore , odds ratios ( or ) with 95% confidence intervals ( ci ) and their significance were calculated . to study mortality between male and female patients , differences between survival curves with a gehan test and appropriate peto & peto modification were carried out . data were tested for goodness of fit between observed and expected genotype frequencies according to hardy - weinberg equilibrium , by tests . significant differences in frequencies among groups were calculated by using test and appropriate tables ( 2 2 and 3 3 tables , etc . significant relationship between genetic variables and pathology risk was analysed using quasi - likely hood binomial models . analysis of variance ( anova ) test ( corrected by bonferroni ) was also utilised to compare positive inflammatory / immune cells between case and control aorta samples . unpaired t - test ( welch corrected ) was , utilised to analyse positive inflammatory / immune cells between pathological and normal case aortas . to identify possible correlations between cd3+cd4+cd8+cd68 + cell number and aorta aneurysm diameter , the same test was utilised to evaluate the correlations between the severity of all examined histopathological abnormalities and patient features or the major risk factors ( i.e. , age , gender , aortic diameter , hypertension , diabetes , and smoking ) and to assess the correlations between the mmp-9 and severity of elastic fragmentation . quantitative values of the cytokines , mmps and crp , were expressed as mean sd . to assess their differences , unpaired t - test their correlations were assessed using spearman 's rank correlation . a p < 0.05 was considered statistically significant . the difference in mean trf length between cases and controls was analysed using an independent sample the difference in mean trf length in subjects with different risk factors was analysed using bivariate correlation for continuous variables and independent samples the independent effect of age , sex , and other risk factors on the mean trf length was analysed using a linear regression model controlling for case control status . the elevated number of sporadic taa men ( 127 versus 34 women ) led us to compare all patient features according to gender . no statistical significant differences were detected , with exception of smoking ( 67 male versus 6 female , p < 0.001 by pearson test , 2 2 table ; or 5.16 ( 1.9216.32 ) , p = 0.0002 by fisher test ) . however , among the major risk factors , hypertension characterised 79% of all patients , opportunely treated with medications like ace inhibitors and beta - blocker , and so forth , during the follow - up and after surgery ( see table 1 ) . according to gender , no significant differences were also observed in evaluating long - term survival ( 80 months ) after surgery . however , or was higher for women ( 4.5 ( 3.26.9 ) versus 2.6 ( 2.22.9 ) for men ) . significant differences were , detected in short - term survival ( 30 days ) . compared with male patients , females showed increased 30 day mortality : 9 of 34 female ( 26% ) versus 10 of 127 male ( 8% ) ( p = 0.049 by gehan test ; or 6.5 ( 1.56.8 ) , p = 0.01 by fisher test ) ( see figure 1 ) . these data correlated with several clinical conditions observed in the major female number after surgical repair ( data not shown ) . no statistical significant differences were detected , with exception of hypertension ( 127 versus 40 p < 0.001 by pearson test , 2 2 table ; see table 1 ) . in order to demonstrate the key role of tlr4-mediated signaling pathway in the pathophysiology of sporadic taa and to support our hypothesis , we compared the allele frequencies of ten selected snps between 161 patients and 128 controls . significant differences were only found for the following snps : rs4986790 tlr4 , rs333 ccr5 , rs2070744 enos , rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 ( see table 2 ) . among these , we interestingly observed that the rs4986790 tlr4 polymorphism confers a higher susceptibility for sporadic taa ( or = 14.4 , p = 0.0008 ) ( see table 2 ) . the protective + 896 g tlr4 allele associated with a low risk of age - related diseases has a frequency only of 0.3% ( 1 ) in cases versus 4% ( 11 ) in controls ( see table 2 ) . in addition , using a quasi - hood binomial statistical model , we obtained that the rs4986790 tlr4 , rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 snps are independent risk factors for sporadic taa ( p = 0.001 ) . considering the biological effects of these snps ( see table s1 ) , we assessed the frequency of + 896atlr4/dace/1562tmmp-9/735tmmp-2 risk genotype in cases and controls . by comparing it with frequency of other combinations , combined risk genotype , high responder genotype , was significantly represented in cases than controls . indeed , 46 patients were carriers of the combined risk genotype versus 10 controls ( p = 0.000009 , by test ; or = 4.7 , p < 0.0001 by fisher 's exact test , see table 3 ) . given the overrepresentation of this combined risk genotype in patients and its strong role in the susceptibility ( or = 4.7 , see table 3 ) for sporadic taa , we evaluated its biological effect in influencing the grade of chronic inflammation . thus , we assessed the eventual significant differences in systemic plasma levels ( aassp levels ) of il-6 , tnf- , crp , and mmp-2 and -9 between all patients and all controls . the same analysis was performed in cases bearing combined risk genotype versus no case carriers and in case carriers versus control carriers . as reported in table 4 , we detected significant differences of all systemic plasma mediators between patients and controls . however , the very interesting and promising datum was the presence of higher levels of all mediators examined in cases bearing combined risk genotype than both cases bearing other genotypes and control carriers of combined risk genotype , as illustrated clearly in table 4 . furthermore , we detected that higher plasma levels of mmp-2 and -9 levels from cases bearing combined risk genotype significantly correlated with the moderate and elevated amounts of mmp-9 detected reciprocally from their tissue aorta samples ( r = 0.397 , p = 0.001 ; r = 0.234 , p = 0.03 by nonparametrical spearman correlation test ; data not shown ) . in particular , cases having combined risk genotype showed a significant association between increased plasma mmp-9 and mmp-2 levels and elevated amounts of mmp-9 ( p = 0.006 by test ; data not shown ) . consistent with these data , positive correlations were detected in the cases with combined risk genotype between the increased plasma levels of mmp-9 and mmp-2 and elastic fragmentation and the elevated amounts of mmp-9 observed in their tissue aorta samples ( r = 0.497 , p = 0.0001 ; r = 0.267 , p = 0.03 , r = 0.342 , p = 0.006 , resp . , by nonparametrical spearman correlation test ; data not shown ) . in verifying the influence and the biological effect of combined risk genotype on levels of systemic inflammatory mediators , and consequently its role in the occurrence of vr , md and their complications , such as sporadic taa , their quantities were compared in control carriers versus no control carriers ( 10 versus 118 , as reported in table 3 ) . as shown in table 5 , this comparison demonstrated significant differences of all systemic inflammatory mediators ( il-6 , tnf- , crp , and mmp-2 and -9 ) in controls bearing the high responder genotype than those with other genotypes . thus , combined risk genotype seems to mediate a crucial biological effect on the levels of systemic inflammatory mediators and , hence , in the evocation of md , vr , and sporadic taa . we also evaluated the potential significant differences of levels of systemic aassp mediators in controls screened only for the presence of rs4986790 tlr4 polymorphism . controls bearing the allele + 896 g , associated with a blunted innate / inflammatory response , showed significant reduced levels of all systemic aassp mediators than carriers of the + 896a tlr4 allele ( see table 5 ) . thus , controls with the tlr4 proinflammatory + 896a allele had significant levels of systemic mediators , but their magnitude was lower than that observed in controls with the combined risk genotype . furthermore , infiltration of lymphocytes and macrophages was also detected in tissue aorta wall samples from patients , control aortas , and normal areas from the same taa tissues . a significant higher infiltrate of lymphocytes and macrophages in tissue aorta wall samples from patients compared with both control aortas and normal areas from the same taa tissues was observed ( see figure 2 ) . interestingly , the infiltration of inflammatory / immune cells was particularly considerable in the vasa vasorum of adventitia from the aorta patient samples . in contrast , a very small infiltrate of these cells was observed in control aortas , which appears to be less significant in respect to that found in normal aorta taa areas . + cell infiltrate was less represented in three groups , even if significant differences were observed by comparing the three cohort tissues ( see figure 2 ) . immunostaining with cd68 antibody also indicated that macrophages were prevalently present in aortas from taa patients in respect to control aortas and normal areas from the same taa tissues ( see figure 2 ) . in order to validate the biological effect mediated of combined risk genotype on the levels of systemic plasma inflammatory mediators , we also compared inflammatory / immune infiltrate between patients bearing high responder genotype and those with other genotypes . according to encouraging data obtained on levels of systemic plasma inflammatory mediators , we assessed a higher inflammatory / immune infiltrate in tissue aorta samples from patients bearing high responder genotype than those bearing other genotypes and control aortas ( see figure 3 ) . positive correlation was identified between the number of cd3+cd4+cd8 + cd68 + cells observed in aorta samples from patients bearing high responder genotype and the histological abnormalities observed through histopathological and immunohistochemical assays and tunel testing ( see table 6 ) . as reported in table 6 , the number of cd3+cd4+cd8 + cd68 + cells also correlated with the increased plasma levels of il-6 , tnf- , crp , and mmp-2 and -9 . in addition , the 46 patients with combined risk genotype showed for the 89% a typical morphological aorta 's phenotype , defined in our previous study as phenotype iii , characterized by elevated cystic md , plurifocal medial apoptosis , and increased mmp-9 amount ( as reported in online supplementary material ) . positive correlations were identified between the severity of histopatological abnormalities characterising this phenotype and hypertension , smoking , and age ( r = 0.179 , p = 0.03 ; r = 0.345 , p = 0.001 ; r = 0.267 , p = 0.02 , resp . consistent with these interesting data , we also evaluated whether the combined risk genotype can likely have a strong role in determining vascular senescence and onset of sporadic taa . to this purpose , we examined the mean of blood leukocyte telomere length using terminal restriction fragment assay ( trf test , a southern blot technique ) and blood samples from 30 patients and 30 controls , using a procedure described in two of our recent studies . thus , we detected that the case group had a mean trf length ( 4.675 0.605 kbp , data not shown ) , significantly lower than that observed in the control group ( 6.218 0.485 kbp , data not shown ) . a difference of 1.543 bp was observed between cases and controls ( 95% confidence interval 68 bp to 201 bp , p = 0.001 ) . there was no significant change in the mean trf length with age in both patients and controls ( 6 bp decrease per year , sd = 4 ; p = 0.25 ) . there was also no significant correlation between aneurysm size and mean trf length ( p = 0.46 ) . there was no significant difference in the mean trf length between male and female patients . comparisons between mean trf length and the risk taa factors including gender , smoking , hypertension , diabetes , and family history were also evaluated . sex as an independent risk factor did not have any significant effect on the mean trf length ( p = 0.76 ) . in contrast , smoking and hypertension in s - taa cases were the only risk factors which were significantly associated with the mean trf length ( p = 0.01 ) . s - taa subjects with smoking and hypertension history had a significantly shorter mean trf length ( 4.491 , sd = 0.237 ) compared to subjects without these risk factors ( 5.997 , sd = 0.302 ) , ( mean difference 132 bp , confidence interval 11 bp to 245 bp , p = 0.01 ) . stratifying the 30 patients having low mean trf length for combined risk genotype , we interestingly observed that 85% ( versus 8% for controls ) were carriers of combined risk genotype ( p = 0.001 by test , 2 2 table ; data not shown ) and had significant higher levels of plasma inflammatory mediators ( il-6 : 13.8 2.3 versus 5.1 1.4 , p < 0.001 ; tnf- : 15.5 1.4 versus 8.2 1.2 , p < 0.001 ; crp : 18.4 1.3 versus 6.6 2.8 , p < 0.0001 ; mmp-9 : 59.9 2.8 versus 11.6 1.8 , p < 0.0001 , and mmp-2 : 57.8 3.5 versus 13.7 1.9 , p < 0.0001 , resp . ; data not shown ) , increased amounts of cd3+cd4+cd8+cd68+cd20 + cells than controls and patients with other genotypes ( p < 0.0001 , by anova test ( corrected by bonferroni ) ) . sporadic taa is predominantly a silent ailment , until rupture or dissection occur , and insidious in its onset and progression . its diagnosis is exclusively based on imaging technologies ( i.e. , echo , ct , or mri ) . accordingly , it is very crucial to early predict , diagnose , and treat sporadic taa , characterised by lack of medical optional treatments and disease biomarkers , such as blood tests . blood tests might consent to detect in general population individuals at sporadic taa risk , to monitor its progression and predict complications . to this purpose recently , it has been suggested that aortic aneurysms , and particularly the sporadic forms , are immune diseases with a strong genetic component we have evidenced this crucial aspect of the complex pathophysiology of this disease in our recent studies [ 16 , 4649 ] . in patients with taad , we observed high levels of immune / inflammatory cells and a significant association of some inflammatory polymorphisms with the taad susceptibility . in line with this , he and colleagues recently observed an increased immune / inflammatory infiltrate in aorta samples of patients with sporadic taa [ 14 , 15 ] . this is leading to identify the inflammatory pathways , which might operate as key link between the onset of sporadic taa and immune system . their recognition should be very imperative in order to translate experimental data in clinical new personalized measures of taa prevention , diagnosis , treatments , and management . considerable and convincing evidence links the pathophysiology of atherosclerosis , cardiac dysfunction , congestive heart failure , and other vascular diseases with the tlr-4-mediated signaling pathway , as amply stressed by frantz and colleagues . recently , the group of pasterkamp also provided an overview of the endogenous molecules , released under cellular cardiovascular stress and damage , which can trigger innate immunity via tlr-4-mediated signaling pathway in cvds . in the specific case of sporadic taa , recent experimental investigations in animal and ex vivo models also emphasise its role in the vascular aorta alterations ( vr and md ) and their complications , such as sporadic taa , by evocating or modulating increased expression and activation of endothelium dysfunction and remodeling aorta pathways [ 23 , 3742 ] . on the other hand , pryshchep and colleagues demonstrated the tlr4-mediated signaling pathway expression in all cells of arterial wall and particularly in ecs and vsmcs . in addition , they also evidenced its functional importance in both mediating physiological aorta homeostasis and maintaining protection , as well as in inducting pathological aorta phenotypes , that is , vr and md . furthermore , song and colleagues demonstrated that signaling via tlr4-mediated signaling pathway and its signal adaptors , that is , myd88 , is responsible for the age - elevated basal il-6 response using vsmcs from aged tlr4 and myd88 mice . eissler and colleagues observed an increased hypertension - related expression of tlr4-mediated signalling pathway in vascular cells of untreated hypertensive rats . the group of golzales - ramos underlined that circulating heat shock protein 70 , associated with an increased cellular aorta 's damage , regulates the profibrotic response of human aorta smcs through increased transforming growth factor type-1 ( tgf-1 ) expression , evocated by tlr4-mediated signaling pathway . in addition , li and colleagues reported the role of tlr4-mediated signaling pathway in regulating the mmp-9 expression in human vsmcs . bucci and colleagues recently emphasized as the vascular thoracic aorta homeostasis and its alteration in rats is based on the activity of tlr4-mediated signaling pathway and its cross talk with other stress and stretch pathways , that is , ace , enos , and mmp pathways . furthermore , a recent study demonstrated in apolipoprotein e - deficient mice that it is possible to limit the inflammatory process by blocking tlr4/c - jun n terminal kinase signaling pathway with rosiglitazone in the initiation stages of aortic aneurysm development . this encouraging and increasing evidence , fruit prevalently of animal investigations , and our recent data on taad [ 16 , 46 , 48 ] , led us to analyze the potential role of genetic variants related to tlr4-mediated signaling pathway in the complex pathophysiology of sporadic taa . until now thus , our study represents the first report which , through a human ex vivo study approach , evidenced as some polymorphisms related to tlr4-mediated signaling pathway significantly modulate the sporadic taa risk : rs4986790 tlr4 , rs333 ccr5 , rs2070744 enos , rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 polymorphisms . among these , the rs4986790 ( + 896a > g ) tlr4 polymorphism confers a higher susceptibility for sporadic taa ( or = 14.4 , p = 0.0008 ) . however , it represents an independent risk taa factor for sporadic taa , as well as the rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 snps . a significant overrepresentation of their combined risk genotype ( + 896atlr4/dace/-1562tmmp-9/-735tmmp-2 ) was observed in cases than controls ( 46 versus 10 , p < 0.000009 ) by comparing it with frequency of other combinations . in addition , it was associated a significant risk for sporadic taa ( or = 4.7 ; p < 0.0001 ) . cases bearing combined risk genotype showed higher systemic inflammatory mediator levels than those with other genotypes and control carriers . in particular , they had higher plasma levels of mmp-9 and -2 which correlated with the amounts of mmp-9 and elastic fragmentation observed in their tissue aorta samples . a higher chronic inflammatory infiltrate was also found in cases bearing combined risk genotype , which positively correlated with histological abnormalities and levels of mediators . in addition , they showed in their tissue aorta samples a typical morphological phenotype , characterized by elevated cystic md , plurifocal medial apoptosis , and increased mmp-9 amounts , and defined in a previous study as phenotype iii . furthermore , we detected that combined risk genotype influences vascular biological ageing , evaluating the gold standard ageing marker , the telomere length , in a small number of cases and controls , selected randomly , but having the same age and gender . it characterized the 85% of the cases examined , which had lower telomere length , higher levels of mediators , increased amount of chronic inflammatory infiltrate . this results concord with the preliminary data reported in a previous study and the recent literature reports [ 5153 ] . these interesting results led us to evaluate the biological effect of the combined risk genotype . controls bearing high responder genotype showed higher levels of systemic mediators than control carriers of only the rs4986790 tlr4 polymorphism . these results are in agreement with our previous data [ 54 , 55 ] . indeed , in clarifying and confirming the biological effects of rs4986790 tlr4 polymorphism and its role in the pathophysiology of age - related diseases , including cvds , alzheimer disease , prostate cancer , diabetes , and longevity , we assessed the levels of il-6 , tnf- , il-10 , and eicosanoids in lps - stimulated whole blood samples in vitro of 50 young healthy sicilians , screened for the presence of rs4986790 tlr4 and 765g > significantly higher levels of both proinflammatory cytokines and eicosanoids were observed in individuals bearing the rs4986790 tlr4 polymorphism . however , their magnitude significantly was more increased in individual 's carriers of their combined genotype . in addition , significantly lower levels of inflammatory mediators were observed in carriers bearing the tlr4 mutation ( the + 896 g allele ) , whereas the anti - inflammatory il-10 values were higher . the same results were detected in this study in the controls carriers of the + 896 g tlr4 allele . thus , these data strengthen our suggestion , stressed for other age - related diseases including alzheimer disease and prostate cancer , that polymorphisms related to tlr4-mediated signaling pathway may have a major influence on the pathophysiology of a disease , such as sporadic taa , when they operate in combination to create a risk profile [ 56 , 57 ] . in the complex , our data seem to suggest the strong relevance of tlr4-mediated signaling pathway in inducing md , age related vr , and their complications , such as sporadic taa . on the other hand , our findings emphasise as a combined risk genotype associated of tlr4-mediated signaling pathway are able to modulate the grade of aorta age - related phenotypical , histological , and systemic abnormalities and consequently vascular aorta ageing , onset , and progression of sporadic taa . they also lead us to suggest that this signaling pathway might also be an optimal target for new therapeutic treatments able to retard or block the typical aorta age - related changes which determine endothelial dysfunction , md , and vr . this might open new perspectives for the prevention of both aortic vr and md and sporadic taa , by using combined risk genotype ( + 896atlr4/dace/1562tmmp-9/735tmmp-2 ) as optimal genetic biomarker for the earlier detection of this silent pathology in preliminary phases and to treat with different and specific therapies depending on individual 's genotypes . consequently , it also leads to view vascular ageing as a modifiable risk factor , particularly for aortic disease . in the specific case of sporadic taa , the therapeutic potential of tlr4-mediated signaling pathway might be defined through the use of its agonists or antagonists , whose effects have been prevalently experimented in mice , rats , and cultures as reported in literature . this might consent to reduce chronic age - related inflammation and limit the dysfunction of ecs and endothelial progenitor cells ( epcs ) , which move towards injured endothelium or inflamed tissues and incorporate into foci of neovascularisation , thereby improving blood flow and tissue repair [ 5860 ] . on the other hand , a recent investigation reports the involvement of tlr4-mediated signaling pathway in maintaining the stem cell phenotype of epcs and enlarging this population . this finding reveals a novel aspect of the multiple - faced tlr-4-mediated signaling pathway biology , and it may open new prospects for using tlr4 agonists in promoting the production of epcs for clinical use . in addition , recent research underlines that mirnas might play important roles in this scenario , modulating tlr-4-mediated signaling pathway activation . mirnas seem , indeed , to have two opposite roles : tlr-4-mediated pathway activation and nf-b signaling inhibition , in a complex scenario where low and chronic inflammation prevails , likely also sustained by cell senescence secretome . mirnas inhibition effect probably belongs to the different levels of anti - inflammatory pathways which have evolved to abate tlr-4 signaling to prevent cell and aorta destruction . based on our findings , we also suggest another possible therapeutic intervention to apply in the preclinical phase in subject 's carriers of combined high risk genotype to the aim to retard to limit the onset and progression of the vascular ageing and its complications , such as sporadic taa . precisely , we propose antibody - mediated stimulation of tam receptors involved in the inhibition of the inflammatory response . the sequential induction of this pathway and its integration with upstream tlr and cytokine signaling networks may impact the evocation of the release of inflammatory mediators limiting the inflammatory response and consent to modulate the telomere / telomerase system reducing the senescence of both the aorta wall cells and the epcs able in repairing aorta injury . on the other hand , patients with cvds exhibit a reduced epc number and function [ 6365 ] . it has become increasingly apparent that these changes may be effected in response to enhanced oxidative stress , possibly as a result of systemic and localized inflammatory responses . the weight of our findings and suggestions might be certainly implemented validating them in a larger sample size , even if our data are the result of a relatively small sample and a very homogenous population . in addition , gene expression analyses , immunohistochemical tlr4 quantification , and soluble tlr4 level detection represent further objectives of our future studies . they should consent to translate with major emphasis our promising data in personalized treatments of a pathology , the sporadic taa , which clinically and predominantly is silent , until rupture or dissection occurs , and insidious in its onset and progression . furthermore , until now its diagnosis is also exclusively based on imaging technologies . finally , our obtained data led us to postulate a potential model about the pathophysiology of sporadic taa , which might be defined as model of the signaling pathway from the double - face , given its features ( see figure 4 ) . we foretell that it can lead several researchers to perform investigations focused to clear the complex puzzle of this pathology .

thoracic aorta shows with advancing age various changes and a progressive deterioration in structure and function . as a result , vascular remodeling ( vr ) and medial degeneration ( md ) occur as pathological entities responsible principally for the sporadic taa onset . little is known about their genetic , molecular , and cellular mechanisms . recent evidence is proposing the strong role of a chronic immune / inflammatory process in their evocation and progression . thus , we evaluated the potential role of toll like receptor- ( tlr- ) 4-mediated signaling pathway and its polymorphisms in sporadic taa . genetic , immunohistochemical , and biochemical analyses were assessed . interestingly , the rs4986790 tlr4 polymorphism confers a higher susceptibility for sporadic taa ( or = 14.4 , p = 0.0008 ) and it represents , together with rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 snps , an independent sporadic taa risk factor . in consistency with these data , a significant association was observed between their combined risk genotype and sporadic taa . cases bearing this risk genotype showed higher systemic inflammatory mediator levels , significant inflammatory / immune infiltrate , a typical md phenotype , lower telomere length , and positive correlations with histopatological abnormalities , hypertension , smoking , and ageing . thus , tlr4 pathway should seem to have a key role in sporadic taa . it might represent a potential useful tool for preventing and monitoring sporadic taa and developing personalized treatments .

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

heart and vascular system , including particularly the large elastic arteries , that is , the aorta , shows with advancing age a multitude of changes at different structural and functional levels [ 15 ] . as a result , vascular remodeling ( vr ) and medial degeneration ( md ) occur [ 2 , 4 ] . at the macroscopic level , these pathological entities induce weakening of aorta wall and a progressive stiffness [ 2 , 4 ] . vr and md are the typical pathological entities of several aorta diseases , including the inherited syndromic and familial forms of thoracic aortic aneurysm ( taa ) and the sporadic forms . aged population shows an increased incidence for sporadic taa with advancing of years , as recently reported by epidemiological studies executed in geographic regions with stable populations with little out- or in - migration , such as in minnesota and sweden [ 9 , 10 ] . however , current research 's interest is enormously increasing , even if the literature data about its genetic , molecular , and cellular mechanisms are inconsistent . in addition , it is growing the opinion to consider thoracic aortic aneurysms , and particularly the sporadic forms , as immune / inflammatory diseases with a strong genetic component . he and colleagues observed an infiltration of inflammatory / immune cells in the media and adventitia from aorta samples of patients with sporadic taa [ 14 , 15 ] . this also leads to supposing that sporadic taa may be the result of a complex combination of factors , including a high genetic propensity , epidemiology factors , age - related vascular alterations , hemodynamic stress , chronic inflammation , and aortic injury . in consistency with this suggestion , we propose that sporadic taa might be the consequence of an active stimulation of a particular inflammatory signaling pathway , the toll - like receptor ( tlr)-4-mediated signaling pathway , able to recognize both pathogens and endogenous ligands . in addition , its strong role in age - related aorta dysfunction , aneurysm 's onset , and its complications ( dissection or rupture ) recently is emerging . recent experimental investigations in animal and ex vivo models also emphasises its role in the vascular aorta alterations ( vr and md ) and their complications , that is , sporadic taa , by inducing or modulating increased expression and activation of endothelium dysfunction and remodeling aorta pathways ( i.e. in particular , polymorphisms of tlr4 gene ( nm-138554.1 ) , and particularly the rs4986790 tlr4 polymorphism , have been associated with the risk for several cvds and other age - related diseases ( i.e. in the light of this current evidence , we sought in the present study to investigate the potential role of tlr4-mediated signaling pathway ( promoter ) in sporadic taa . precisely , we analysed the weight of ten genetic variants related to tlr4-mediated signaling pathway in disease susceptibility , evocation of aorta abnormalities , systemic inflammation ( arterial - associated senescence secretor phenotype - aassp ) , and vascular biological ageing . thus , associations between their combined risk genotypes and typical pathological tissue phenotypes , systemic inflammatory mediator levels , inflammatory / immune infiltrate , apoptosis of vsmc cells , tissue mmp-9 amounts , and attrition of telomeres were also evaluated . in addition , the following histological features were evaluated : ( 1 ) fibrosis ( defined as an increase in interstitial collagen ) ; ( 2 ) medionecrosis ( defined as a focal loss of smooth muscle cell nuclei in the media ) ; ( 3 ) cystic medial necrosis ( defined as mucoid material accumulation ) ; ( 4 ) focal or plurifocal medial apoptosis ; ( 5 ) elastic fragmentation ( defined as focal fragmentation of elastic lamellae in the media ) ; ( 6 ) amounts of mmp-9 ; ( 7 ) inflammatory / immune cell infiltration . they were genotyped for ten snps located in promoter and coding regions of selected candidate genes codifying molecules related to tlr4-mediated signaling pathway and with biological effects able to modulate the susceptibility for several cvds , such as sporadic taa . information about these snps was acquired from dbsnp ncbi , the ensembl database ( http://www.ensembl.org/index.html ) , and the ucsc genome browser website ( http://genome.ucsc.edu ) and reported in table s1 of online supplementary material . , age , gender , aortic diameter , hypertension , diabetes , and smoking ) and to assess the correlations between the mmp-9 and severity of elastic fragmentation . no statistical significant differences were detected , with exception of smoking ( 67 male versus 6 female , p < 0.001 by pearson test , 2 2 table ; or 5.16 ( 1.9216.32 ) , p = 0.0002 by fisher test ) . compared with male patients , females showed increased 30 day mortality : 9 of 34 female ( 26% ) versus 10 of 127 male ( 8% ) ( p = 0.049 by gehan test ; or 6.5 ( 1.56.8 ) , p = 0.01 by fisher test ) ( see figure 1 ) . in order to demonstrate the key role of tlr4-mediated signaling pathway in the pathophysiology of sporadic taa and to support our hypothesis , we compared the allele frequencies of ten selected snps between 161 patients and 128 controls . significant differences were only found for the following snps : rs4986790 tlr4 , rs333 ccr5 , rs2070744 enos , rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 ( see table 2 ) . among these , we interestingly observed that the rs4986790 tlr4 polymorphism confers a higher susceptibility for sporadic taa ( or = 14.4 , p = 0.0008 ) ( see table 2 ) . in addition , using a quasi - hood binomial statistical model , we obtained that the rs4986790 tlr4 , rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 snps are independent risk factors for sporadic taa ( p = 0.001 ) . indeed , 46 patients were carriers of the combined risk genotype versus 10 controls ( p = 0.000009 , by test ; or = 4.7 , p < 0.0001 by fisher 's exact test , see table 3 ) . given the overrepresentation of this combined risk genotype in patients and its strong role in the susceptibility ( or = 4.7 , see table 3 ) for sporadic taa , we evaluated its biological effect in influencing the grade of chronic inflammation . thus , we assessed the eventual significant differences in systemic plasma levels ( aassp levels ) of il-6 , tnf- , crp , and mmp-2 and -9 between all patients and all controls . the same analysis was performed in cases bearing combined risk genotype versus no case carriers and in case carriers versus control carriers . however , the very interesting and promising datum was the presence of higher levels of all mediators examined in cases bearing combined risk genotype than both cases bearing other genotypes and control carriers of combined risk genotype , as illustrated clearly in table 4 . furthermore , we detected that higher plasma levels of mmp-2 and -9 levels from cases bearing combined risk genotype significantly correlated with the moderate and elevated amounts of mmp-9 detected reciprocally from their tissue aorta samples ( r = 0.397 , p = 0.001 ; r = 0.234 , p = 0.03 by nonparametrical spearman correlation test ; data not shown ) . in particular , cases having combined risk genotype showed a significant association between increased plasma mmp-9 and mmp-2 levels and elevated amounts of mmp-9 ( p = 0.006 by test ; data not shown ) . consistent with these data , positive correlations were detected in the cases with combined risk genotype between the increased plasma levels of mmp-9 and mmp-2 and elastic fragmentation and the elevated amounts of mmp-9 observed in their tissue aorta samples ( r = 0.497 , p = 0.0001 ; r = 0.267 , p = 0.03 , r = 0.342 , p = 0.006 , resp . in verifying the influence and the biological effect of combined risk genotype on levels of systemic inflammatory mediators , and consequently its role in the occurrence of vr , md and their complications , such as sporadic taa , their quantities were compared in control carriers versus no control carriers ( 10 versus 118 , as reported in table 3 ) . thus , combined risk genotype seems to mediate a crucial biological effect on the levels of systemic inflammatory mediators and , hence , in the evocation of md , vr , and sporadic taa . we also evaluated the potential significant differences of levels of systemic aassp mediators in controls screened only for the presence of rs4986790 tlr4 polymorphism . thus , controls with the tlr4 proinflammatory + 896a allele had significant levels of systemic mediators , but their magnitude was lower than that observed in controls with the combined risk genotype . interestingly , the infiltration of inflammatory / immune cells was particularly considerable in the vasa vasorum of adventitia from the aorta patient samples . in order to validate the biological effect mediated of combined risk genotype on the levels of systemic plasma inflammatory mediators , we also compared inflammatory / immune infiltrate between patients bearing high responder genotype and those with other genotypes . according to encouraging data obtained on levels of systemic plasma inflammatory mediators , we assessed a higher inflammatory / immune infiltrate in tissue aorta samples from patients bearing high responder genotype than those bearing other genotypes and control aortas ( see figure 3 ) . in addition , the 46 patients with combined risk genotype showed for the 89% a typical morphological aorta 's phenotype , defined in our previous study as phenotype iii , characterized by elevated cystic md , plurifocal medial apoptosis , and increased mmp-9 amount ( as reported in online supplementary material ) . positive correlations were identified between the severity of histopatological abnormalities characterising this phenotype and hypertension , smoking , and age ( r = 0.179 , p = 0.03 ; r = 0.345 , p = 0.001 ; r = 0.267 , p = 0.02 , resp . consistent with these interesting data , we also evaluated whether the combined risk genotype can likely have a strong role in determining vascular senescence and onset of sporadic taa . to this purpose , we examined the mean of blood leukocyte telomere length using terminal restriction fragment assay ( trf test , a southern blot technique ) and blood samples from 30 patients and 30 controls , using a procedure described in two of our recent studies . a difference of 1.543 bp was observed between cases and controls ( 95% confidence interval 68 bp to 201 bp , p = 0.001 ) . comparisons between mean trf length and the risk taa factors including gender , smoking , hypertension , diabetes , and family history were also evaluated . sex as an independent risk factor did not have any significant effect on the mean trf length ( p = 0.76 ) . in contrast , smoking and hypertension in s - taa cases were the only risk factors which were significantly associated with the mean trf length ( p = 0.01 ) . stratifying the 30 patients having low mean trf length for combined risk genotype , we interestingly observed that 85% ( versus 8% for controls ) were carriers of combined risk genotype ( p = 0.001 by test , 2 2 table ; data not shown ) and had significant higher levels of plasma inflammatory mediators ( il-6 : 13.8 2.3 versus 5.1 1.4 , p < 0.001 ; tnf- : 15.5 1.4 versus 8.2 1.2 , p < 0.001 ; crp : 18.4 1.3 versus 6.6 2.8 , p < 0.0001 ; mmp-9 : 59.9 2.8 versus 11.6 1.8 , p < 0.0001 , and mmp-2 : 57.8 3.5 versus 13.7 1.9 , p < 0.0001 , resp . sporadic taa is predominantly a silent ailment , until rupture or dissection occur , and insidious in its onset and progression . in patients with taad , we observed high levels of immune / inflammatory cells and a significant association of some inflammatory polymorphisms with the taad susceptibility . in line with this , he and colleagues recently observed an increased immune / inflammatory infiltrate in aorta samples of patients with sporadic taa [ 14 , 15 ] . in the specific case of sporadic taa , recent experimental investigations in animal and ex vivo models also emphasise its role in the vascular aorta alterations ( vr and md ) and their complications , such as sporadic taa , by evocating or modulating increased expression and activation of endothelium dysfunction and remodeling aorta pathways [ 23 , 3742 ] . furthermore , song and colleagues demonstrated that signaling via tlr4-mediated signaling pathway and its signal adaptors , that is , myd88 , is responsible for the age - elevated basal il-6 response using vsmcs from aged tlr4 and myd88 mice . bucci and colleagues recently emphasized as the vascular thoracic aorta homeostasis and its alteration in rats is based on the activity of tlr4-mediated signaling pathway and its cross talk with other stress and stretch pathways , that is , ace , enos , and mmp pathways . furthermore , a recent study demonstrated in apolipoprotein e - deficient mice that it is possible to limit the inflammatory process by blocking tlr4/c - jun n terminal kinase signaling pathway with rosiglitazone in the initiation stages of aortic aneurysm development . this encouraging and increasing evidence , fruit prevalently of animal investigations , and our recent data on taad [ 16 , 46 , 48 ] , led us to analyze the potential role of genetic variants related to tlr4-mediated signaling pathway in the complex pathophysiology of sporadic taa . until now thus , our study represents the first report which , through a human ex vivo study approach , evidenced as some polymorphisms related to tlr4-mediated signaling pathway significantly modulate the sporadic taa risk : rs4986790 tlr4 , rs333 ccr5 , rs2070744 enos , rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 polymorphisms . among these , the rs4986790 ( + 896a > g ) tlr4 polymorphism confers a higher susceptibility for sporadic taa ( or = 14.4 , p = 0.0008 ) . however , it represents an independent risk taa factor for sporadic taa , as well as the rs1799752 ace , rs3918242 mmp-9 , and rs2285053 mmp-2 snps . a significant overrepresentation of their combined risk genotype ( + 896atlr4/dace/-1562tmmp-9/-735tmmp-2 ) was observed in cases than controls ( 46 versus 10 , p < 0.000009 ) by comparing it with frequency of other combinations . in addition , it was associated a significant risk for sporadic taa ( or = 4.7 ; p < 0.0001 ) . cases bearing combined risk genotype showed higher systemic inflammatory mediator levels than those with other genotypes and control carriers . a higher chronic inflammatory infiltrate was also found in cases bearing combined risk genotype , which positively correlated with histological abnormalities and levels of mediators . in addition , they showed in their tissue aorta samples a typical morphological phenotype , characterized by elevated cystic md , plurifocal medial apoptosis , and increased mmp-9 amounts , and defined in a previous study as phenotype iii . furthermore , we detected that combined risk genotype influences vascular biological ageing , evaluating the gold standard ageing marker , the telomere length , in a small number of cases and controls , selected randomly , but having the same age and gender . controls bearing high responder genotype showed higher levels of systemic mediators than control carriers of only the rs4986790 tlr4 polymorphism . indeed , in clarifying and confirming the biological effects of rs4986790 tlr4 polymorphism and its role in the pathophysiology of age - related diseases , including cvds , alzheimer disease , prostate cancer , diabetes , and longevity , we assessed the levels of il-6 , tnf- , il-10 , and eicosanoids in lps - stimulated whole blood samples in vitro of 50 young healthy sicilians , screened for the presence of rs4986790 tlr4 and 765g > significantly higher levels of both proinflammatory cytokines and eicosanoids were observed in individuals bearing the rs4986790 tlr4 polymorphism . thus , these data strengthen our suggestion , stressed for other age - related diseases including alzheimer disease and prostate cancer , that polymorphisms related to tlr4-mediated signaling pathway may have a major influence on the pathophysiology of a disease , such as sporadic taa , when they operate in combination to create a risk profile [ 56 , 57 ] . in the complex , our data seem to suggest the strong relevance of tlr4-mediated signaling pathway in inducing md , age related vr , and their complications , such as sporadic taa . on the other hand , our findings emphasise as a combined risk genotype associated of tlr4-mediated signaling pathway are able to modulate the grade of aorta age - related phenotypical , histological , and systemic abnormalities and consequently vascular aorta ageing , onset , and progression of sporadic taa . this might open new perspectives for the prevention of both aortic vr and md and sporadic taa , by using combined risk genotype ( + 896atlr4/dace/1562tmmp-9/735tmmp-2 ) as optimal genetic biomarker for the earlier detection of this silent pathology in preliminary phases and to treat with different and specific therapies depending on individual 's genotypes . in the specific case of sporadic taa , the therapeutic potential of tlr4-mediated signaling pathway might be defined through the use of its agonists or antagonists , whose effects have been prevalently experimented in mice , rats , and cultures as reported in literature . this finding reveals a novel aspect of the multiple - faced tlr-4-mediated signaling pathway biology , and it may open new prospects for using tlr4 agonists in promoting the production of epcs for clinical use . based on our findings , we also suggest another possible therapeutic intervention to apply in the preclinical phase in subject 's carriers of combined high risk genotype to the aim to retard to limit the onset and progression of the vascular ageing and its complications , such as sporadic taa . they should consent to translate with major emphasis our promising data in personalized treatments of a pathology , the sporadic taa , which clinically and predominantly is silent , until rupture or dissection occurs , and insidious in its onset and progression .

findings from many cross - sectional and longitudinal population studies have shown that diabetes is associated with higher prevalence of global cognitive impairment and an increased risk of dementia ( 14 ) . the underlying mechanisms of the relationship between diabetes and cerebral disease are still unclear ( 5 ) . however , brain imaging studies using mri have provided important insights into structural correlates of cognitive dysfunction in people with type 2 diabetes pointing to a convincing evidence for an association between diabetes and structural brain abnormalities such as cerebral atrophy and lacunar infarcts ( 610 ) . besides studying the possible physiological contribution of diabetes to late - age brain pathology , another important research topic , which has received much less attention , is the influence of diabetes disease management strategies on brain structure . the memory in diabetes ( mind ) substudy , embedded within the action to control cardiovascular risk in diabetes ( accord ) trial ( 11,12 ) , was designed to test the effects of early glycemic intervention on brain outcomes in older people with type 2 diabetes . previously published baseline results from the accord - mind trial have shown that lower levels of baseline cognitive performance are associated with higher levels of hba1c and diabetes duration ( 13 ) . however the primary mri analyses of the trial showed that although people in a intensive glycemic treatment group had less longitudinal decline in total brain volume at 40 months compared with those receiving a standard treatment strategy ( p = 0.0007 ) , no significant differences were found in the cognitive outcomes between the two treatment groups ( 14 ) . this difference in findings may reflect the effect of several factors with a basis ranging from methodological to biological . previous analyses investigated relatively global measures of brain volume changes , and possibly these changes were relatively restricted to regions mediating cognitive functions other than those tested in the trial . also possible is that treatment - related changes in brain volume loss precede cognitive changes and are therefore detected first . the current study investigated whether there are spatially heterogeneous patterns of structural brain changes that vary by diabetes characteristics and by glycemic treatment strategy . advanced computer - based imaging pattern analysis methods , including volume - preserving spatial normalization that allows for accurate quantification of very localized brain changes ( regional analysis of volumes examined in normalized space [ ravens ] ) ( 1517 ) and a new voxel - based analysis framework ( optimally - discriminative voxel - based analysis [ odvba ] ) ( 18 ) , were applied on longitudinal mri data from 488 accord - mind participants to detect imaging patterns that highlight potential spatial differences in the relationship between images and subject groups . our general hypothesis is that specific areas of the brain are particularly more vulnerable to diabetes effects . in this report we investigate diabetes duration and hba1c levels , two main clinical variables relevant to diabetes and shown to be related to cognition ( 13 ) . we also hypothesize that there are spatially specific patterns of longitudinal change that differ between the intensive and standard glycemic treatment arms and that those regions displaying longitudinal treatment effects will show similarities with those vulnerable to diabetes effects . accord , described in detail elsewhere ( 11 ) , was a randomized , multicenter , double two - by - two factorial parallel - treatment trial that tested the effect of treatment strategies to control blood glucose , blood pressure , and blood lipid concentrations on cardiovascular disease events . participants targeted by accord were aged 4579 years and had type 2 diabetes , high hba1c concentrations ( > 7.5% [ > 58 mmol / mol ] ) , and a high risk for cardiovascular disease events suggested by significant atherosclerosis , albuminuria , left ventricular hypertrophy , or at least two additional risk factors for cardiovascular disease . key exclusion criteria were frequent or recent serious hypoglycemic events , unwillingness to monitor glucose at home or inject insulin , bmi greater than 45 kg / m , serum creatinine level greater than 1.5 mg / dl ( 133 mol / l ) , or other serious illness . all accord participants were randomly assigned to receive intensive glycemic treatment targeting hba1c to less than 6.0% ( 42 mmol / mol ) or standard glycemic treatment targeting hba1c to 7.07.9% ( 5363 mmol / mol ) . the accord - mind study design has been described elsewhere ( 12 ) . from within the overall accord study population , 2,977 participants who had been randomly assigned to treatment groups were recruited into the accord - mind . a cognitive test battery was administered to mind participants at baseline and at 20 months and 40 months after randomization . the cognitive battery tested for verbal memory ( rey auditory learning test [ ravlt ] , mean number of recalled words over five trials ) , processing speed ( digit symbol substitution test [ dsst ] , number of cells correctly filled in ) , and executive function ( modified stroop test ( stroop ) , time to finish the interference challenge ) . a total of 614 participants had a successful baseline scan , from which 503 had a successful 40-month follow - up scan . after processing of image data , 15 participants were excluded from the analysis based on visual quality control ( qc ) due to insufficient quality of the final image maps . reasons for missing and excluded scans were similarly distributed across treatment groups ( supplementary table 1 ) . the treatment groups had similar baseline characteristics and cardiovascular risk factors ( supplementary table 2 ) . the standardized mri scan protocol ( 12 ) included axial , coronal , and sagittal gradient echo scout views that served as localizers ; a three - dimensional fast spoiled gradient - echo t1-weighted sequence ( repetition time [ tr ] , 21 ; flip angle , 30 ; echo time [ te ] , 8) to image brain structure , and a two - dimensional axial fast spin - echo ( fse ) fluid - attenuated inversion recovery ( tr , 8,000 ; inversion time , 2,000 ; te , 100 ) and proton - density / t2-weighted ( tr , 3,200 ; te1 , 27 ; te2 , 120 ) sequences to image pathology . voxel size was 1.5 0.9 0.9 mm for the three - dimensional t1 sequence and 3.0 0.9 0.9 mm for the two - dimensional sequences . scanners at the four mri sites had identical field strength ( 1.5 tesla ) but were from three different manufacturers . the procedures followed the american college of radiology s ( acr ) mri qc program , which is based on the analysis of data acquired from scanning an acr national electrical manufacturers association qc phantom . each field center was responsible for keeping its accord scanners within acr performance specifications and for sending monthly digital images to the mri qc center for in - house review . according to acr phantom analyses , mri scanner performance was stable across the mri sites and over the duration of the study . in vivo confirmation of scanner performance over time is reflected by the stability of subject intracranial volumes ( icv ) over time ( baseline mean icv , 1,132.34 cm ; follow - up mean icv , 1,132.32 cm ; p = 0.47 by paired t test ) . the preprocessing steps included 1 ) alignment to the anterior commissure posterior commissure plane ; 2 ) removal of extracranial material ( skull - stripping ) and cerebellum ; 3 ) n3 bias correction ( 19 ) ; 4 ) tissue segmentation into gray matter ( gm ) , white matter ( wm ) , cerebrospinal fluid ( csf ) , and lateral ventricles ( vn ) ( 20 ) ; 5 ) nonlinear image warping ( 21 ) to a common brain atlas using a montreal neurological institute template , as previously described ( 22 ) ; and 6 ) formation of regional volumetric maps , called ravens maps ( 1517 ) , using tissue - preserving image warping to enable comparative analysis of tissue volumes in the common template space . ravens map intensity values quantify the regional distribution of gm , wm , and ventricular csf , with one ravens map for each tissue type . in particular , ravens values in the template s ( stereotaxic ) space are directly proportional to the volume of the respective structures in the original brain scan . therefore , regional volumetric measurements and comparisons are performed by measurements and comparisons of the respective ravens maps . for example , patterns of relatively lower regional gm volume in the temporal lobe are quantified by patterns of ravens decrease in the temporal lobe in the template space . the ravens algorithm has been extensively validated and applied to a variety of studies ( 15,23 ) . the ravens maps were normalized by individual icv to adjust for global between - person differences in intracranial size and down - sampled to 2 2 2 mm . estimates of region - specific longitudinal volumetric change were obtained from the baseline and follow - up ravens maps by calculating the intensity difference at each voxel between the two time points . these rates of change are denoted as dravens . for the pattern analysis of the imaging data , we used the diabetes duration and hba1c levels at baseline as dependent variables for the cross - sectional analysis , and the glycemic intervention arm ( i.e. , standard or intensive treatment ) as the dependent variable for the longitudinal analysis . to investigate regional patterns of brain change associated with longitudinal change in cognition , an additional analysis was done using cognitive test scores as dependent variables . in our initial statistical analysis on baseline data using the standard voxel - based morphometry ( vbm ) approach , we identified trends for diabetes duration ( e.g. , regions with significance p < 0.01 ) , but no regions showed significance after false discovery rate ( fdr ) correction ( 24 ) using a significance threshold of q 0.05 . accordingly , we applied a new voxel - based analysis methodology , odvba ( 18 ) , taking into account the superiority of odvba to the classical vbm approach in sensitivity and spatial specificity , which has been demonstrated on both structural and functional mri ( 25,26 ) . to leverage the strengths of odvba , which is a discriminative method , the values for diabetes duration and hba1c were dichotomized and used in the analysis as class variables by grouping the subjects with values lower than the 20th percentile and with values higher than the 80th percentile of each variable into the low and high groups , respectively ( 27 ) the low diabetes duration group included subjects with 04 years of diabetes ( average 2.5 1.3 ) , compared with 1540 years ( average 21 6.3 ) for the high diabetes duration group . participants in the low hba1c group had hba1c values between 5.5% ( 37 mmol / mol ) and 7.4% ( 57 mmol / mol ) , with an average 7.1% 0.3% ( 54 3 mmol / mol ) , whereas the high hba1c group had values between 8.8% ( 73 mmol / mol ) and 11.8% ( 105 mmol / mol ) , with an average 9.6% 0.7% ( 81 8 mmol / mol ) . in the cross - sectional analysis , odvba was applied on baseline imaging values of gm , wm , and vn ravens maps . before applying odvba , ravens maps were corrected for age , sex , and systolic blood pressure by fitting a voxel - by - voxel generalized linear model to the ravens values against the covariates and by calculating the residual of the regression at each voxel . odvba constructed a voxel - by - voxel image of significance derived from the optimized spatial adaptive filtering applied to the data , which highlights potential spatial differences in the relationship between images and subject groups . because many statistical tests ( i.e. , one on each individual voxel ) were being conducted , the final maps were corrected for multiple comparisons using fdr correction . the clusters that exceeded a significance threshold of q 0.05 after correction for multiple comparisons are reported . in the longitudinal analysis , odvba was applied on gm , wm , and vn dravens maps , which capture longitudinal rate of regional change in tissue volume in a given subject s brain at each voxel , for detecting imaging patterns of volume change associated with group differences between subjects in intensive and standard glycemic treatment arms . fdr - corrected values are reported at a significance level of q 0.05 . for the analysis of cognition , the longitudinal rate of change of each participant in each cognitive test was estimated by linear regression using baseline , 20-month , and 40-month test scores for dsst , ravlt , and stroop . the subjects were then stratified into declining and nondeclining cognition categories based on the 20th and 80th percentiles , similar to what was done for diabetes duration and hba1c . odvba was applied using gm , wm , and vn dravens maps of participants in the two groups for each test . accord , described in detail elsewhere ( 11 ) , was a randomized , multicenter , double two - by - two factorial parallel - treatment trial that tested the effect of treatment strategies to control blood glucose , blood pressure , and blood lipid concentrations on cardiovascular disease events . participants targeted by accord were aged 4579 years and had type 2 diabetes , high hba1c concentrations ( > 7.5% [ > 58 mmol / mol ] ) , and a high risk for cardiovascular disease events suggested by significant atherosclerosis , albuminuria , left ventricular hypertrophy , or at least two additional risk factors for cardiovascular disease . key exclusion criteria were frequent or recent serious hypoglycemic events , unwillingness to monitor glucose at home or inject insulin , bmi greater than 45 kg / m , serum creatinine level greater than 1.5 mg / dl ( 133 mol / l ) , or other serious illness . all accord participants were randomly assigned to receive intensive glycemic treatment targeting hba1c to less than 6.0% ( 42 mmol / mol ) or standard glycemic treatment targeting hba1c to 7.07.9% ( 5363 mmol / mol ) . the accord - mind study design has been described elsewhere ( 12 ) . from within the overall accord study population , 2,977 participants who had been randomly assigned to treatment groups were recruited into the accord - mind . a cognitive test battery was administered to mind participants at baseline and at 20 months and 40 months after randomization . the cognitive battery tested for verbal memory ( rey auditory learning test [ ravlt ] , mean number of recalled words over five trials ) , processing speed ( digit symbol substitution test [ dsst ] , number of cells correctly filled in ) , and executive function ( modified stroop test ( stroop ) , time to finish the interference challenge ) . a total of 614 participants had a successful baseline scan , from which 503 had a successful 40-month follow - up scan . after processing of image data , 15 participants were excluded from the analysis based on visual quality control ( qc ) due to insufficient quality of the final image maps . reasons for missing and excluded scans were similarly distributed across treatment groups ( supplementary table 1 ) . the treatment groups had similar baseline characteristics and cardiovascular risk factors ( supplementary table 2 ) . the standardized mri scan protocol ( 12 ) included axial , coronal , and sagittal gradient echo scout views that served as localizers ; a three - dimensional fast spoiled gradient - echo t1-weighted sequence ( repetition time [ tr ] , 21 ; flip angle , 30 ; echo time [ te ] , 8) to image brain structure , and a two - dimensional axial fast spin - echo ( fse ) fluid - attenuated inversion recovery ( tr , 8,000 ; inversion time , 2,000 ; te , 100 ) and proton - density / t2-weighted ( tr , 3,200 ; te1 , 27 ; te2 , 120 ) sequences to image pathology . voxel size was 1.5 0.9 0.9 mm for the three - dimensional t1 sequence and 3.0 0.9 0.9 mm for the two - dimensional sequences . scanners at the four mri sites had identical field strength ( 1.5 tesla ) but were from three different manufacturers . the procedures followed the american college of radiology s ( acr ) mri qc program , which is based on the analysis of data acquired from scanning an acr national electrical manufacturers association qc phantom . each field center was responsible for keeping its accord scanners within acr performance specifications and for sending monthly digital images to the mri qc center for in - house review . according to acr phantom analyses , mri scanner performance was stable across the mri sites and over the duration of the study . in vivo confirmation of scanner performance over time is reflected by the stability of subject intracranial volumes ( icv ) over time ( baseline mean icv , 1,132.34 cm ; follow - up mean icv , 1,132.32 cm ; p = 0.47 by paired t test ) . the preprocessing steps included 1 ) alignment to the anterior commissure posterior commissure plane ; 2 ) removal of extracranial material ( skull - stripping ) and cerebellum ; 3 ) n3 bias correction ( 19 ) ; 4 ) tissue segmentation into gray matter ( gm ) , white matter ( wm ) , cerebrospinal fluid ( csf ) , and lateral ventricles ( vn ) ( 20 ) ; 5 ) nonlinear image warping ( 21 ) to a common brain atlas using a montreal neurological institute template , as previously described ( 22 ) ; and 6 ) formation of regional volumetric maps , called ravens maps ( 1517 ) , using tissue - preserving image warping to enable comparative analysis of tissue volumes in the common template space . ravens map intensity values quantify the regional distribution of gm , wm , and ventricular csf , with one ravens map for each tissue type . in particular , ravens values in the template s ( stereotaxic ) space are directly proportional to the volume of the respective structures in the original brain scan . therefore , regional volumetric measurements and comparisons are performed by measurements and comparisons of the respective ravens maps . for example , patterns of relatively lower regional gm volume in the temporal lobe are quantified by patterns of ravens decrease in the temporal lobe in the template space . the ravens algorithm has been extensively validated and applied to a variety of studies ( 15,23 ) . the ravens maps were normalized by individual icv to adjust for global between - person differences in intracranial size and down - sampled to 2 2 2 mm . estimates of region - specific longitudinal volumetric change were obtained from the baseline and follow - up ravens maps by calculating the intensity difference at each voxel between the two time points . for the pattern analysis of the imaging data , we used the diabetes duration and hba1c levels at baseline as dependent variables for the cross - sectional analysis , and the glycemic intervention arm ( i.e. , standard or intensive treatment ) as the dependent variable for the longitudinal analysis . to investigate regional patterns of brain change associated with longitudinal change in cognition , an additional analysis was done using cognitive test scores as dependent variables . in our initial statistical analysis on baseline data using the standard voxel - based morphometry ( vbm ) approach , we identified trends for diabetes duration ( e.g. , regions with significance p < 0.01 ) , but no regions showed significance after false discovery rate ( fdr ) correction ( 24 ) using a significance threshold of q 0.05 . accordingly , we applied a new voxel - based analysis methodology , odvba ( 18 ) , taking into account the superiority of odvba to the classical vbm approach in sensitivity and spatial specificity , which has been demonstrated on both structural and functional mri ( 25,26 ) . to leverage the strengths of odvba , which is a discriminative method , the values for diabetes duration and hba1c were dichotomized and used in the analysis as class variables by grouping the subjects with values lower than the 20th percentile and with values higher than the 80th percentile of each variable into the the low diabetes duration group included subjects with 04 years of diabetes ( average 2.5 1.3 ) , compared with 1540 years ( average 21 6.3 ) for the high diabetes duration group . participants in the low hba1c group had hba1c values between 5.5% ( 37 mmol / mol ) and 7.4% ( 57 mmol / mol ) , with an average 7.1% 0.3% ( 54 3 mmol / mol ) , whereas the high hba1c group had values between 8.8% ( 73 mmol / mol ) and 11.8% ( 105 mmol / mol ) , with an average 9.6% 0.7% ( 81 8 mmol / mol ) . in the cross - sectional analysis , odvba was applied on baseline imaging values of gm , wm , and vn ravens maps . before applying odvba , ravens maps were corrected for age , sex , and systolic blood pressure by fitting a voxel - by - voxel generalized linear model to the ravens values against the covariates and by calculating the residual of the regression at each voxel . odvba constructed a voxel - by - voxel image of significance derived from the optimized spatial adaptive filtering applied to the data , which highlights potential spatial differences in the relationship between images and subject groups . because many statistical tests ( i.e. , one on each individual voxel ) were being conducted , the final maps were corrected for multiple comparisons using fdr correction . the clusters that exceeded a significance threshold of q 0.05 after correction for multiple comparisons are reported . in the longitudinal analysis , odvba was applied on gm , wm , and vn dravens maps , which capture longitudinal rate of regional change in tissue volume in a given subject s brain at each voxel , for detecting imaging patterns of volume change associated with group differences between subjects in intensive and standard glycemic treatment arms . fdr - corrected values are reported at a significance level of q 0.05 . for the analysis of cognition , the longitudinal rate of change of each participant in each cognitive test was estimated by linear regression using baseline , 20-month , and 40-month test scores for dsst , ravlt , and stroop . the subjects were then stratified into declining and nondeclining cognition categories based on the 20th and 80th percentiles , similar to what was done for diabetes duration and hba1c . odvba was applied using gm , wm , and vn dravens maps of participants in the two groups for each test . regional pattern analysis using odvba was applied on baseline gm , wm , and vn ravens maps , which were corrected for age , sex , and systolic blood pressure , to identify group differences between subject categories for low and high values of diabetes duration and hba1c . the baseline characteristics of participants with low and high diabetes duration are given in table 1 . longer diabetes duration ( more than 14 years ) , compared with relatively short duration of diabetes ( less than 5 years ) , was correlated with a relatively lower regional gm volume , particularly in the frontal lobes , left temporal lobe , right parietal lobe , and limbic cortex . the specific regions that were affected were the middle frontal gyrus , precentral gyrus , and inferior frontal gyrus left and right ; middle temporal gyrus , inferior temporal gyrus , and lateral occipitotemporal gyrus left ; angular gyrus right ; and parietal cingulate region right and left . the areas of significant group differences between gm ravens maps and short versus long diabetes duration after fdr correction ( q < 0.05 ) are shown in fig . a trend of positive association ( p < 0.05 ) of lateral ventricular enlargement and diabetes duration was detected , but no voxels survived the fdr correction ( supplementary fig . baseline characteristics comparing low diabetes duration and high diabetes duration groups data are mean sd or n ( % ) . mmse , mini - mental state examination . * between short - duration and long - duration groups . * * defined as patient health questionnaire score > 10 . a : gm ravens maps in relationship with short vs. long diabetes duration at baseline . subjects with long diabetes duration ( n = 100 ) had lower ravens values ( i.e. , lower regional gm volume , in the highlighted areas ) compared with subjects with short diabetes duration ( n = 100 ) . subjects in the intensive treatment arm ( n = 221 ) had lower longitudinal decrease in gm tissue volume in the highlighted areas compared with subjects in the standard treatment arm ( n = 267 ) . the green color indicates the detected significant regions with fdr - corrected q < 0.05 . the hot color indicates the trends toward significance characterized by the log ( p ) values shown in the color bar . there was no statistically significant correlation of low versus high baseline hba1c levels to the gm , wm , and vn ravens maps . the longitudinal analysis using odvba has been applied for identifying group differences in regional volume loss patterns between subjects in the intensive glycemic treatment arm and those in the standard treatment arm . estimates of region - specific longitudinal volumetric change were obtained from the baseline and follow - up ravens maps by calculating the intensity difference at each voxel between the two time points . intensive treatment preserved gm volume more than the standard treatment ( fdr - corrected q < 0.05 ) in certain cortical areas , specifically in the left and right superior temporal gyrus , left pre- and postcentral gyri , left and right medial front - orbital gyrus , and left and right frontal cingulate regions ( fig . 1b ) . there was a partial overlap between the areas associated with treatment differences and those associated with diabetes duration . importantly , the regions that showed longitudinal differences were mostly adjacent to regions displaying significant cross - sectional relationship with diabetes duration and tended to show just a trend at baseline . summary statistics of cognitive decline and nondecline categories for each cognitive test are given in table 2 . a positive association between higher gm volume loss in certain cortical areas of the brain and cognitive decline was detected for all three cognitive tests ; however , after fdr correction , significant group differences were found only for ravlt ( fig . 2 ) . odvba detected trends in right occipitotemporal regions for dsst , a global cortical trend for ravlt with significant effects on frontotemporal regions , and trends mostly in parietal regions for stroop . summary statistics of cognitive categories included in the pattern analysis * change in score per month , estimated by linear regression to baseline , 20-month , and 40-month test scores . * * contrary to dsst and ravlt , a higher score in stroop test means lower performance . three - dimensional surface renderings of odvba results for longitudinal change in cognitive test scores . group differences on gm dravens maps between the group with declining cognition ( n = 100 ) vs. nondeclining cognition ( n = 100 ) are shown for dsst ( a ) , ravlt ( b ) , and stroop ( c ) tests . for each three tests , subjects in the group with declining cognition had a higher longitudinal decrease in gm volume in the highlighted areas compared with subjects in the group with nondeclining cognition . the green color indicates the detected significant regions with fdr - corrected q < 0.05 . the hot color indicates the trends toward significance characterized by the log ( p ) values shown in the color bar . regional pattern analysis using odvba was applied on baseline gm , wm , and vn ravens maps , which were corrected for age , sex , and systolic blood pressure , to identify group differences between subject categories for low and high values of diabetes duration and hba1c . the baseline characteristics of participants with low and high diabetes duration are given in table 1 . longer diabetes duration ( more than 14 years ) , compared with relatively short duration of diabetes ( less than 5 years ) , was correlated with a relatively lower regional gm volume , particularly in the frontal lobes , left temporal lobe , right parietal lobe , and limbic cortex . the specific regions that were affected were the middle frontal gyrus , precentral gyrus , and inferior frontal gyrus left and right ; middle temporal gyrus , inferior temporal gyrus , and lateral occipitotemporal gyrus left ; angular gyrus right ; and parietal cingulate region right and left . the areas of significant group differences between gm ravens maps and short versus long diabetes duration after fdr correction ( q < 0.05 ) are shown in fig . a trend of positive association ( p < 0.05 ) of lateral ventricular enlargement and diabetes duration was detected , but no voxels survived the fdr correction ( supplementary fig . baseline characteristics comparing low diabetes duration and high diabetes duration groups data are mean sd or n ( % ) . mmse , mini - mental state examination . * between short - duration and long - duration groups . * * defined as patient health questionnaire score > 10 . a : gm ravens maps in relationship with short vs. long diabetes duration at baseline . subjects with long diabetes duration ( n = 100 ) had lower ravens values ( i.e. , lower regional gm volume , in the highlighted areas ) compared with subjects with short diabetes duration ( n = 100 ) . subjects in the intensive treatment arm ( n = 221 ) had lower longitudinal decrease in gm tissue volume in the highlighted areas compared with subjects in the standard treatment arm ( n = 267 ) . the green color indicates the detected significant regions with fdr - corrected q < 0.05 . the hot color indicates the trends toward significance characterized by the log ( p ) values shown in the color bar . there was no statistically significant correlation of low versus high baseline hba1c levels to the gm , wm , and vn ravens maps . the longitudinal analysis using odvba has been applied for identifying group differences in regional volume loss patterns between subjects in the intensive glycemic treatment arm and those in the standard treatment arm . estimates of region - specific longitudinal volumetric change were obtained from the baseline and follow - up ravens maps by calculating the intensity difference at each voxel between the two time points . intensive treatment preserved gm volume more than the standard treatment ( fdr - corrected q < 0.05 ) in certain cortical areas , specifically in the left and right superior temporal gyrus , left pre- and postcentral gyri , left and right medial front - orbital gyrus , and left and right frontal cingulate regions ( fig . there was a partial overlap between the areas associated with treatment differences and those associated with diabetes duration . importantly , the regions that showed longitudinal differences were mostly adjacent to regions displaying significant cross - sectional relationship with diabetes duration and tended to show just a trend at baseline . summary statistics of cognitive decline and nondecline categories for each cognitive test are given in table 2 . a positive association between higher gm volume loss in certain cortical areas of the brain and cognitive decline was detected for all three cognitive tests ; however , after fdr correction , significant group differences were found only for ravlt ( fig . 2 ) . odvba detected trends in right occipitotemporal regions for dsst , a global cortical trend for ravlt with significant effects on frontotemporal regions , and trends mostly in parietal regions for stroop . summary statistics of cognitive categories included in the pattern analysis * change in score per month , estimated by linear regression to baseline , 20-month , and 40-month test scores . * * contrary to dsst and ravlt , a higher score in stroop test means lower performance . three - dimensional surface renderings of odvba results for longitudinal change in cognitive test scores . group differences on gm dravens maps between the group with declining cognition ( n = 100 ) vs. nondeclining cognition ( n = 100 ) are shown for dsst ( a ) , ravlt ( b ) , and stroop ( c ) tests . for each three tests , subjects in the group with declining cognition had a higher longitudinal decrease in gm volume in the highlighted areas compared with subjects in the group with nondeclining cognition . the green color indicates the detected significant regions with fdr - corrected q < 0.05 . the hot color indicates the trends toward significance characterized by the log ( p ) values shown in the color bar . to the best of our knowledge , this is the first study to use pattern analysis methods to investigate the spatial specificity of patterns of brain volume loss in relation to diabetes duration and hba1c . moreover , this is the first study to apply these methods to investigate the spatial patterns of treatment effects on brain structure in a large diabetes clinical trial and using state - of - the - art pattern analysis methodology . we applied a new vbm approach , odvba ( 18 ) , in this analysis . in classical vbm methods , gaussian smoothing of images , which is applied to account for registration errors and to integrate imaging signals from a region , has also become a limitation of these methods , because it is often chosen empirically and lacks spatial adaptivity to the shape and spatial extent of the region of interest , such as a region of volume loss . in contrast , odvba , using machine - learning techniques on local image neighborhoods , determines the spatially adaptive smoothing kernel whose coefficients define the optimally discriminative direction between two groups ( e.g. , patients and control subjects ) . information from all neighborhoods that contain a given voxel is then composed to produce the statistic for each voxel , and permutation tests are used to obtain a statistical parametric map of group differences . experimental results on three sets of previously published data from studies in schizophrenia , mild cognitive impairment , and alzheimer disease suggest that odvba is considerably more sensitive in detecting group differences and performs better than classical vbm methods in the spatial extent of detected area and agreement of anatomical boundary ( 25 ) . we found , at baseline , that participants with longer diabetes duration had significantly reduced gm volumes in a number of brain regions . the cortical patterns of decreased gm volume showed similarities with previously reported regions in mri studies comparing diabetic and normal participants ( 8,9,2836 ) . in a similar approach , brundel et al . ( 9 ) detected region - specific group differences between healthy controls and patients using cortical area and volume and thickness values , imaging measures complementary to those we used in our analysis . consistent with our findings , they concluded that the cortical atrophy in type 2 diabetic patients was not equally distributed across the entire brain but showed spatially specific patterns . also , detected patterns for the change , particularly in cortical area and volume , largely overlapped with those found in our analysis , showing large effects in middle temporal and frontal areas . in contrast to our findings , this study reported smaller cortical thickness values for diabetic patients in the hippocampal region . however , the authors noted that separating gm and wm in the hippocampal region with their present technique is difficult , which may affect the reliability of the cortical thickness and volume measurements . previous studies also reported enlarged vns in diabetic patients compared with healthy controls ( 37,38 ) . although we lost significance after fdr correction , our uncorrected significance values ( p < 0.05 ) showed a trend of positive association of lateral vn enlargement and diabetes duration ( supplementary fig . an important finding is that intensive glycemic treatment displays relative preservation of some but not all brain regions . the finding of a differential treatment effect is consistent with previous findings ( 14 ) that preservation of the brain volume was significantly better in the participants in the intensive treatment arm during the 40-month follow - up period compared with the participants in the standard arm . the current analyses go beyond the volumetric measures by providing information on how the volume loss is spatially distributed and , hence , provides more information on how the disease and the intervention interact to produce the outcomes . we found a heterogeneous pattern affecting specific brain regions to a greater degree than others . in particular , the strongest treatment effects were found in brain regions around the sylvian fissure as well in the medial - frontal cortex . interestingly , the regions displaying a differential treatment effect of reduced gm volume loss were adjacent to but almost nonoverlapping with those regions that displayed significant association with diabetes duration at baseline . therefore , intensive treatment appears to slow the spatial spreading of gm volume loss from regions mostly affected by diabetes duration toward adjacent brain regions . additional studies must be performed , potentially by using individualized longitudinal analyses , to better measure this apparent slowing of spatial progression interestingly , the areas that were associated to change in cognition were different from those affected from the treatment , which may explain the previous finding that there were no significant differences in the cognitive outcomes between the two treatment groups ( 14 ) . nonetheless , areas detected for ravlt , although showing a global cortical effect , have also shown partial overlap with areas affected from the treatment as well as with areas affected from diabetes duration . verbal learning tests have been considered a useful tool for the early diagnosis of cognitive decline and alzheimer disease ( 39,40 ) . as such , these overlapping regions might be particularly important for investigating the relationship between diabetes , its treatment , and dementia . neuropathological correlates of the macroscopic volumetric reduction are heterogeneous and can include not only neuronal loss but also cortical thinning , subcortical vascular pathology , wm rarefaction , or other reasons ( 9 ) . results from animal models ( 41,42 ) have presented the associations of diabetes with neuronal changes in specific brain regions , with the aim of understanding underlying mechanisms of structural brain changes . however , these mechanisms are still unclear , as findings propose the involvement of multiple factors in the development of diabetes related brain changes ( 43 ) . consequently , the etiology of potentially relatively higher vulnerability of certain brain regions to diabetes duration can not be elucidated by the current study and might relate to the differences in the underlying neuronal types and their sensitivity to insulin , underlying effects of diabetes in brain tissue perfusion , or might be effects of insulin resistance and its potential effect on brain connections . a growing body of work during the past decade has demonstrated that insulin - sensitive glucose transporters are localized to the same regions supporting memory , suggesting that insulin and its metabolism may contribute to normal cognitive functioning and that insulin abnormalities may exacerbate cognitive impairments , such as those associated with type 2 diabetes and even alzheimer disease . the regional differences identified here in the cross - sectional and longitudinal analyses are similar to some of those brain regions associated with cognitive health ( 44,45 ) . no brain region was found to display significant correlation with low versus high hba1c . that no brain region was significantly correlated with hba1c levels was against our initial expectations , which were based on the central diagnostic / disease management role of hba1c in diabetes and on earlier findings in the larger sample participating in the cognitive component of accord - mind ( 13 ) . there , we found an association of increasing hba1c and reduced performance on the cognitive tests of speed , memory , and executive function . because the mri sample was smaller than the cognitive sample , we may not have had enough power to detect differences in a measure such as hba1c level , which only reflect blood glucose levels during the relatively short period of 3 months , whereas diabetes duration reflects the cumulative effect of the disease over years . however , we can not rule out that other pathologic conditions associated with diabetes , such as hypertension , might have a relatively more deleterious effect on the brain than glucose levels . finally , hypoglycemia might be associated with physiologic ( e.g. , perfusion ) but not structural brain changes . our findings on the relationship between diabetes duration and extent of brain volume loss are relatively conservative because they were obtained after correcting for age . diabetes duration and age were somewhat correlated ( r = 0.216 ) , albeit not very strongly . new studies , including nondiabetic control subjects , would be necessary to allow us to correct only for patterns of brain volume loss explained by normal aging and independently from diabetes duration . several factors might have contributed to the dissociation between the treatment effects on brain structure and on cognition ( launer et al cognitive tests are known to be variable and subject to the learning effect ; hence , imaging measurements , especially the pattern analysis adopted in this study , might be able to detect more subtle effects . moreover , brain changes are likely to precede cognitive changes , which would further explain this dissociation . however , our results suggest a spatially heterogeneous effect of diabetes duration , as well as of its treatment , thereby pointing the way to future refinement of cognitive tests to tease out functions mediated by those brain regions that are more vulnerable to diabetes and more responsive to treatment .

objectiveunderstanding the effect of diabetes as well as of alternative treatment strategies on cerebral structure is critical for the development of targeted interventions against accelerated neurodegeneration in type 2 diabetes . we investigated whether diabetes characteristics were associated with spatially specific patterns of brain changes and whether those patterns were affected by intensive versus standard glycemic treatment.research design and methodsusing baseline mris of 488 participants with type 2 diabetes from the action to control cardiovascular risk in diabetes - memory in diabetes ( accord - mind ) study , we applied a new voxel - based analysis methodology to identify spatially specific patterns of gray matter and white matter volume loss related to diabetes duration and hba1c . the longitudinal analysis used 40-month follow - up data to evaluate differences in progression of volume loss between intensive and standard glycemic treatment arms.resultsparticipants with longer diabetes duration had significantly lower gray matter volumes , primarily in certain regions in the frontal and temporal lobes . the longitudinal analysis of treatment effects revealed a heterogeneous pattern of decelerated loss of gray matter volume associated with intensive glycemic treatment . intensive treatment decelerated volume loss , particularly in regions adjacent to those cross - sectionally associated with diabetes duration . no significant relationship between low versus high baseline hba1c levels and brain changes was found . finally , regions in which cognitive change was associated with longitudinal volume loss had only small overlap with regions related to diabetes duration and to treatment effects.conclusionsapplying advanced quantitative image pattern analysis methods on longitudinal mri data of a large sample of patients with type 2 diabetes , we demonstrate that there are spatially specific patterns of brain changes that vary by diabetes characteristics and that the progression of gray matter volume loss is slowed by intensive glycemic treatment , particularly in regions adjacent to areas affected by diabetes .

Introduction Research Design and Methods Participants MRI Scan Protocol Image Processing Statistical Analyses Results Patterns of Regional Volume Change Associated With Diabetes Characteristics Patterns of Regional Volume Change Associated With Glycemia Treatment Patterns of Regional Volume Change Associated With Longitudinal Change in Cognition Conclusions Supplementary Material

however , brain imaging studies using mri have provided important insights into structural correlates of cognitive dysfunction in people with type 2 diabetes pointing to a convincing evidence for an association between diabetes and structural brain abnormalities such as cerebral atrophy and lacunar infarcts ( 610 ) . the memory in diabetes ( mind ) substudy , embedded within the action to control cardiovascular risk in diabetes ( accord ) trial ( 11,12 ) , was designed to test the effects of early glycemic intervention on brain outcomes in older people with type 2 diabetes . previously published baseline results from the accord - mind trial have shown that lower levels of baseline cognitive performance are associated with higher levels of hba1c and diabetes duration ( 13 ) . however the primary mri analyses of the trial showed that although people in a intensive glycemic treatment group had less longitudinal decline in total brain volume at 40 months compared with those receiving a standard treatment strategy ( p = 0.0007 ) , no significant differences were found in the cognitive outcomes between the two treatment groups ( 14 ) . the current study investigated whether there are spatially heterogeneous patterns of structural brain changes that vary by diabetes characteristics and by glycemic treatment strategy . advanced computer - based imaging pattern analysis methods , including volume - preserving spatial normalization that allows for accurate quantification of very localized brain changes ( regional analysis of volumes examined in normalized space [ ravens ] ) ( 1517 ) and a new voxel - based analysis framework ( optimally - discriminative voxel - based analysis [ odvba ] ) ( 18 ) , were applied on longitudinal mri data from 488 accord - mind participants to detect imaging patterns that highlight potential spatial differences in the relationship between images and subject groups . in this report we investigate diabetes duration and hba1c levels , two main clinical variables relevant to diabetes and shown to be related to cognition ( 13 ) . we also hypothesize that there are spatially specific patterns of longitudinal change that differ between the intensive and standard glycemic treatment arms and that those regions displaying longitudinal treatment effects will show similarities with those vulnerable to diabetes effects . accord , described in detail elsewhere ( 11 ) , was a randomized , multicenter , double two - by - two factorial parallel - treatment trial that tested the effect of treatment strategies to control blood glucose , blood pressure , and blood lipid concentrations on cardiovascular disease events . participants targeted by accord were aged 4579 years and had type 2 diabetes , high hba1c concentrations ( > 7.5% [ > 58 mmol / mol ] ) , and a high risk for cardiovascular disease events suggested by significant atherosclerosis , albuminuria , left ventricular hypertrophy , or at least two additional risk factors for cardiovascular disease . all accord participants were randomly assigned to receive intensive glycemic treatment targeting hba1c to less than 6.0% ( 42 mmol / mol ) or standard glycemic treatment targeting hba1c to 7.07.9% ( 5363 mmol / mol ) . from within the overall accord study population , 2,977 participants who had been randomly assigned to treatment groups were recruited into the accord - mind . a total of 614 participants had a successful baseline scan , from which 503 had a successful 40-month follow - up scan . the preprocessing steps included 1 ) alignment to the anterior commissure posterior commissure plane ; 2 ) removal of extracranial material ( skull - stripping ) and cerebellum ; 3 ) n3 bias correction ( 19 ) ; 4 ) tissue segmentation into gray matter ( gm ) , white matter ( wm ) , cerebrospinal fluid ( csf ) , and lateral ventricles ( vn ) ( 20 ) ; 5 ) nonlinear image warping ( 21 ) to a common brain atlas using a montreal neurological institute template , as previously described ( 22 ) ; and 6 ) formation of regional volumetric maps , called ravens maps ( 1517 ) , using tissue - preserving image warping to enable comparative analysis of tissue volumes in the common template space . estimates of region - specific longitudinal volumetric change were obtained from the baseline and follow - up ravens maps by calculating the intensity difference at each voxel between the two time points . for the pattern analysis of the imaging data , we used the diabetes duration and hba1c levels at baseline as dependent variables for the cross - sectional analysis , and the glycemic intervention arm ( i.e. , standard or intensive treatment ) as the dependent variable for the longitudinal analysis . to investigate regional patterns of brain change associated with longitudinal change in cognition , an additional analysis was done using cognitive test scores as dependent variables . in our initial statistical analysis on baseline data using the standard voxel - based morphometry ( vbm ) approach , we identified trends for diabetes duration ( e.g. accordingly , we applied a new voxel - based analysis methodology , odvba ( 18 ) , taking into account the superiority of odvba to the classical vbm approach in sensitivity and spatial specificity , which has been demonstrated on both structural and functional mri ( 25,26 ) . to leverage the strengths of odvba , which is a discriminative method , the values for diabetes duration and hba1c were dichotomized and used in the analysis as class variables by grouping the subjects with values lower than the 20th percentile and with values higher than the 80th percentile of each variable into the low and high groups , respectively ( 27 ) the low diabetes duration group included subjects with 04 years of diabetes ( average 2.5 1.3 ) , compared with 1540 years ( average 21 6.3 ) for the high diabetes duration group . odvba constructed a voxel - by - voxel image of significance derived from the optimized spatial adaptive filtering applied to the data , which highlights potential spatial differences in the relationship between images and subject groups . in the longitudinal analysis , odvba was applied on gm , wm , and vn dravens maps , which capture longitudinal rate of regional change in tissue volume in a given subject s brain at each voxel , for detecting imaging patterns of volume change associated with group differences between subjects in intensive and standard glycemic treatment arms . for the analysis of cognition , the longitudinal rate of change of each participant in each cognitive test was estimated by linear regression using baseline , 20-month , and 40-month test scores for dsst , ravlt , and stroop . the subjects were then stratified into declining and nondeclining cognition categories based on the 20th and 80th percentiles , similar to what was done for diabetes duration and hba1c . accord , described in detail elsewhere ( 11 ) , was a randomized , multicenter , double two - by - two factorial parallel - treatment trial that tested the effect of treatment strategies to control blood glucose , blood pressure , and blood lipid concentrations on cardiovascular disease events . participants targeted by accord were aged 4579 years and had type 2 diabetes , high hba1c concentrations ( > 7.5% [ > 58 mmol / mol ] ) , and a high risk for cardiovascular disease events suggested by significant atherosclerosis , albuminuria , left ventricular hypertrophy , or at least two additional risk factors for cardiovascular disease . all accord participants were randomly assigned to receive intensive glycemic treatment targeting hba1c to less than 6.0% ( 42 mmol / mol ) or standard glycemic treatment targeting hba1c to 7.07.9% ( 5363 mmol / mol ) . from within the overall accord study population , 2,977 participants who had been randomly assigned to treatment groups were recruited into the accord - mind . a total of 614 participants had a successful baseline scan , from which 503 had a successful 40-month follow - up scan . the preprocessing steps included 1 ) alignment to the anterior commissure posterior commissure plane ; 2 ) removal of extracranial material ( skull - stripping ) and cerebellum ; 3 ) n3 bias correction ( 19 ) ; 4 ) tissue segmentation into gray matter ( gm ) , white matter ( wm ) , cerebrospinal fluid ( csf ) , and lateral ventricles ( vn ) ( 20 ) ; 5 ) nonlinear image warping ( 21 ) to a common brain atlas using a montreal neurological institute template , as previously described ( 22 ) ; and 6 ) formation of regional volumetric maps , called ravens maps ( 1517 ) , using tissue - preserving image warping to enable comparative analysis of tissue volumes in the common template space . estimates of region - specific longitudinal volumetric change were obtained from the baseline and follow - up ravens maps by calculating the intensity difference at each voxel between the two time points . for the pattern analysis of the imaging data , we used the diabetes duration and hba1c levels at baseline as dependent variables for the cross - sectional analysis , and the glycemic intervention arm ( i.e. , standard or intensive treatment ) as the dependent variable for the longitudinal analysis . to investigate regional patterns of brain change associated with longitudinal change in cognition , an additional analysis was done using cognitive test scores as dependent variables . in our initial statistical analysis on baseline data using the standard voxel - based morphometry ( vbm ) approach , we identified trends for diabetes duration ( e.g. accordingly , we applied a new voxel - based analysis methodology , odvba ( 18 ) , taking into account the superiority of odvba to the classical vbm approach in sensitivity and spatial specificity , which has been demonstrated on both structural and functional mri ( 25,26 ) . to leverage the strengths of odvba , which is a discriminative method , the values for diabetes duration and hba1c were dichotomized and used in the analysis as class variables by grouping the subjects with values lower than the 20th percentile and with values higher than the 80th percentile of each variable into the the low diabetes duration group included subjects with 04 years of diabetes ( average 2.5 1.3 ) , compared with 1540 years ( average 21 6.3 ) for the high diabetes duration group . odvba constructed a voxel - by - voxel image of significance derived from the optimized spatial adaptive filtering applied to the data , which highlights potential spatial differences in the relationship between images and subject groups . in the longitudinal analysis , odvba was applied on gm , wm , and vn dravens maps , which capture longitudinal rate of regional change in tissue volume in a given subject s brain at each voxel , for detecting imaging patterns of volume change associated with group differences between subjects in intensive and standard glycemic treatment arms . for the analysis of cognition , the longitudinal rate of change of each participant in each cognitive test was estimated by linear regression using baseline , 20-month , and 40-month test scores for dsst , ravlt , and stroop . the subjects were then stratified into declining and nondeclining cognition categories based on the 20th and 80th percentiles , similar to what was done for diabetes duration and hba1c . regional pattern analysis using odvba was applied on baseline gm , wm , and vn ravens maps , which were corrected for age , sex , and systolic blood pressure , to identify group differences between subject categories for low and high values of diabetes duration and hba1c . longer diabetes duration ( more than 14 years ) , compared with relatively short duration of diabetes ( less than 5 years ) , was correlated with a relatively lower regional gm volume , particularly in the frontal lobes , left temporal lobe , right parietal lobe , and limbic cortex . there was no statistically significant correlation of low versus high baseline hba1c levels to the gm , wm , and vn ravens maps . the longitudinal analysis using odvba has been applied for identifying group differences in regional volume loss patterns between subjects in the intensive glycemic treatment arm and those in the standard treatment arm . estimates of region - specific longitudinal volumetric change were obtained from the baseline and follow - up ravens maps by calculating the intensity difference at each voxel between the two time points . intensive treatment preserved gm volume more than the standard treatment ( fdr - corrected q < 0.05 ) in certain cortical areas , specifically in the left and right superior temporal gyrus , left pre- and postcentral gyri , left and right medial front - orbital gyrus , and left and right frontal cingulate regions ( fig . there was a partial overlap between the areas associated with treatment differences and those associated with diabetes duration . importantly , the regions that showed longitudinal differences were mostly adjacent to regions displaying significant cross - sectional relationship with diabetes duration and tended to show just a trend at baseline . regional pattern analysis using odvba was applied on baseline gm , wm , and vn ravens maps , which were corrected for age , sex , and systolic blood pressure , to identify group differences between subject categories for low and high values of diabetes duration and hba1c . longer diabetes duration ( more than 14 years ) , compared with relatively short duration of diabetes ( less than 5 years ) , was correlated with a relatively lower regional gm volume , particularly in the frontal lobes , left temporal lobe , right parietal lobe , and limbic cortex . there was no statistically significant correlation of low versus high baseline hba1c levels to the gm , wm , and vn ravens maps . the longitudinal analysis using odvba has been applied for identifying group differences in regional volume loss patterns between subjects in the intensive glycemic treatment arm and those in the standard treatment arm . estimates of region - specific longitudinal volumetric change were obtained from the baseline and follow - up ravens maps by calculating the intensity difference at each voxel between the two time points . intensive treatment preserved gm volume more than the standard treatment ( fdr - corrected q < 0.05 ) in certain cortical areas , specifically in the left and right superior temporal gyrus , left pre- and postcentral gyri , left and right medial front - orbital gyrus , and left and right frontal cingulate regions ( fig . there was a partial overlap between the areas associated with treatment differences and those associated with diabetes duration . importantly , the regions that showed longitudinal differences were mostly adjacent to regions displaying significant cross - sectional relationship with diabetes duration and tended to show just a trend at baseline . to the best of our knowledge , this is the first study to use pattern analysis methods to investigate the spatial specificity of patterns of brain volume loss in relation to diabetes duration and hba1c . moreover , this is the first study to apply these methods to investigate the spatial patterns of treatment effects on brain structure in a large diabetes clinical trial and using state - of - the - art pattern analysis methodology . we applied a new vbm approach , odvba ( 18 ) , in this analysis . in classical vbm methods , gaussian smoothing of images , which is applied to account for registration errors and to integrate imaging signals from a region , has also become a limitation of these methods , because it is often chosen empirically and lacks spatial adaptivity to the shape and spatial extent of the region of interest , such as a region of volume loss . we found , at baseline , that participants with longer diabetes duration had significantly reduced gm volumes in a number of brain regions . consistent with our findings , they concluded that the cortical atrophy in type 2 diabetic patients was not equally distributed across the entire brain but showed spatially specific patterns . also , detected patterns for the change , particularly in cortical area and volume , largely overlapped with those found in our analysis , showing large effects in middle temporal and frontal areas . the finding of a differential treatment effect is consistent with previous findings ( 14 ) that preservation of the brain volume was significantly better in the participants in the intensive treatment arm during the 40-month follow - up period compared with the participants in the standard arm . in particular , the strongest treatment effects were found in brain regions around the sylvian fissure as well in the medial - frontal cortex . interestingly , the regions displaying a differential treatment effect of reduced gm volume loss were adjacent to but almost nonoverlapping with those regions that displayed significant association with diabetes duration at baseline . therefore , intensive treatment appears to slow the spatial spreading of gm volume loss from regions mostly affected by diabetes duration toward adjacent brain regions . additional studies must be performed , potentially by using individualized longitudinal analyses , to better measure this apparent slowing of spatial progression interestingly , the areas that were associated to change in cognition were different from those affected from the treatment , which may explain the previous finding that there were no significant differences in the cognitive outcomes between the two treatment groups ( 14 ) . nonetheless , areas detected for ravlt , although showing a global cortical effect , have also shown partial overlap with areas affected from the treatment as well as with areas affected from diabetes duration . as such , these overlapping regions might be particularly important for investigating the relationship between diabetes , its treatment , and dementia . however , these mechanisms are still unclear , as findings propose the involvement of multiple factors in the development of diabetes related brain changes ( 43 ) . consequently , the etiology of potentially relatively higher vulnerability of certain brain regions to diabetes duration can not be elucidated by the current study and might relate to the differences in the underlying neuronal types and their sensitivity to insulin , underlying effects of diabetes in brain tissue perfusion , or might be effects of insulin resistance and its potential effect on brain connections . a growing body of work during the past decade has demonstrated that insulin - sensitive glucose transporters are localized to the same regions supporting memory , suggesting that insulin and its metabolism may contribute to normal cognitive functioning and that insulin abnormalities may exacerbate cognitive impairments , such as those associated with type 2 diabetes and even alzheimer disease . the regional differences identified here in the cross - sectional and longitudinal analyses are similar to some of those brain regions associated with cognitive health ( 44,45 ) . no brain region was found to display significant correlation with low versus high hba1c . that no brain region was significantly correlated with hba1c levels was against our initial expectations , which were based on the central diagnostic / disease management role of hba1c in diabetes and on earlier findings in the larger sample participating in the cognitive component of accord - mind ( 13 ) . because the mri sample was smaller than the cognitive sample , we may not have had enough power to detect differences in a measure such as hba1c level , which only reflect blood glucose levels during the relatively short period of 3 months , whereas diabetes duration reflects the cumulative effect of the disease over years . however , we can not rule out that other pathologic conditions associated with diabetes , such as hypertension , might have a relatively more deleterious effect on the brain than glucose levels . our findings on the relationship between diabetes duration and extent of brain volume loss are relatively conservative because they were obtained after correcting for age . new studies , including nondiabetic control subjects , would be necessary to allow us to correct only for patterns of brain volume loss explained by normal aging and independently from diabetes duration . several factors might have contributed to the dissociation between the treatment effects on brain structure and on cognition ( launer et al cognitive tests are known to be variable and subject to the learning effect ; hence , imaging measurements , especially the pattern analysis adopted in this study , might be able to detect more subtle effects . however , our results suggest a spatially heterogeneous effect of diabetes duration , as well as of its treatment , thereby pointing the way to future refinement of cognitive tests to tease out functions mediated by those brain regions that are more vulnerable to diabetes and more responsive to treatment .

nickel is a toxic transition metal and is widely used in many industries , including electroplating and the manufacture of steel , some special alloys , batteries , and electronic devices . epidemiological studies have indicated that chronic occupational exposure to nickel ( ni ) compounds increases the incidence of certain human cancers , such as lung and nasal cancers ( 1,2 ) . nickel exposure - induced generation of reactive oxygen species ( ros ) has been considered a pivotal step in nickel - induced carcinogenesis ( 3 ) . recent studies also show that ros production induced by nickel exposure is involved in nickel - induced apoptosis ( 4,5 ) . apoptosis is originally viewed as a normal physiologic process , removing cells carrying abnormal genetic information to maintain the functional integrity of the cell populations . in the case of metal - induced apoptosis , in contrast , it might allow the escape of cells with potentially carcinogenic ability from apoptosis under certain conditions , due to an abnormal apoptotic response ( 6 ) . therefore , investigation of nickel - induced apoptosis is necessary to understand the overall mechanism of nickel - induced carcinogenesis . one of the possible mechanisms of nickel - induced apoptosis and carcinogenesis is the induction of ros by nickel compounds . ros , such as superoxide anion ( o2 ) , hydrogen peroxide ( h2o2 ) , and the hydroxyl radical ( ho ) , have numerous effects on essential biological processes , including normal cell growth , induction and maintenance of the transformed state , programmed cell death , and cellular senescence ( 7 ) . an aberrant increase in the level of ros may result in transient or permanent cellular alterations , such as irreversible oxidative damage on dna , causing genomic instability and the consequent malignant transformation of the cells ( 8) . thus , ros are thought to play multiple roles in tumor initiation , progression , and maintenance . various studies have demonstrated that ros induced by toxic metals are critical in metal - induced apoptosis and carcinogenesis ( 4,6,9 ) . ros generation has been shown to be involved in the akt ( protein kinase b ) signaling pathway ( 10,11 ) . akt serine / threonine kinases are one of the essential regulators of cell survival function in response to growth factor stimulations ( 12,13 ) . it is generally believed that akt kinases are antiapoptotic through phosphorylation and inhibition of a number of apoptosis regulatory proteins ( 14 ) . apoptosis signal - regulating kinase 1 ( ask1 ) has been reported to be phosphorylated by akt at serine 83 ( ser83 ) and its activity reduced ( 1517 ) . ask1 is a serine - threonine kinase that was initially discovered as a mitogen - activated protein kinase kinase kinase ( mapkkk ) in the c - jun n - terminal kinase / stress - activated protein kinase ( jnk / sapk ) and p38 mapk signaling cascades ( 18,19 ) . these stimuli include serum or trophic factor withdrawal , tnf- , ros , microtubule - interfering agents , and genotoxic stress . some of these stress signals induce thr838 ( corresponding to thr845 in mice ) phosphorylation and activation of the ask1 ( 20,21 ) . activated ask1 will result in activation of the downstream kinases , leading to cell apoptosis ( 18,19,22 ) . to date , the majority of research on akt has focused on its role in cell growth promotion . the present study demonstrates that nickel - induced generation of ros activated akt , which activated ask1 through thr838 phosphorylation , leading to downstream activation of p38 mapk , eventually causing cell apoptosis . human bronchial epithelial cells cells ( beas-2b ) were cultured in dulbecco s modified eagle s medium ( dmem , calbiochem ) supplemented with 10% fetal bovine serum ( fbs ) , 5% penicillin / streptomycin , and 2 mm l - glutamine ( invitrogen ) at 37 c in a humidified atmosphere with 5% co2 . nickel subsulfide ( ni3s2 ) , n - acetyl - l - cysteine ( nac ) , and vitamin e ( -tocopherol ) were purchased from sigma ; catalase was from roche applied science co. dihydroethidium ( dhe ) and 5-(and-6)-chloromethyl-2 , 7-dichlorodihydrofluorescein diacetate acetyl ester ( cm - h2dcfda ) were from invitrogen ( eugene , or ) . antibodies against akt , phospho - jnk , jnk , and -actin were purchased from santa cruz biotechnology . bcl - xl , phospho - akt specific for ser473 phosphorylation , phospho - ask1 specific for thr838 phosphorylation , phospho - ask1 specific for ser83 phosphorylation , ask1 , phospho - p38 , and p38 were purchased from cell signaling . bcl-2 was purchased from dako , anticatalase antibody was from novus biologicals , inc . , and anti - cu / zn sod and anti - mnsod antibody were from upstate biotechnology . all primary antibodies were diluted at 1:1000 , except 1:2000 for actin and 1:200 for phospho - p38 , and secondary antibodies were diluted at 1:4000 . ros were detected by staining the cells with dhe or cm - h2dcfda ( molecular probes ) . dhe is oxidized to red fluorescent ethidium by o2 , and cm - h2dcfda is oxidized to green fluorescent dcf ( dichlorofluorescein ) by h2o2(23 ) . cells were loaded with 10 m dhe and 5 m cm - h2dcfda for 30 min , respectively , at 37 c , and 5% co2 in pbs and then were washed with pbs and returned to media for a 30 min recovery period . the mean fluorescence intensity was determined as ros generation by flow cytometry facs calibur ( bd bioscience , san jose , ca ) . beas-2b cells were seeded in each well of six well plates overnight and then treated without or with various doses of ni3s2 for 72 h or with ni3s2 2 g / cm for various time points as indicated . after treatments , cells were washed by pbs and trypsinized , and then , cell counting was carried out using beckman coulter . for annexin v / propidium iodide ( pi ) assays , beas-2b cells were stained with annexin v - fitc and pi and then evaluated for apoptosis by flow cytometry according to the manufacturer s protocol ( bd pharmingen ) . briefly , 1 10 cells were washed twice with cold pbs and stained with 5 l of annexin v - fitc and 8 l of pi ( 5 g / ml ) in 1 binding buffer [ 10 mmol / l hepes ( ph 7.4 ) , 140 mmol / l naoh , and 2.5 mmol / l cacl2 ] for 10 min at room temperature in the dark . the apoptotic cells were determined using a becton dickinson facscan cytofluorometer . both early apoptotic ( annexin v - positive and pi - negative ) and late apoptotic ( annexin v - positive and pi - positive ) cells were included in cell death determinations . western blot analysis was performed using the nupage bis - tris electrophoresis system ( invitrogen ) . the total cellular samples were washed once with ice cold pbs and lysed in 1 ripa buffer supplemented with 50 mmol / l dtt ( fisher biotech ) and then loaded with nupage lds sample buffer . the total cellular protein extracts were separated by sds - page and transferred to nitrocellulose membrane in 20 mmol / l tris - hcl ( ph 8.0 ) containing 150 mmol / l glycine and 20% ( v / v ) methanol . membranes were blocked with 5% fat - free dry milk in 1 tbs containing 0.05% tween 20 and incubated with antibodies . protein bands were detected by incubation with horseradish peroxidase - conjugated antibodies ( kirkegaard and perry laboratories ) and visualized with enhanced chemiluminescence reagent ( perkin - elmer life sciences ) . band densities in the western blots were all analyzed with alphaimager hp ( alpha innotech ) . the control and specific small interference rna targeting ask1 ( sirna ask1 , which inhibits expression of ask1 ) was purchased from santa cruz co. sirna akt ( which inhibits expression of akt1 and 2 , not akt3 ) and corresponding sirna control were purchased from cell signaling co. to block ask1 or akt signal , cells were transfected with the indicated sirna , respectively , using lipofectamine rnaimax from invitrogen co. the transfection procedure was followed by the protocol provided by the transfection reagent manufacturer . briefly , control sirna and sirna ask1 or akt were incubated with lipofectamine rnaimax in opti - mem i for 30 min at room temperature and then added to cells in maintenance media without antibiotics ( the final concentration of both control sirna and ask1 or akt sirna was 100 nm each ) . media were replaced with maintenance media with antibiotics 24 h later after transfection , and then , nickel was added to the media . experiments were performed approximately 72 h following transfection . for analysis of apoptosis and cell counting , beas-2b cells were treated with ni3s2 for 48 h followed by cell apoptosis analyses using flow cytometry . cell apoptosis was increased by 11.1 , 14.7 , and 29.5% at the concentrations of 1 , 2 , and 4 g / cm of ni3s2 treatment , respectively , whereas only 4.8% of the control cells were apoptotic ( figure 1a , b ) . figure 1c shows that the cell number was also decreased with increased nickel concentration and treatment time , suggesting that cell growth arrest was also induced by nickel treatment . other studies have shown the inhibitory effect of nickel on cell proliferation through interfering cell cycle progression . have demonstrated that up - regulation of cyclin b1 is responsible for nickel - induced m phase arrest and cell growth inhibition ( 24 ) . others revealed that soluble nickel compounds caused cell growth arrest and cyclin d1 degradation throught ikk -dependent pathway ( 25 ) . figure 1d shows that nickel treatment , in addition to decreasing cell number , also induced concomitant morphological changes of the beas-2b cells . the majority of nickel - treated beas-2b cells that originally had an epithelial cell - like appearance became elongated and resembled fibroblasts ( figure 1d ) , as observed and reported by others ( 26 ) . the elongation developed in the first 24 h of nickel exposure and persisted throughout the remaining 48 h of treatment . ( a ) beas-2b cells ( 4 10 ) were seeded in a 60 mm dish overnight , then treated without or with various concentrations of ni3s2 for 48 h , and then stained with annexin v / pi . data were obtained from annexin v / pi assays and are represented as means ses of three separate experiments . ( c ) beas-2b cells ( 1.5 10 ) were seeded in each well of six well plates overnight and then treated without or with various doses of ni3s2 for 72 h or with 2 g / cm ni3s2 for various time points as indicated , and then , cell counting was carried out . beas-2b cells were seeded in 60 mm dishes . after they were cultured at 37 c overnight , the cells were treated without or with 2 g / cm ni3s2 for 48 h. pictures were taken using a phase contrast microscope . ( e ) down - regulation of bcl-2 and bcl - xl is involved in nickel - induced apoptosis . beas-2b cells were seeded in each 100 mm dish and cultured in 10% fbs / dmem at 37 c . when the cell density reached 7080% , the cells were exposed to different concentrations of ni3s2 for 48 h. after treatments , total cellular extracts were prepared and subjected to western blot assay using antibodies against bcl-2 , bcl - xl , and -actin . blots were subsequently stripped and reprobed with antibody against -actin to ensure equivalent loading and transfer . all means marked with * ( p < 0.05 ) are significantly different from the control . bcl-2-family proteins are evolutionarily conserved regulators of apoptosis ( 27,28 ) . within this family , bc1 - 2 and bcl - xl proteins are potent antiapoptotic proteins that inhibit a mitochondria - operated pathway of apoptosis in many types of cells . both bcl-2 and bcl - xl were down - regulated by nickel treatment ( figure 1e , f ) . it has been reported that nickel may induce ros generation of the cells under some circumstances ( 2931 ) . to study the relationship between ros generation and apoptosis , nickel - induced ros production was determined by staining the cells with cm - h2dcfda and dhe , fluorescent dyes for h2o2 and o2 , respectively . figure 2a shows that cells treated with ni3s2 stimulated generation of h2o2 , whereas there was no apparent alteration in o2 generation ( figure 2b ) . the addition of catalase , a scavenger of h2o2 , also inhibited ros generation ( figure 2d ) . vitamin e , another well - established antioxidant , was also used to evaluate effect on ros generation stimulated by nickel . as shown in figure 2e , pretreatment of beas-2b cells with vitamin e reduced nickel - induced ros generation . beas-2b ( 4 10 ) cells were seeded in a 60 mm dish overnight and then treated without or with different concentrations of ni3s2 for 48 h. the cells were labeled with cm - h2dcfda and dhe , respectively , followed by flow cytometry as described in the materials and methods . the rightward shift of the overlay reflected the ros generation . beas-2b cells ( 4 10 ) were seeded in a 60 mm dish overnight and then pretreated with or without nac ( 10 mm ) or catalase ( 2000 units ) for 2 h , respectively , and then treated without or with 2 g / cm ni3s2 for 48 h. the cells were stained with cm - h2dcfda and measured by flow cytometry . beas-2b cells ( 4 10 ) were seeded in a 60 mm dish overnight and then pretreated with or without vitamin e ( 20 m ) for 2 h and then treated without or with 2 g / cm ni3s2 for 48 h. the cells were stained with cm - h2dcfda and measured by flow cytometry . each bar represents the mean se of the three independent experiments . * p < 0.05 between the indicated two groups . beas-2b cells ( 4 10 ) were seeded in a 60 mm dish overnight , then pretreated with or without nac ( 10 mm ) for 2 h , and then treated without or with 4 g / cm ni3s2 for 48 h. the cells were stained with annexin v / pi , and apoptosis was determined using flow cytometry as described in the materials and methods . ( g ) nickel decreased the protein expression of catalase , not cu / zn sod or mn sod . beas-2b cells were treated without or with various concentrations of ni3s2 for 48 h as indicated and then measured by western blot assay . band densities of catalase in the western blots were normalized against -actin ( shown on right panel ) . all means marked with * ( p < 0.05 ) are significantly different from the control . to investigate the possible role of ros in nickel - induced apoptosis , the effects of specific modifiers of ros on apoptosis were determined . the results show that pretreatment of the cells with nac attenuated nickel - induced apoptosis ( p < 0.05 ) ( figure 2f ) . we also pretreated beas-2b cells with antioxidant vitamin e , and our result shows that apoptosis induced by nickel was also ameliorated by vitamin e treatment ( data not shown ) . in addition , the protein level of catalase was decreased with the stimulation of ni3s2 , while the protein level of cu / zn sod ( sod1 ) and mn sod ( sod2 ) remained the same ( figure 2 g ) . accordingly , h2o2 is likely the main ros induced by nickel treatment . since its discovery by ichijo et al . in 1997 ( 19 ) , ask1 has drawn much attention in cell apoptosis , especially in oxidative stress - induced cell apoptosis through thr838 ( corresponding to thr845 in mice ) phosphorylation ( 20,21 ) . since nickel induced ros generation , we speculated that ask1 could be involved in nickel - induced apoptosis . by performing immunoblotting analysis , our results showed that ask1 phosphorylation at thr838 , which is correlated with its activity , was increased with the nickel treatment ( figure 3a ) , whereas phosphorylation at ser83 , which attenuates its activity and promotes cell survival ( 15 ) , remained unchangeable ( figure 3a ) . since ask1 is located upstream of the sek1/mkk4-jnk / sapk and mkk3/mkk6-p38 pathways ( 19 ) , we examined the activation of the multiple downstream protein kinases by western blot using phospho - specific antibodies ( jnk and p38 ) . figure 3b shows that treatment with nickel resulted in the activation of p38 mapk but not jnk . ( a ) nickel treatment increased protein phosphorylation of ask1 at thr838 , not ser83 . beas-2b cells were treated without or with different concentrations of ni3s2 for 48 h. after treatments , a western blot assay was performed using antibodies against ask1 at thr838 or ser83 , respectively . blots were subsequently stripped and reprobed with antibody against sk1 to ensure equivalent loading and transfer . band densities of ask1 phosphorylation at thr838 in the western blots were normalized against ask1 ( shown on lower panel ) . all means marked with * ( p < 0.05 ) are significantly different from the control . beas-2b cells were treated without or with different concentrations of ni3s2 for 48 h. after treatments , a western blot assay was performed using antibodies against phosphor - p38 or phosphor - jnk , respectively . blots were subsequently stripped and reprobed with antibody against p38 or jnk to ensure equivalent loading and transfer . band densities of p38 phosphorylation in the western blots were normalized against p38 ( shown on lower panel ) . all means marked with * ( p < 0.05 ) are significantly different from the control . beas-2b cells were transfected with 100 nm ask1 sirna and control sirna , and 24 h later , cells were treated with 2 g / cm ni3s2 for 48 h. the protein expression was measured by western blotting . band densities of ask1 and p38 phosphorylation in the western blots were normalized against actin and p38 , respectively ( shown on right panel ) . all means marked with * ( p < 0.05 ) are significantly different from the control . ( d ) sb203580 , a p38 mapk inhibitor , has no effect on ask1 phosphorylation at thr838 . beas-2b cells were pretreated with or without sb203580 ( 10 m ) for 2 h and then coincubated with 2 g / cm ni3s2 for 48 h. the protein levels were measured by western blot assays and represented from three separate experiments . blots were late stripped and reprobed with antibody against ask1 to ensure equivalent loading and transfer . to investigate the role of ask1 in regulating p38 mapk , we used sirna that specifically silences ask1 , an approach of loss - of - function analysis utilizing rna interference . expression of sirnas is able to silence gene expression and allows the functional inactivation of the targeted gene ( 32,33 ) . both sirna control and sirna ask1 products that we used here are from santa cruz co. and have been tested to reduce protein expression in human cells . by western blotting analysis , our results show that ask1 was down - regulated after transfection with sirna specific to ask1 ( figure 3c ) . figure 3c shows that sirna ask1 decreased nickel - induced activation of p38 mapk , suggesting that ask1 mediated nickel - induced p38 mapk activation . utilizing a pharmacological inhibition method , we checked the effect of p38 mapk inhibition through sb203580 , a widely used p38 inhibitor , on ask1 activation , to see whether ask1 activation is inversely regulated by p38 mapk . immunoblot results show that activation of ask1 induced by nickel was not altered by sb203580 ( figure 3d ) . akt has been revealed by many researches to play an essential role in promoting cell survival , inhibiting apoptosis , and so on . by immunoblotting , we observed a pronounced activation of akt by nickel treatment ( figure 4a ) . it has been reported that akt can phosphorylate ask1 on ser83 and inactivates the apoptotic function of ask1 , leading to the enhancement of cell survival ( 15 ) . akt is activated and involved in ask1/p38 mapk signaling pathway in nickel - induced apoptosis . beas-2b cells were treated with different concentrations of ni3s2 for 48 h and followed by immunoblot analysis of phospho - akt at ser473 . ( b ) small interfering rnas designed to target akt mrna ( akt sirna ) decreased both akt phosphorylation at ser473 and akt expression . beas-2b cells were transfected with 100 nm akt sirna and control sirna for 24 h and then treated with 2 g / cm ni3s2 for 48 h. the protein expression was measured by western blotting . ( c ) akt sirnas attenuated nickel - induced protein phosphorylation of ask1 and p38 ( experimental conditions are the same as in panel b described above ) . quantification of phospho - ask1 at thr 838 and phospho - p38 was normalized to ask1 and p38 , respectively . * ( p < 0.05 ) means significantly different from the control , or # ( p < 0.05 ) means significantly different between the indicated two groups . ( d ) without nickel stimulation , sirna akt has no effect on ask1 phosphorylation and p38 phosphorylation . beas-2b cells were transfected with 100 nm akt sirna and control sirna , and 72 h later , the protein expression was measured by western blotting . beas-2b cells were transfected with 100 nm akt sirna and control sirna , and 24 h later , cells were treated with 2 g / cm ni3s2 for 48 h. apoptosis was measured by flow cytometry as described in the materials and methods . ( p < 0.05 ) represents a significant difference between the two indicated groups . to obtain direct evidence for the involvement of akt in mediating the ask1/p38 pathway in nickel - induced apoptosis , we used sirna that specifically silences akt . both sirna control and sirna akt products that we used here are from cell signaling co. and have been tested in - house and shown to reduce protein expression of akt . as shown in figure 4b , both expression of akt and phosphorylatd akt at ser473 were all decreased by sirna specific to akt but not the control sirna . as compared with sirna control , protein levels of phosphorylated akt and akt after sirna akt were decreased by almost 70 and 60% , respectively , through quantitative analysis ( data not shown here ) . as shown in figure 4c , activation of ask1 and downstream kinase p38 was attenuated by sirna akt . in a control experiment , we transfected beas-2b cells with sirna control and sirna akt . all conditions and procedures are exactly the same as before except omitting nickel stimulation . our results showed that , in the absence of nickel stimulation , sirna akt had no effect on ask1 phosphorylation at both thr838 and ser83 and p38 mapk phosphoryltion demonstrated by western blot analysis ( figure 4d ) . flow cytometric analysis further indicated that apoptosis induced by nickel was decreased by akt - specific sirna ( figure 4e ) . accordingly , these observations demonstrate that akt plays a role in mediating ask1/p38 pathway and apoptosis induced by nickel . as signal molecules , ros our results have already demonstrated that nickel could induce ros generation ( figure 2 ) . here , to dissect the role of ros in mediating signal transduction pathways in nickel - induced apoptosis , beas-2b cells were preincubated with nac and catalase for 2 h , and then , the cells were used to study the alteration of signaling pathway in response to nickel . as shown in figure 5ac , treatment of nac and catalase attenuated nickel - induced phosphorylation of akt , ask1 , and downstream p38 mapk . the effects of these ros modifiers on signaling changes are in agreement with their effects on nickel - induced apoptosis ( figure 2f ) . thus , the results show that the generation of ros stimulated by nickel is involved in nickel - induced apoptotic signaling pathway . ( a ) effects of nac and catalase on the akt phosphorylation at ser473 and ask1 phosphorylation at thr838 . beas-2b cells were pretreated with or without nac ( 10 mm ) and catalase ( 2000 units / ml ) for 2 h and then coincubated with 2 g / cm ni3s2 for 48 h. the protein levels were measured by western blot assays and were from three separate experiments . ( b ) quantitative analyses of the blots are normalized to akt and ask1 , respectively . * beas-2b cells were similarly treated with the agents indicated above , and p38 phosphorylation was determined by western blotting . quantitative analyses of the blots are normalized to p38 and displayed on lower panel . * the present study addressed the importance of ros in mediating akt / ask1/p38 signal cascades in nickel - induced apoptosis . , very limited information is available with regard to the mechanisms of nickel - induced apoptosis and related signaling pathways . the nickel - induced apoptosis was first reported in chinese hamster ovary cells ( 34 ) . the phenomenon of apoptosis was also observed in other cell lines , for example , t cell hybridoma cells , jurkat cells , and mouse epidermal jb6 cells ( 3537 ) . fas ligand ( fasl ) expression , cell cycle alteration , and activation of c - myc through the erk pathway are reportedly involved in nickel - induced apoptosis ( 35,36 ) . down - regulation of bcl-2 and bcl - xl , two of the central players of the bcl-2 family members , was involved in nickel - induced apoptosis . the bcl-2 family proteins are the principal regulators of apoptosis , which consist of two groups , antiapoptotic members , including bcl-2 , bcl - xl , bcl - w , and mcl-1 , and pro - apoptotic members , including bax and the bh3-only families ( 38 ) . bcl-2 down - regulation is reportedly involved in arsenic - induced apoptosis ( 39 ) . our study shows that nickel could down - regulate expression of both bcl-2 and bcl - xl proteins . accompanied by apoptosis under stimulation of nickel is the cell morphological alteration from epithelial cell - like appearance to becoming elongated and fibroblast - like . ros are generated in various biological systems and are well - known to be important determinants in regulation of cell signaling pathways involved in proliferation , apoptosis , and senescence ( 40,41 ) . the generation of ros in response to certain metals has been implicated in the triggering of apoptosis ( 4244 ) . nickel compounds have been reported to induce oxidative damage resulting from an increase of ros production ( 45 ) . results from the present study demonstrate that nickel exposure caused ros production and cell apoptosis . by using molecular probes cm - h2dcfda and dhe , we found that nickel mainly induced h2o2 generation , for no apparent increase of o2 was observed , which is in agreement with the data of protein expression of catalase and sod after nickel treatment . among antioxidant defense mechanisms in mammalian cells , antioxidant enzymes , such as catalase , sod1 , and sod2 , play key roles in the detoxification of h2o2 and o2 . our study showed that nickel treatment decreased protein expression of catalase , whereas sod1 and sod2 remained unchanged . these results indicate that h2o2 is likely the main ros involved in nickel - induced apoptosis . moreover , pretreatment of beas-2b cells with nac , vitamin e , or catalase all reduced nickel - induced ros generation , respectively . even though the exact mechanism of ros generation stimulated by nickel is unknown , several sources of ros generation could exist in cells , including cyclooxygenase , lipoxygenase , mitochondrial electron transfer system , and cytochrome p450 . the major source of ros has been suggested to be the nadph oxidase ( nox ) ( 46 ) . ros induced by nickel may act as upstream - mediating molecules of the akt / ask1/p38 signaling pathway in nickel - induced apoptosis in beas-2b cells . the akt kinases play critical roles in regulating growth , proliferation , survival , metabolism , and other cellular activities . akt can phosphorylate a number of pro - apoptotic proteins , including glycogen synthase kinase 3 ( gsk-3 ) , bad , caspase 9 , and forkhead transcription factors , to suppress apoptosis ( 4751 ) . a study by others shows that nickel compounds can activate the akt pathway to induce hypoxia - inducible factor transactivation and cap43 expression ( 52 ) . however , in contrast to its well - established survival - promoting role , we found here that akt also plays a pro - apoptotic role in nickel - induced apoptosis . the same concentration of nickel treatment leading to cell apoptosis also induced activation of akt . in agreement with our study , recent researches performed by others also indicate that akt is not just a single - function kinase . under certain conditions , showed that akt activation increases oxidative stress , which in turn further increases akt phosphorylation and renders cells susceptible to ros - triggered cell senescence or death ( 53 ) . additionally , anticancer drugs , such as methotrexate , docetaxel , and doxorubicin , can also activate the akt / cdk2 ( cyclin dependent kinase ) pathway to promote , rather than suppress , cell death ( 54 ) . apoptin , a viral protein , has also been reported to selectively kill cancer cell death through akt activation followed by cdk activation ( 55 ) . furthermore , in the case of the death receptor pathway , activation of akt by fas ligand stimulation leads to apoptosis in epidermal c141 cells ( 56 ) . activation of akt in nickel - induced apoptosis has also been observed in jb6 cells by another group ( 37 ) . our study demonstrates that nickel induced beas-2b cell apoptosis through the akt - mediated ask1/p38 pathway . ask1 is one of the map3k that activates p38 and jnk via activating the map2ks , mkk4/mkk7 and mkk3/mkk6 ( 18,19 ) . ask1 is activated by a variety of stresses including calcium influx , endoplasmic reticulum ( er ) stress , lipopolysaccharide ( lps ) , ros , and tumor necrosis factor ( 5759 ) . these stresses induce activation of ask1 through thr838 ( thr845 in mice ) phosphorylation ( 20,21 ) . in recent years , researches have revealed that activation of ask1 plays pivotal roles in a wide variety of cellular responses , such as cell differentiation , apoptosis , and immune response , with special focus on oxidative stress - induced apoptosis ( 60 ) . our results show that nickel treatment induced ask1 phosphorylation at thr838 , not ser83 , which decreases ask1 activity . activation of ask1 can selectively activate jnk and p38 map kinases , leading to apoptosis . here , we found that nickel treatment caused p38 mapk phosphorylation , not jnk . to obtain evidence that ask1 regulates p38 mapk , we employed sirna method to specifically down - regulate ask1 expression . however , inhibition of p38 mapk with pharmacological inhibitor , sb203580 , had no effect on phosphorylation of ask1 at thr838 , probably implicating that ask1 was not reversely regulated by p38 mapk . it has been shown that ask1 activity can be regulated by a number of ask1-interacting proteins . among them , thioredoxin ( trx ) and 14 - 3 - 3 can directly bind to ask1 leading to inhibition of ask1 activity ( 59,61 ) . protein phosphatase 5 ( pp5 ) is also capable of binding to and dephosphorylating thr838 ( thr845 in mice ) to inactivate ask1 in response to oxidative stress ( 62 ) . previous studies show akt that can also act as an upstream kinase of ask1 to phosphorylate and negatively regulate ask1 at the site of ser83 ( 1517 ) . nevertheless , in nickel - induced apoptosis in beas-2b cells , both akt and ask1 were all activated . to elucidate the direct regulation of akt on ask1 , we down - regulated akt by utilizing sirna specific to akt . our study shows that under the stimulation of nickel , phosphorylation of ask1 at thr838 and p38 mapk , downstream of ask1 , were all attenuated by sirna akt . in addition , sirna akt also partially ameliorated nickel - induced apoptosis . on the contrary , in the absence of nickel stimulation , sirna akt showed no effect on ask1 phosphorylation at thr838 and ser83 as well as p38 phosphorylation . these observations suggest that akt acted upstream of ask1/p38 mapk pathway in the nickel - induced beas-2b cell apoptosis . given the results that we obtained in sirna ask1 ( figure 3c ) that p38 phosphorylation was almost completely inhibited by sirna ask1 , we propose that akt may phosphorylate ask1 at thr838 first , followed by p38 phosphorylation . regulation of p38 activity by akt through ask1 was also demonstrated by others ( 17,63 ) . in addition , a number of substrates have been shown to be downstream of p38 mapk activation that are involved in regulating diverse cellular functions . among them , the p38/p53 pathway is reportedly involved in g1-s arrest induced by loss of centrosome integrity ( 64 ) . activation of stat1 by p38 mapk has been shown to be important for lps - induced death of macrophages ( 65 ) . whether those substrates of p38 mapk mentioned above are involved in nickel - induced apoptosis remains to be investigated . there is increasing evidence within the literature that ros contribute to apoptosis stimulated by diverse stimuli . our study also showed that nickel - induced apoptosis was attenuated by ros scavenger , indicating that ros are probably involved in nickel - induced apoptosis in beas-2b cells . furthermore , activation of signaling kinases including akt , ask1 , and downstream p38 mapk were all ameliorated by scavenging ros , implying that ros mediated the akt / ask1/p38 pathway in nickel - induced cell apoptosis . several lines of evidence have indicated the involvement of ros in these pathways ( 66 ) . for instance , ros mediate pi3k / akt signaling and apoptosis induced by fasl(56 ) . ros are also required for lipopolysaccharide ( lps)-induced activation of ask1/p38 pathway and so on ( 57 ) . however , we found that neither nac nor sirna akt could completely block nickel - induced apoptosis pathway . it suggests that there are some other factors or pathways contributing to nickel - induced apoptosis . apoptosis is a complicated process . in mammalian cells , there are two major apoptotic pathways , the death - receptor pathway and the mitochondrial pathway . in the case of nickel - induced apoptosis , increased fasl expression , cell cycle alteration , activation of c - myc through erk pathway , caspase-8/aif - mediated pathways , and so on have been reportedly involved ( 24,3537 ) . also of note is the fact that some other unknown pathways are probably also involved in nickel - induced apoptosis . in summary , the present study has demonstrated that ros induced by nickel probably play a role in nickel - induced apoptosis . ros mediates nickel - induced apoptosis through the akt - ask1-p38 axis ( figure 6 ) . considering the important role of the akt signaling pathway in cell transformation and cancer , understanding the mechanism of nickel - induced apoptosis through the akt signaling pathway will be important in understanding the mechanism of nickel - induced carcinogenesis . schematic model for the role of ros / akt / ask1/p38 signaling pathway in nickel - induced apoptosis . nickel - generated ros stimulate the phosphorylation of akt , which in turn activates the ask1 by phosphorylating ask1 on thr838 , then followed by p38 activation , leading to an apoptotic effect .

nickel compounds are carcinogenic to humans , possibly through induction of reactive oxygen species ( ros ) that damage macromolecules including dna and proteins . the aim of the present study is to elucidate the role of the ros - mediated akt / apoptosis - regulating signal kinase ( ask ) 1/p38 pathway in nickel - induced apoptosis . exposure of human bronchial epithelial cells ( beas-2b ) to nickel compounds induced the generation of ros and activation of akt that is associated with the activation of ask1 and p38 mitogen - activated protein kinase . immunoblotting suggested a down - regulation of several antiapoptotic proteins , including bcl-2 and bcl - xl in the nickel compound - treated cells . indeed , a notable cell apoptosis following nickel compound treatment is evident as revealed by flow cytometry analysis . n - acetyl - l - cysteine ( nac , a general antioxidant ) and vitamin e or catalase ( a specific h2o2 inhibitor ) all decreased nickel - induced ros generation . scavenging of nickel - induced ros by nac or catalase attenuated akt , ask1 , and p38 mapk activation and apoptosis , which implies involvement of ros in the akt / ask1/p38 pathway . in addition , nickel - induced activation of p38 mapk was attenuated by a small interference of rna specific to ask1 ( sirna ask1 ) , implying that p38 mapk was downstream of ask1 , while ask1 activation was not reversely regulated by the inhibition of p38 mapk by sb203580 , a widely used p38 mapk inhibitor . silencing akt by sirna reduced the activation of ask1 and p38 mapk and cell apoptosis , whereas without nickel stimulation , sirna akt had no effect on the activation of ask1 and p38 mapk . thus , these results suggest that the ros - dependent akt - ask1-p38 axis is important for nickel - induced apoptosis .

Introduction Materials and Methods Results Discussion

nickel is a toxic transition metal and is widely used in many industries , including electroplating and the manufacture of steel , some special alloys , batteries , and electronic devices . nickel exposure - induced generation of reactive oxygen species ( ros ) has been considered a pivotal step in nickel - induced carcinogenesis ( 3 ) . recent studies also show that ros production induced by nickel exposure is involved in nickel - induced apoptosis ( 4,5 ) . in the case of metal - induced apoptosis , in contrast , it might allow the escape of cells with potentially carcinogenic ability from apoptosis under certain conditions , due to an abnormal apoptotic response ( 6 ) . therefore , investigation of nickel - induced apoptosis is necessary to understand the overall mechanism of nickel - induced carcinogenesis . one of the possible mechanisms of nickel - induced apoptosis and carcinogenesis is the induction of ros by nickel compounds . ros , such as superoxide anion ( o2 ) , hydrogen peroxide ( h2o2 ) , and the hydroxyl radical ( ho ) , have numerous effects on essential biological processes , including normal cell growth , induction and maintenance of the transformed state , programmed cell death , and cellular senescence ( 7 ) . an aberrant increase in the level of ros may result in transient or permanent cellular alterations , such as irreversible oxidative damage on dna , causing genomic instability and the consequent malignant transformation of the cells ( 8) . ros generation has been shown to be involved in the akt ( protein kinase b ) signaling pathway ( 10,11 ) . apoptosis signal - regulating kinase 1 ( ask1 ) has been reported to be phosphorylated by akt at serine 83 ( ser83 ) and its activity reduced ( 1517 ) . ask1 is a serine - threonine kinase that was initially discovered as a mitogen - activated protein kinase kinase kinase ( mapkkk ) in the c - jun n - terminal kinase / stress - activated protein kinase ( jnk / sapk ) and p38 mapk signaling cascades ( 18,19 ) . some of these stress signals induce thr838 ( corresponding to thr845 in mice ) phosphorylation and activation of the ask1 ( 20,21 ) . the present study demonstrates that nickel - induced generation of ros activated akt , which activated ask1 through thr838 phosphorylation , leading to downstream activation of p38 mapk , eventually causing cell apoptosis . human bronchial epithelial cells cells ( beas-2b ) were cultured in dulbecco s modified eagle s medium ( dmem , calbiochem ) supplemented with 10% fetal bovine serum ( fbs ) , 5% penicillin / streptomycin , and 2 mm l - glutamine ( invitrogen ) at 37 c in a humidified atmosphere with 5% co2 . nickel subsulfide ( ni3s2 ) , n - acetyl - l - cysteine ( nac ) , and vitamin e ( -tocopherol ) were purchased from sigma ; catalase was from roche applied science co. dihydroethidium ( dhe ) and 5-(and-6)-chloromethyl-2 , 7-dichlorodihydrofluorescein diacetate acetyl ester ( cm - h2dcfda ) were from invitrogen ( eugene , or ) . bcl - xl , phospho - akt specific for ser473 phosphorylation , phospho - ask1 specific for thr838 phosphorylation , phospho - ask1 specific for ser83 phosphorylation , ask1 , phospho - p38 , and p38 were purchased from cell signaling . the control and specific small interference rna targeting ask1 ( sirna ask1 , which inhibits expression of ask1 ) was purchased from santa cruz co. sirna akt ( which inhibits expression of akt1 and 2 , not akt3 ) and corresponding sirna control were purchased from cell signaling co. to block ask1 or akt signal , cells were transfected with the indicated sirna , respectively , using lipofectamine rnaimax from invitrogen co. the transfection procedure was followed by the protocol provided by the transfection reagent manufacturer . cell apoptosis was increased by 11.1 , 14.7 , and 29.5% at the concentrations of 1 , 2 , and 4 g / cm of ni3s2 treatment , respectively , whereas only 4.8% of the control cells were apoptotic ( figure 1a , b ) . have demonstrated that up - regulation of cyclin b1 is responsible for nickel - induced m phase arrest and cell growth inhibition ( 24 ) . the majority of nickel - treated beas-2b cells that originally had an epithelial cell - like appearance became elongated and resembled fibroblasts ( figure 1d ) , as observed and reported by others ( 26 ) . ( e ) down - regulation of bcl-2 and bcl - xl is involved in nickel - induced apoptosis . within this family , bc1 - 2 and bcl - xl proteins are potent antiapoptotic proteins that inhibit a mitochondria - operated pathway of apoptosis in many types of cells . both bcl-2 and bcl - xl were down - regulated by nickel treatment ( figure 1e , f ) . to study the relationship between ros generation and apoptosis , nickel - induced ros production was determined by staining the cells with cm - h2dcfda and dhe , fluorescent dyes for h2o2 and o2 , respectively . as shown in figure 2e , pretreatment of beas-2b cells with vitamin e reduced nickel - induced ros generation . the rightward shift of the overlay reflected the ros generation . beas-2b cells ( 4 10 ) were seeded in a 60 mm dish overnight and then pretreated with or without nac ( 10 mm ) or catalase ( 2000 units ) for 2 h , respectively , and then treated without or with 2 g / cm ni3s2 for 48 h. the cells were stained with cm - h2dcfda and measured by flow cytometry . beas-2b cells ( 4 10 ) were seeded in a 60 mm dish overnight and then pretreated with or without vitamin e ( 20 m ) for 2 h and then treated without or with 2 g / cm ni3s2 for 48 h. the cells were stained with cm - h2dcfda and measured by flow cytometry . beas-2b cells ( 4 10 ) were seeded in a 60 mm dish overnight , then pretreated with or without nac ( 10 mm ) for 2 h , and then treated without or with 4 g / cm ni3s2 for 48 h. the cells were stained with annexin v / pi , and apoptosis was determined using flow cytometry as described in the materials and methods . to investigate the possible role of ros in nickel - induced apoptosis , the effects of specific modifiers of ros on apoptosis were determined . the results show that pretreatment of the cells with nac attenuated nickel - induced apoptosis ( p < 0.05 ) ( figure 2f ) . in addition , the protein level of catalase was decreased with the stimulation of ni3s2 , while the protein level of cu / zn sod ( sod1 ) and mn sod ( sod2 ) remained the same ( figure 2 g ) . in 1997 ( 19 ) , ask1 has drawn much attention in cell apoptosis , especially in oxidative stress - induced cell apoptosis through thr838 ( corresponding to thr845 in mice ) phosphorylation ( 20,21 ) . since nickel induced ros generation , we speculated that ask1 could be involved in nickel - induced apoptosis . by performing immunoblotting analysis , our results showed that ask1 phosphorylation at thr838 , which is correlated with its activity , was increased with the nickel treatment ( figure 3a ) , whereas phosphorylation at ser83 , which attenuates its activity and promotes cell survival ( 15 ) , remained unchangeable ( figure 3a ) . since ask1 is located upstream of the sek1/mkk4-jnk / sapk and mkk3/mkk6-p38 pathways ( 19 ) , we examined the activation of the multiple downstream protein kinases by western blot using phospho - specific antibodies ( jnk and p38 ) . figure 3b shows that treatment with nickel resulted in the activation of p38 mapk but not jnk . band densities of ask1 and p38 phosphorylation in the western blots were normalized against actin and p38 , respectively ( shown on right panel ) . ( d ) sb203580 , a p38 mapk inhibitor , has no effect on ask1 phosphorylation at thr838 . to investigate the role of ask1 in regulating p38 mapk , we used sirna that specifically silences ask1 , an approach of loss - of - function analysis utilizing rna interference . by western blotting analysis , our results show that ask1 was down - regulated after transfection with sirna specific to ask1 ( figure 3c ) . figure 3c shows that sirna ask1 decreased nickel - induced activation of p38 mapk , suggesting that ask1 mediated nickel - induced p38 mapk activation . utilizing a pharmacological inhibition method , we checked the effect of p38 mapk inhibition through sb203580 , a widely used p38 inhibitor , on ask1 activation , to see whether ask1 activation is inversely regulated by p38 mapk . immunoblot results show that activation of ask1 induced by nickel was not altered by sb203580 ( figure 3d ) . akt has been revealed by many researches to play an essential role in promoting cell survival , inhibiting apoptosis , and so on . akt is activated and involved in ask1/p38 mapk signaling pathway in nickel - induced apoptosis . ( c ) akt sirnas attenuated nickel - induced protein phosphorylation of ask1 and p38 ( experimental conditions are the same as in panel b described above ) . quantification of phospho - ask1 at thr 838 and phospho - p38 was normalized to ask1 and p38 , respectively . ( d ) without nickel stimulation , sirna akt has no effect on ask1 phosphorylation and p38 phosphorylation . beas-2b cells were transfected with 100 nm akt sirna and control sirna , and 24 h later , cells were treated with 2 g / cm ni3s2 for 48 h. apoptosis was measured by flow cytometry as described in the materials and methods . to obtain direct evidence for the involvement of akt in mediating the ask1/p38 pathway in nickel - induced apoptosis , we used sirna that specifically silences akt . as shown in figure 4b , both expression of akt and phosphorylatd akt at ser473 were all decreased by sirna specific to akt but not the control sirna . as shown in figure 4c , activation of ask1 and downstream kinase p38 was attenuated by sirna akt . our results showed that , in the absence of nickel stimulation , sirna akt had no effect on ask1 phosphorylation at both thr838 and ser83 and p38 mapk phosphoryltion demonstrated by western blot analysis ( figure 4d ) . here , to dissect the role of ros in mediating signal transduction pathways in nickel - induced apoptosis , beas-2b cells were preincubated with nac and catalase for 2 h , and then , the cells were used to study the alteration of signaling pathway in response to nickel . as shown in figure 5ac , treatment of nac and catalase attenuated nickel - induced phosphorylation of akt , ask1 , and downstream p38 mapk . thus , the results show that the generation of ros stimulated by nickel is involved in nickel - induced apoptotic signaling pathway . * beas-2b cells were similarly treated with the agents indicated above , and p38 phosphorylation was determined by western blotting . * the present study addressed the importance of ros in mediating akt / ask1/p38 signal cascades in nickel - induced apoptosis . , very limited information is available with regard to the mechanisms of nickel - induced apoptosis and related signaling pathways . the nickel - induced apoptosis was first reported in chinese hamster ovary cells ( 34 ) . fas ligand ( fasl ) expression , cell cycle alteration , and activation of c - myc through the erk pathway are reportedly involved in nickel - induced apoptosis ( 35,36 ) . down - regulation of bcl-2 and bcl - xl , two of the central players of the bcl-2 family members , was involved in nickel - induced apoptosis . the bcl-2 family proteins are the principal regulators of apoptosis , which consist of two groups , antiapoptotic members , including bcl-2 , bcl - xl , bcl - w , and mcl-1 , and pro - apoptotic members , including bax and the bh3-only families ( 38 ) . bcl-2 down - regulation is reportedly involved in arsenic - induced apoptosis ( 39 ) . our study shows that nickel could down - regulate expression of both bcl-2 and bcl - xl proteins . the generation of ros in response to certain metals has been implicated in the triggering of apoptosis ( 4244 ) . results from the present study demonstrate that nickel exposure caused ros production and cell apoptosis . these results indicate that h2o2 is likely the main ros involved in nickel - induced apoptosis . moreover , pretreatment of beas-2b cells with nac , vitamin e , or catalase all reduced nickel - induced ros generation , respectively . even though the exact mechanism of ros generation stimulated by nickel is unknown , several sources of ros generation could exist in cells , including cyclooxygenase , lipoxygenase , mitochondrial electron transfer system , and cytochrome p450 . ros induced by nickel may act as upstream - mediating molecules of the akt / ask1/p38 signaling pathway in nickel - induced apoptosis in beas-2b cells . akt can phosphorylate a number of pro - apoptotic proteins , including glycogen synthase kinase 3 ( gsk-3 ) , bad , caspase 9 , and forkhead transcription factors , to suppress apoptosis ( 4751 ) . however , in contrast to its well - established survival - promoting role , we found here that akt also plays a pro - apoptotic role in nickel - induced apoptosis . the same concentration of nickel treatment leading to cell apoptosis also induced activation of akt . furthermore , in the case of the death receptor pathway , activation of akt by fas ligand stimulation leads to apoptosis in epidermal c141 cells ( 56 ) . activation of akt in nickel - induced apoptosis has also been observed in jb6 cells by another group ( 37 ) . our study demonstrates that nickel induced beas-2b cell apoptosis through the akt - mediated ask1/p38 pathway . in recent years , researches have revealed that activation of ask1 plays pivotal roles in a wide variety of cellular responses , such as cell differentiation , apoptosis , and immune response , with special focus on oxidative stress - induced apoptosis ( 60 ) . activation of ask1 can selectively activate jnk and p38 map kinases , leading to apoptosis . however , inhibition of p38 mapk with pharmacological inhibitor , sb203580 , had no effect on phosphorylation of ask1 at thr838 , probably implicating that ask1 was not reversely regulated by p38 mapk . among them , thioredoxin ( trx ) and 14 - 3 - 3 can directly bind to ask1 leading to inhibition of ask1 activity ( 59,61 ) . nevertheless , in nickel - induced apoptosis in beas-2b cells , both akt and ask1 were all activated . to elucidate the direct regulation of akt on ask1 , we down - regulated akt by utilizing sirna specific to akt . our study shows that under the stimulation of nickel , phosphorylation of ask1 at thr838 and p38 mapk , downstream of ask1 , were all attenuated by sirna akt . in addition , sirna akt also partially ameliorated nickel - induced apoptosis . on the contrary , in the absence of nickel stimulation , sirna akt showed no effect on ask1 phosphorylation at thr838 and ser83 as well as p38 phosphorylation . these observations suggest that akt acted upstream of ask1/p38 mapk pathway in the nickel - induced beas-2b cell apoptosis . given the results that we obtained in sirna ask1 ( figure 3c ) that p38 phosphorylation was almost completely inhibited by sirna ask1 , we propose that akt may phosphorylate ask1 at thr838 first , followed by p38 phosphorylation . in addition , a number of substrates have been shown to be downstream of p38 mapk activation that are involved in regulating diverse cellular functions . activation of stat1 by p38 mapk has been shown to be important for lps - induced death of macrophages ( 65 ) . whether those substrates of p38 mapk mentioned above are involved in nickel - induced apoptosis remains to be investigated . our study also showed that nickel - induced apoptosis was attenuated by ros scavenger , indicating that ros are probably involved in nickel - induced apoptosis in beas-2b cells . furthermore , activation of signaling kinases including akt , ask1 , and downstream p38 mapk were all ameliorated by scavenging ros , implying that ros mediated the akt / ask1/p38 pathway in nickel - induced cell apoptosis . however , we found that neither nac nor sirna akt could completely block nickel - induced apoptosis pathway . it suggests that there are some other factors or pathways contributing to nickel - induced apoptosis . in the case of nickel - induced apoptosis , increased fasl expression , cell cycle alteration , activation of c - myc through erk pathway , caspase-8/aif - mediated pathways , and so on have been reportedly involved ( 24,3537 ) . also of note is the fact that some other unknown pathways are probably also involved in nickel - induced apoptosis . in summary , the present study has demonstrated that ros induced by nickel probably play a role in nickel - induced apoptosis . ros mediates nickel - induced apoptosis through the akt - ask1-p38 axis ( figure 6 ) . considering the important role of the akt signaling pathway in cell transformation and cancer , understanding the mechanism of nickel - induced apoptosis through the akt signaling pathway will be important in understanding the mechanism of nickel - induced carcinogenesis . schematic model for the role of ros / akt / ask1/p38 signaling pathway in nickel - induced apoptosis . nickel - generated ros stimulate the phosphorylation of akt , which in turn activates the ask1 by phosphorylating ask1 on thr838 , then followed by p38 activation , leading to an apoptotic effect .

the symptoms that patients are experiencing during acute coronary events are a key component in health care providers ' decision to initiate medical treatment and in further evaluation . in addition , patients ' earlier recognition of symptoms will facilitate the need for treatment seeking.1 ) a study reported that chest pain was the sole symptom in only 35% of cases and chest pain was accompanied by other symptoms in 57% of cases.2 ) overall , patients experienced a mean of 4.75 symptoms as part of the acute event of acute myocardial infarction ( ami).3 ) the absence of chest pain and the vagueness of symptoms might not be recognized as an indication of a cardiac problem , resulting in a delay in seeking medical care . in addition , this atypical presentation predicted lower use of thrombolytic therapy and was associated with hospital complications compared with the typical presentation.4)5 ) symptom clusters can be defined as multiple symptoms that are related to each other and are experienced concurrently.6)7 ) the clusters share a common etiology and a measurement of frequency , and severity of symptoms within clusters typically correlate with each other.8 ) they may also be useful in formulating criteria and evaluating patterns of symptoms . it is worthwhile to know how individual symptoms cluster by demographic and clinical characteristics of the subgroups in order to provide key information for early detection and diagnosis.9)10 ) identification of symptom clusters can be a clinically relevant way to detect specific demographic groups that are most likely to experience similar symptoms with readily identifiable characteristics.11)12 ) previous cluster studies on prehospital symptoms provided a useful grouping of acute coronary syndrome ( acs ) patients based on their experienced symptoms.9)-12 ) the clarification of prospective clinical outcomes for major adverse cardiac events ( mace ) should be helpful to health care providers in planning treatment strategies depending on the clusters . therefore , this study examined symptom clusters during acute events and clinical outcomes at 1-year follow - up by symptom cluster in patients with first - time ami . this clinical trial should enable health care providers to educate , more efficiently , individuals in the target populations at risk for cardiovascular disease ( cvd ) , to reduce prehospital delay time in korea , and to provide a useful treatment strategy for different symptomatic groups . the data for this study were obtained from first - time ami patients who were hospitalized for revascularization . subjects were recruited from the pool of patients hospitalized in chonnam national university hospital ( cnuh ) between 2007 and 2009 . inclusion criteria were : 1 ) patients who had undergone a first attack of st - elevation myocardial infarction ( stemi ) or non - st - elevation myocardial infarction ( nstemi ) , 2 ) verbally communicable , and 3 ) those who agreed to participate in the study . after obtaining approval of the institutional review board of the hospital , the principal investigator ( pi ) contacted eligible patients who were hospitalized in a cardiovascular care unit to ask them to participate in the study . this study was approved by the clinical ethics committee at cnuh before data collection was carried out . the pi interviewed the patients who had undergone a percutaneous coronary intervention using a semi - structured questionnaire . the interview was done in the education room with a research assistant ( ra ) . electronic medical records ( emr ) were also reviewed to collect medical treatment histories and laboratory data . each patient was asked about his chief complaints and associated symptoms that he experienced during the acute phase of an ami . if needed , figured descriptors were given for patients to choose and the pi or the ras checked their symptoms . patients ' pain or discomfort anywhere in the chest was coded as a chest pain . severity of chest pain or discomfort during the acute phase was asked and recoded on a 1 - 10 scale . patients were also asked about risk factors , their chronic disease , and past / family history questions . data for clinical outcomes including mace and mortality were also obtained at the 1-year follow - up period . all clinical events were verified through the emr or telephone follow - up calls by a research nurse . all acute symptoms of patients were coded as a categorical variable ; 1 point was given for ' present ' and 0 points for ' none ' . the coded symptoms in the statistical package for the social sciences ( spss , spss inc . , chicago , il , usa ) data set were analyzed by latent class ( lc ) cluster analysis using latent gold software , version 3.0.13 ) cluster analysis is defined as the classification of similar objects into the form of groups , which refers to the parameters of cluster ; that is , to its cluster - specific means , variances , and covariances that have a geometrical interpretation.13 ) lc cluster analysis is a statistical model - based clustering technique and can include covariates to predict class membership . an individual 's posterior class - membership probabilities are computed from the estimated model parameters and his observed scores using the maximum likelihood method . the most popular set of model selection tools in lc cluster analysis are information criteria such as akaike information criterion ( aic ) and the bayesian information criterion ( bic).14 ) in this study , decreases in the values of bic and aic were minimal between the three- and four - cluster models . the cluster membership of 3 clusters was conversed to an spss data set to identify the membership and associated factors . bivariate analyses using -tests , t - tests , and analysis of variance were conducted to identify the relationship between cluster assignment and patients ' characteristics . cox proportional hazards regression analysis was used to test a cluster membership as an independent predictor of 1-year mortality and maces . age was included and controlled for in the multivariate models of mortality and maces due to aging 's known prognostic importance . the data for this study were obtained from first - time ami patients who were hospitalized for revascularization . subjects were recruited from the pool of patients hospitalized in chonnam national university hospital ( cnuh ) between 2007 and 2009 . inclusion criteria were : 1 ) patients who had undergone a first attack of st - elevation myocardial infarction ( stemi ) or non - st - elevation myocardial infarction ( nstemi ) , 2 ) verbally communicable , and 3 ) those who agreed to participate in the study . after obtaining approval of the institutional review board of the hospital , the principal investigator ( pi ) contacted eligible patients who were hospitalized in a cardiovascular care unit to ask them to participate in the study . this study was approved by the clinical ethics committee at cnuh before data collection was carried out . the pi interviewed the patients who had undergone a percutaneous coronary intervention using a semi - structured questionnaire . the interview was done in the education room with a research assistant ( ra ) . electronic medical records ( emr ) were also reviewed to collect medical treatment histories and laboratory data . each patient was asked about his chief complaints and associated symptoms that he experienced during the acute phase of an ami . if needed , figured descriptors were given for patients to choose and the pi or the ras checked their symptoms . patients ' pain or discomfort anywhere in the chest was coded as a chest pain . severity of chest pain or discomfort during the acute phase was asked and recoded on a 1 - 10 scale . patients were also asked about risk factors , their chronic disease , and past / family history questions . data for clinical outcomes including mace and mortality were also obtained at the 1-year follow - up period . all clinical events were verified through the emr or telephone follow - up calls by a research nurse . all acute symptoms of patients were coded as a categorical variable ; 1 point was given for ' present ' and 0 points for ' none ' . the coded symptoms in the statistical package for the social sciences ( spss , spss inc . , chicago , il , usa ) data set were analyzed by latent class ( lc ) cluster analysis using latent gold software , version 3.0.13 ) cluster analysis is defined as the classification of similar objects into the form of groups , which refers to the parameters of cluster ; that is , to its cluster - specific means , variances , and covariances that have a geometrical interpretation.13 ) lc cluster analysis is a statistical model - based clustering technique and can include covariates to predict class membership . an individual 's posterior class - membership probabilities are computed from the estimated model parameters and his observed scores using the maximum likelihood method . the most popular set of model selection tools in lc cluster analysis are information criteria such as akaike information criterion ( aic ) and the bayesian information criterion ( bic).14 ) in this study , decreases in the values of bic and aic were minimal between the three- and four - cluster models . the cluster membership of 3 clusters was conversed to an spss data set to identify the membership and associated factors . bivariate analyses using -tests , t - tests , and analysis of variance were conducted to identify the relationship between cluster assignment and patients ' characteristics . cox proportional hazards regression analysis was used to test a cluster membership as an independent predictor of 1-year mortality and maces . age was included and controlled for in the multivariate models of mortality and maces due to aging 's known prognostic importance . the majority of patients ( 71.1% ) were men and the average age was 63.810.3 years ( range 26 - 89 ) . about 52% of the patients 49.1% had hypertension and 32.2% had diabetes ( average lengths of periods 8.47.3 and 11.89.7 years ) . about one third of patients ( 35.3% ) had a dyslipidemia and 38.6% were current smokers . about 12% of the patients had a past history of angina or stroke , and 19.7% had a family history of cvd including hypertension , stroke or ischemic heart disease ( table 1 ) . the size of cluster 1 was the biggest ( n=223 , 57.0% ) , followed by cluster 2 ( n=109 , 27.9% ) and cluster 3 ( n=59 , 15.1% ) . all cluster membership within each symptom cluster was significantly different ( p<0.01 ) except palpitation ( p=0.527 ) . the patients in cluster 1 and cluster 2 experienced a high frequency of chest pain / discomfort ( 100% and 94.5% , respectively ) and a moderate cold sweating . among the three groups , cluster 3 had the least chest pain and moderate frequencies of shortness of breath and weakness or fatigue . we named cluster 1 , 2 , and 3 as typical chest pain , multiple symptoms , and atypical symptom clusters , respectively . bivariate analyses showed that patients of cluster 2 were the youngest ( 58.112.8 years ) and those of cluster 3 were the oldest ( 69.312.3 years ) among the three clusters ( p<0.001 ) . as for cvd risk factors , the patients of cluster 2 were more likely to be overweight bmi > 25 kg / m ( p=0.002 ) and current smokers ( p=0.031 ) , and the patients of cluster 3 were more likely to have diabetes ( p<0.001 ) . 94.3% of cluster 3 reported that they did not perceive their symptoms as cardiac in origin and they had the fewest complaints of acute symptoms and the lowest self - reported intensity score of chest pain among the three groups ( p<0.001 ) . the patients of cluster 3 had the highest value for high sensitivity c - reactive protein ( hs - crp ) and the lowest in left ventricular ejection fraction ( lvef ) , which were checked immediately after hospital admission ; they had the longest stay at hospital among the three groups ( p<0.001 ) ( table 2 ) . bivariate analysis revealed that there was no significant difference among clustered groups in the prevalence of maces including an ami re - attack and total vessel or lesion revascularization . however , the prevalence of cardiac or non - cardiac deaths turned out to be significantly different by clusters ( p=0.022 ) ( table 3 ) . multivariate cox proportional hazards regression analysis demonstrated that when age , marital status and monthly income were adjusted for , patients in cluster 3 predicted a significantly higher risk of 1-year mortality compared to those in cluster 1 , a typical chest pain cluster ( hazard ratio 3.288 , 95% confidence interval 1.087 - 9.943 , p=0.035 ) ( table 4 ) . the majority of patients ( 71.1% ) were men and the average age was 63.810.3 years ( range 26 - 89 ) . about 52% of the patients 49.1% had hypertension and 32.2% had diabetes ( average lengths of periods 8.47.3 and 11.89.7 years ) . about one third of patients ( 35.3% ) had a dyslipidemia and 38.6% were current smokers . about 12% of the patients had a past history of angina or stroke , and 19.7% had a family history of cvd including hypertension , stroke or ischemic heart disease ( table 1 ) . the size of cluster 1 was the biggest ( n=223 , 57.0% ) , followed by cluster 2 ( n=109 , 27.9% ) and cluster 3 ( n=59 , 15.1% ) . all cluster membership within each symptom cluster was significantly different ( p<0.01 ) except palpitation ( p=0.527 ) . the patients in cluster 1 and cluster 2 experienced a high frequency of chest pain / discomfort ( 100% and 94.5% , respectively ) and a moderate cold sweating . among the three groups , cluster 3 had the least chest pain and moderate frequencies of shortness of breath and weakness or fatigue . we named cluster 1 , 2 , and 3 as typical chest pain , multiple symptoms , and atypical symptom clusters , respectively . bivariate analyses showed that patients of cluster 2 were the youngest ( 58.112.8 years ) and those of cluster 3 were the oldest ( 69.312.3 years ) among the three clusters ( p<0.001 ) . as for cvd risk factors , the patients of cluster 2 were more likely to be overweight bmi > 25 kg / m ( p=0.002 ) and current smokers ( p=0.031 ) , and the patients of cluster 3 were more likely to have diabetes ( p<0.001 ) . 94.3% of cluster 3 reported that they did not perceive their symptoms as cardiac in origin and they had the fewest complaints of acute symptoms and the lowest self - reported intensity score of chest pain among the three groups ( p<0.001 ) . the patients of cluster 3 had the highest value for high sensitivity c - reactive protein ( hs - crp ) and the lowest in left ventricular ejection fraction ( lvef ) , which were checked immediately after hospital admission ; they had the longest stay at hospital among the three groups ( p<0.001 ) ( table 2 ) . bivariate analysis revealed that there was no significant difference among clustered groups in the prevalence of maces including an ami re - attack and total vessel or lesion revascularization . however , the prevalence of cardiac or non - cardiac deaths turned out to be significantly different by clusters ( p=0.022 ) ( table 3 ) . multivariate cox proportional hazards regression analysis demonstrated that when age , marital status and monthly income were adjusted for , patients in cluster 3 predicted a significantly higher risk of 1-year mortality compared to those in cluster 1 , a typical chest pain cluster ( hazard ratio 3.288 , 95% confidence interval 1.087 - 9.943 , p=0.035 ) ( table 4 ) . three clusters of acute symptoms in this study were consistent with previous studies of 1 ) 1270 ami women that was made up of older / silent asymptomatic , diverse / mildly symptomatic , and younger / multiple distressing symptom groups,10 ) and of 2 ) 247 acs elderly people that had classic , weary ( severe fatigue and shortness of breath ) , and diffuse symptoms.11 ) however , four or five groupings were also reported and this grouping differed by a target population , number of examined symptoms , or statistical methods.9)15 ) about 97% ( 56/57 ) of patients in cluster 3 , the atypical symptom cluster , did not experience any pain or discomfort anywhere but the chest . in addition , they reported the least pain intensity among the three clusters , and instead were more likely to have gastrointestinal symptoms , weakness and syncope compared to the other two clusters . it was also challenging for clinicians in triage to decide on a cardiac diagnosis.4)5 ) this delayed decision to seek care in this study was supported by the fact that the majority of the patients in cluster 3 did not attribute their symptoms to a cardiac origin until they went to the hospital . failure to recognize the symptoms correctly as cardiac problems was consistently recognized as a significant predicting factor for prehospital delay by lots of nursing researchers.16)-18 ) in this study , 16.1% of the sample had no chest pain , while previous studies showed that about 9 - 20% of an international sample9)19 ) and 9% of a korean sample experienced no chest pain.20 ) atypical symptoms at the hospital presentation were found in many clinical studies as a significant factor predicting lower use of thrombolytic therapy and adverse hospital outcomes.4)5 ) the present study also supports the view that the rate of all - causes of death ( 7/59 , 12.3% ) in cluster 3 is significantly higher than that of the other clusters ( < 6% ) . the patients who belonged to cluster 3 were significantly older than those of the other two clusters . only 5% of them referred to chest pain as a chief complaint at hospital presentation . this was consistent with a previous study that older ami patients were less likely to report classic pain and used fewer words to describe their discomfort compared with younger patients.4 ) this result was also supported by a recent study on older adults ( > 65 years ) based on symptoms they had experienced for a week prior to an acute event showing that fatigue emerged in all three clusters with an overall prevalence of 76.1% , while chest pain was experienced by only 56% of patients.11 ) the characteristics represented in cluster 3 were similar to the diffuse symptom cluster that was identified among 331 patients with acs by riegel and her colleagues.15 ) they reported that the diffuse symptom group with a low frequency of symptoms tended to be older than other groups . this finding supports the need for an increased index of supervision for patients with an atypical symptom presentation . the 10.3% ( 21/203 ) of patients with st - elevation myocardial infarction ( stemi ) belonged to cluster 3 , the atypical symptom group , in this study . we should have greater concern for these stemi patients with atypical presentation to increase their awareness of the benefit of rapid reperfusion therapy . further research would then be needed to examine the associations between symptom clusters and characteristics especially for stemi patients , to provide a useful guideline for early recognition of the symptoms during an acute coronary event . about 51% of the patients in cluster 3 had a history of diabetes and this was significantly higher compared to the other two clusters . this finding was supported by previous studies demonstrating that ami patients with atypical symptoms were more likely to be older , female , and diabetic.1)5)21 ) therefore , older adults at high risk for cvd , especially those who have diabetes , should pay more attention to their risks of developing ami and should engage in health - seeking behaviors when unusual symptoms occur . the values of hs - crp , lvef , and killip class were significantly increased and total length of hospital stay was significantly longer in those in the atypical symptom cluster . when age and socio - economic status were adjusted for , although the prevalence of maces was not different , the atypical symptom cluster predicted a 3.3 times higher risk of 1-year mortality compared with the typical chest pain cluster . this is consistent with a previous cluster study showing that the patients in the diffuse symptom group had significantly higher mortality at 2 years than those with the other three cluster groups in acs patients ( 17% vs. 2 - 5%).15 ) these findings support the view that intensive medical treatments are needed for ami patients with atypical presentations , especially for those who are old and diabetic . however , riegel 's study used a secondary data set , which was obtained from 5 multi - centers during a 2-year follow - up period for known cvd patients including history of ami and care of a cardiologist.15 ) this limits direct comparisons with our study , which was conducted in first - time ami patients through a direct interview at one hospital . further longitudinal research is needed to replicate and verify the differences in clinical outcome among symptom cluster groups . classic symptoms characterized by chest pain and associated symptoms were presented dominantly by clusters 1 and 2 , and the patients of cluster 2 who were the youngest complained of the largest number of acute symptoms . this finding was consistent with previous observational and cluster studies showing that classic chest symptom was a predictive factor of acs in younger patients < 70 years.1)9)21 ) they were more likely to be obese , current smokers , and to have dyslipidemia compared to other cluster groups . this supports the conclusion that it is essential to manage individuals with those risk factors to prevent cvd in younger adults . therefore , the study population can not be considered as representative of all ami patients in korea . other limitations include the retrospective nature of the study and the fact that patient information involved personal recollections . we have identified three distinct clusters among 14 acute symptoms ; typical chest pain , multiple symptom , and atypical symptom clusters . the patients with the atypical symptom cluster , which contained older and more diabetic patients , had worse clinical markers at hospital presentation and significantly higher mortality rates within the 1-year of follow - up compared to those in the other two clusters . this study poses a challenge to healthcare providers : how to utilize the symptom cluster to identify common symptom patterns and to develop educational strategies that may facilitate rapid identification of ami . also , we recommend that clinicians treat more intensively older ami patients who have vague or atypical symptoms at hospital presentation . therefore , the study population can not be considered as representative of all ami patients in korea . other limitations include the retrospective nature of the study and the fact that patient information involved personal recollections . we have identified three distinct clusters among 14 acute symptoms ; typical chest pain , multiple symptom , and atypical symptom clusters . the patients with the atypical symptom cluster , which contained older and more diabetic patients , had worse clinical markers at hospital presentation and significantly higher mortality rates within the 1-year of follow - up compared to those in the other two clusters . this study poses a challenge to healthcare providers : how to utilize the symptom cluster to identify common symptom patterns and to develop educational strategies that may facilitate rapid identification of ami . also , we recommend that clinicians treat more intensively older ami patients who have vague or atypical symptoms at hospital presentation .

background and objectivesidentifying symptom clusters of acute myocardial infarction ( ami ) and their clinical significance may be useful in guiding treatment seeking behaviors and in planning treatment strategy . the aim of this study was to identify clusters of acute symptoms and their associated factors that manifested in patients with first - time ami , and to compare clinical outcomes among cluster groups within 1-year of follow-up.subjects and methodsa total of 391 ami patients were interviewed individually using a structured questionnaire for acute and associated symptoms between march 2008 and june 2009 in korea.resultsamong 14 acute symptoms , three distinct clusters were identified by latent class cluster analysis : typical chest symptom ( 57.0% ) , multiple symptom ( 27.9% ) , and atypical symptom ( 15.1% ) clusters . the cluster with atypical symptoms was characterized by the least chest pain ( 3.4% ) and moderate frequencies ( 31 - 61% ) of gastrointestinal symptoms , weakness or fatigue , and shortness of breath ; they were more likely to be older , diabetic and to have worse clinical markers at hospital presentation compared with those with other clusters . cox proportional hazards regression analysis showed that , when age and gender were adjusted for , the atypical symptom cluster significantly predicted a higher risk of 1-year mortality compared to the typical chest pain cluster ( hazard ratio 3.288 , 95% confidence interval 1.087 - 9.943 , p=0.035).conclusionclusters of symptoms can be utilized in guiding a rapid identification of symptom patterns and in detecting higher risk patients . intensive treatment should be considered for older and diabetic patients with atypical presentation .

Introduction Subjects and Methods Study sample Data collection Statistical analysis Results Baseline clinical characteristics Symptom clusters and associated variables Clinical outcome by symptom clusters during 1-year of follow-up Discussion Limitations of this study Conclusions

the symptoms that patients are experiencing during acute coronary events are a key component in health care providers ' decision to initiate medical treatment and in further evaluation . in addition , patients ' earlier recognition of symptoms will facilitate the need for treatment seeking.1 ) a study reported that chest pain was the sole symptom in only 35% of cases and chest pain was accompanied by other symptoms in 57% of cases.2 ) overall , patients experienced a mean of 4.75 symptoms as part of the acute event of acute myocardial infarction ( ami).3 ) the absence of chest pain and the vagueness of symptoms might not be recognized as an indication of a cardiac problem , resulting in a delay in seeking medical care . in addition , this atypical presentation predicted lower use of thrombolytic therapy and was associated with hospital complications compared with the typical presentation.4)5 ) symptom clusters can be defined as multiple symptoms that are related to each other and are experienced concurrently.6)7 ) the clusters share a common etiology and a measurement of frequency , and severity of symptoms within clusters typically correlate with each other.8 ) they may also be useful in formulating criteria and evaluating patterns of symptoms . it is worthwhile to know how individual symptoms cluster by demographic and clinical characteristics of the subgroups in order to provide key information for early detection and diagnosis.9)10 ) identification of symptom clusters can be a clinically relevant way to detect specific demographic groups that are most likely to experience similar symptoms with readily identifiable characteristics.11)12 ) previous cluster studies on prehospital symptoms provided a useful grouping of acute coronary syndrome ( acs ) patients based on their experienced symptoms.9)-12 ) the clarification of prospective clinical outcomes for major adverse cardiac events ( mace ) should be helpful to health care providers in planning treatment strategies depending on the clusters . therefore , this study examined symptom clusters during acute events and clinical outcomes at 1-year follow - up by symptom cluster in patients with first - time ami . this clinical trial should enable health care providers to educate , more efficiently , individuals in the target populations at risk for cardiovascular disease ( cvd ) , to reduce prehospital delay time in korea , and to provide a useful treatment strategy for different symptomatic groups . the data for this study were obtained from first - time ami patients who were hospitalized for revascularization . inclusion criteria were : 1 ) patients who had undergone a first attack of st - elevation myocardial infarction ( stemi ) or non - st - elevation myocardial infarction ( nstemi ) , 2 ) verbally communicable , and 3 ) those who agreed to participate in the study . after obtaining approval of the institutional review board of the hospital , the principal investigator ( pi ) contacted eligible patients who were hospitalized in a cardiovascular care unit to ask them to participate in the study . this study was approved by the clinical ethics committee at cnuh before data collection was carried out . the pi interviewed the patients who had undergone a percutaneous coronary intervention using a semi - structured questionnaire . each patient was asked about his chief complaints and associated symptoms that he experienced during the acute phase of an ami . patients were also asked about risk factors , their chronic disease , and past / family history questions . all acute symptoms of patients were coded as a categorical variable ; 1 point was given for ' present ' and 0 points for ' none ' . , chicago , il , usa ) data set were analyzed by latent class ( lc ) cluster analysis using latent gold software , version 3.0.13 ) cluster analysis is defined as the classification of similar objects into the form of groups , which refers to the parameters of cluster ; that is , to its cluster - specific means , variances , and covariances that have a geometrical interpretation.13 ) lc cluster analysis is a statistical model - based clustering technique and can include covariates to predict class membership . the most popular set of model selection tools in lc cluster analysis are information criteria such as akaike information criterion ( aic ) and the bayesian information criterion ( bic).14 ) in this study , decreases in the values of bic and aic were minimal between the three- and four - cluster models . the cluster membership of 3 clusters was conversed to an spss data set to identify the membership and associated factors . bivariate analyses using -tests , t - tests , and analysis of variance were conducted to identify the relationship between cluster assignment and patients ' characteristics . cox proportional hazards regression analysis was used to test a cluster membership as an independent predictor of 1-year mortality and maces . the data for this study were obtained from first - time ami patients who were hospitalized for revascularization . inclusion criteria were : 1 ) patients who had undergone a first attack of st - elevation myocardial infarction ( stemi ) or non - st - elevation myocardial infarction ( nstemi ) , 2 ) verbally communicable , and 3 ) those who agreed to participate in the study . after obtaining approval of the institutional review board of the hospital , the principal investigator ( pi ) contacted eligible patients who were hospitalized in a cardiovascular care unit to ask them to participate in the study . this study was approved by the clinical ethics committee at cnuh before data collection was carried out . the pi interviewed the patients who had undergone a percutaneous coronary intervention using a semi - structured questionnaire . each patient was asked about his chief complaints and associated symptoms that he experienced during the acute phase of an ami . severity of chest pain or discomfort during the acute phase was asked and recoded on a 1 - 10 scale . patients were also asked about risk factors , their chronic disease , and past / family history questions . all acute symptoms of patients were coded as a categorical variable ; 1 point was given for ' present ' and 0 points for ' none ' . , chicago , il , usa ) data set were analyzed by latent class ( lc ) cluster analysis using latent gold software , version 3.0.13 ) cluster analysis is defined as the classification of similar objects into the form of groups , which refers to the parameters of cluster ; that is , to its cluster - specific means , variances , and covariances that have a geometrical interpretation.13 ) lc cluster analysis is a statistical model - based clustering technique and can include covariates to predict class membership . the most popular set of model selection tools in lc cluster analysis are information criteria such as akaike information criterion ( aic ) and the bayesian information criterion ( bic).14 ) in this study , decreases in the values of bic and aic were minimal between the three- and four - cluster models . the cluster membership of 3 clusters was conversed to an spss data set to identify the membership and associated factors . bivariate analyses using -tests , t - tests , and analysis of variance were conducted to identify the relationship between cluster assignment and patients ' characteristics . cox proportional hazards regression analysis was used to test a cluster membership as an independent predictor of 1-year mortality and maces . the majority of patients ( 71.1% ) were men and the average age was 63.810.3 years ( range 26 - 89 ) . about 12% of the patients had a past history of angina or stroke , and 19.7% had a family history of cvd including hypertension , stroke or ischemic heart disease ( table 1 ) . the size of cluster 1 was the biggest ( n=223 , 57.0% ) , followed by cluster 2 ( n=109 , 27.9% ) and cluster 3 ( n=59 , 15.1% ) . all cluster membership within each symptom cluster was significantly different ( p<0.01 ) except palpitation ( p=0.527 ) . the patients in cluster 1 and cluster 2 experienced a high frequency of chest pain / discomfort ( 100% and 94.5% , respectively ) and a moderate cold sweating . among the three groups , cluster 3 had the least chest pain and moderate frequencies of shortness of breath and weakness or fatigue . we named cluster 1 , 2 , and 3 as typical chest pain , multiple symptoms , and atypical symptom clusters , respectively . bivariate analyses showed that patients of cluster 2 were the youngest ( 58.112.8 years ) and those of cluster 3 were the oldest ( 69.312.3 years ) among the three clusters ( p<0.001 ) . as for cvd risk factors , the patients of cluster 2 were more likely to be overweight bmi > 25 kg / m ( p=0.002 ) and current smokers ( p=0.031 ) , and the patients of cluster 3 were more likely to have diabetes ( p<0.001 ) . 94.3% of cluster 3 reported that they did not perceive their symptoms as cardiac in origin and they had the fewest complaints of acute symptoms and the lowest self - reported intensity score of chest pain among the three groups ( p<0.001 ) . the patients of cluster 3 had the highest value for high sensitivity c - reactive protein ( hs - crp ) and the lowest in left ventricular ejection fraction ( lvef ) , which were checked immediately after hospital admission ; they had the longest stay at hospital among the three groups ( p<0.001 ) ( table 2 ) . however , the prevalence of cardiac or non - cardiac deaths turned out to be significantly different by clusters ( p=0.022 ) ( table 3 ) . multivariate cox proportional hazards regression analysis demonstrated that when age , marital status and monthly income were adjusted for , patients in cluster 3 predicted a significantly higher risk of 1-year mortality compared to those in cluster 1 , a typical chest pain cluster ( hazard ratio 3.288 , 95% confidence interval 1.087 - 9.943 , p=0.035 ) ( table 4 ) . the majority of patients ( 71.1% ) were men and the average age was 63.810.3 years ( range 26 - 89 ) . about 12% of the patients had a past history of angina or stroke , and 19.7% had a family history of cvd including hypertension , stroke or ischemic heart disease ( table 1 ) . the size of cluster 1 was the biggest ( n=223 , 57.0% ) , followed by cluster 2 ( n=109 , 27.9% ) and cluster 3 ( n=59 , 15.1% ) . all cluster membership within each symptom cluster was significantly different ( p<0.01 ) except palpitation ( p=0.527 ) . the patients in cluster 1 and cluster 2 experienced a high frequency of chest pain / discomfort ( 100% and 94.5% , respectively ) and a moderate cold sweating . among the three groups , cluster 3 had the least chest pain and moderate frequencies of shortness of breath and weakness or fatigue . we named cluster 1 , 2 , and 3 as typical chest pain , multiple symptoms , and atypical symptom clusters , respectively . bivariate analyses showed that patients of cluster 2 were the youngest ( 58.112.8 years ) and those of cluster 3 were the oldest ( 69.312.3 years ) among the three clusters ( p<0.001 ) . as for cvd risk factors , the patients of cluster 2 were more likely to be overweight bmi > 25 kg / m ( p=0.002 ) and current smokers ( p=0.031 ) , and the patients of cluster 3 were more likely to have diabetes ( p<0.001 ) . 94.3% of cluster 3 reported that they did not perceive their symptoms as cardiac in origin and they had the fewest complaints of acute symptoms and the lowest self - reported intensity score of chest pain among the three groups ( p<0.001 ) . the patients of cluster 3 had the highest value for high sensitivity c - reactive protein ( hs - crp ) and the lowest in left ventricular ejection fraction ( lvef ) , which were checked immediately after hospital admission ; they had the longest stay at hospital among the three groups ( p<0.001 ) ( table 2 ) . however , the prevalence of cardiac or non - cardiac deaths turned out to be significantly different by clusters ( p=0.022 ) ( table 3 ) . multivariate cox proportional hazards regression analysis demonstrated that when age , marital status and monthly income were adjusted for , patients in cluster 3 predicted a significantly higher risk of 1-year mortality compared to those in cluster 1 , a typical chest pain cluster ( hazard ratio 3.288 , 95% confidence interval 1.087 - 9.943 , p=0.035 ) ( table 4 ) . three clusters of acute symptoms in this study were consistent with previous studies of 1 ) 1270 ami women that was made up of older / silent asymptomatic , diverse / mildly symptomatic , and younger / multiple distressing symptom groups,10 ) and of 2 ) 247 acs elderly people that had classic , weary ( severe fatigue and shortness of breath ) , and diffuse symptoms.11 ) however , four or five groupings were also reported and this grouping differed by a target population , number of examined symptoms , or statistical methods.9)15 ) about 97% ( 56/57 ) of patients in cluster 3 , the atypical symptom cluster , did not experience any pain or discomfort anywhere but the chest . in addition , they reported the least pain intensity among the three clusters , and instead were more likely to have gastrointestinal symptoms , weakness and syncope compared to the other two clusters . it was also challenging for clinicians in triage to decide on a cardiac diagnosis.4)5 ) this delayed decision to seek care in this study was supported by the fact that the majority of the patients in cluster 3 did not attribute their symptoms to a cardiac origin until they went to the hospital . failure to recognize the symptoms correctly as cardiac problems was consistently recognized as a significant predicting factor for prehospital delay by lots of nursing researchers.16)-18 ) in this study , 16.1% of the sample had no chest pain , while previous studies showed that about 9 - 20% of an international sample9)19 ) and 9% of a korean sample experienced no chest pain.20 ) atypical symptoms at the hospital presentation were found in many clinical studies as a significant factor predicting lower use of thrombolytic therapy and adverse hospital outcomes.4)5 ) the present study also supports the view that the rate of all - causes of death ( 7/59 , 12.3% ) in cluster 3 is significantly higher than that of the other clusters ( < 6% ) . only 5% of them referred to chest pain as a chief complaint at hospital presentation . this was consistent with a previous study that older ami patients were less likely to report classic pain and used fewer words to describe their discomfort compared with younger patients.4 ) this result was also supported by a recent study on older adults ( > 65 years ) based on symptoms they had experienced for a week prior to an acute event showing that fatigue emerged in all three clusters with an overall prevalence of 76.1% , while chest pain was experienced by only 56% of patients.11 ) the characteristics represented in cluster 3 were similar to the diffuse symptom cluster that was identified among 331 patients with acs by riegel and her colleagues.15 ) they reported that the diffuse symptom group with a low frequency of symptoms tended to be older than other groups . this finding supports the need for an increased index of supervision for patients with an atypical symptom presentation . the 10.3% ( 21/203 ) of patients with st - elevation myocardial infarction ( stemi ) belonged to cluster 3 , the atypical symptom group , in this study . we should have greater concern for these stemi patients with atypical presentation to increase their awareness of the benefit of rapid reperfusion therapy . further research would then be needed to examine the associations between symptom clusters and characteristics especially for stemi patients , to provide a useful guideline for early recognition of the symptoms during an acute coronary event . about 51% of the patients in cluster 3 had a history of diabetes and this was significantly higher compared to the other two clusters . this finding was supported by previous studies demonstrating that ami patients with atypical symptoms were more likely to be older , female , and diabetic.1)5)21 ) therefore , older adults at high risk for cvd , especially those who have diabetes , should pay more attention to their risks of developing ami and should engage in health - seeking behaviors when unusual symptoms occur . the values of hs - crp , lvef , and killip class were significantly increased and total length of hospital stay was significantly longer in those in the atypical symptom cluster . when age and socio - economic status were adjusted for , although the prevalence of maces was not different , the atypical symptom cluster predicted a 3.3 times higher risk of 1-year mortality compared with the typical chest pain cluster . this is consistent with a previous cluster study showing that the patients in the diffuse symptom group had significantly higher mortality at 2 years than those with the other three cluster groups in acs patients ( 17% vs. 2 - 5%).15 ) these findings support the view that intensive medical treatments are needed for ami patients with atypical presentations , especially for those who are old and diabetic . however , riegel 's study used a secondary data set , which was obtained from 5 multi - centers during a 2-year follow - up period for known cvd patients including history of ami and care of a cardiologist.15 ) this limits direct comparisons with our study , which was conducted in first - time ami patients through a direct interview at one hospital . further longitudinal research is needed to replicate and verify the differences in clinical outcome among symptom cluster groups . classic symptoms characterized by chest pain and associated symptoms were presented dominantly by clusters 1 and 2 , and the patients of cluster 2 who were the youngest complained of the largest number of acute symptoms . this finding was consistent with previous observational and cluster studies showing that classic chest symptom was a predictive factor of acs in younger patients < 70 years.1)9)21 ) they were more likely to be obese , current smokers , and to have dyslipidemia compared to other cluster groups . this supports the conclusion that it is essential to manage individuals with those risk factors to prevent cvd in younger adults . therefore , the study population can not be considered as representative of all ami patients in korea . we have identified three distinct clusters among 14 acute symptoms ; typical chest pain , multiple symptom , and atypical symptom clusters . the patients with the atypical symptom cluster , which contained older and more diabetic patients , had worse clinical markers at hospital presentation and significantly higher mortality rates within the 1-year of follow - up compared to those in the other two clusters . this study poses a challenge to healthcare providers : how to utilize the symptom cluster to identify common symptom patterns and to develop educational strategies that may facilitate rapid identification of ami . also , we recommend that clinicians treat more intensively older ami patients who have vague or atypical symptoms at hospital presentation . therefore , the study population can not be considered as representative of all ami patients in korea . we have identified three distinct clusters among 14 acute symptoms ; typical chest pain , multiple symptom , and atypical symptom clusters . the patients with the atypical symptom cluster , which contained older and more diabetic patients , had worse clinical markers at hospital presentation and significantly higher mortality rates within the 1-year of follow - up compared to those in the other two clusters . this study poses a challenge to healthcare providers : how to utilize the symptom cluster to identify common symptom patterns and to develop educational strategies that may facilitate rapid identification of ami . also , we recommend that clinicians treat more intensively older ami patients who have vague or atypical symptoms at hospital presentation .

quiescent hematopoietic stem cells ( hscs ) reside in the bone marrow ( bm ) near the endosteum , in specific microenvironments called endosteal niches [ 13 ] . low numbers of hscs leave the bm and enter the blood stream in steady state conditions [ 4 , 5 ] , thereafter establishing in extramedullary sites of hematopoiesis like the spleen and liver , or returning to the bm [ 6 , 7 ] . recent studies involve bone resorbing osteoclasts in the homeostasis and mobilization of hsc and hematopoietic progenitor cells ( hspc ) in conditions of stress , although this function remains controversial [ 811 ] . indeed , stimulation of osteoclasts activity by receptor activator of nuclear factor kappa - b ligand ( rankl ) increases hspc mobilization through secretion of cathepsin k , which cleaves c - kit - ligand and stromal cell - derived factor 1 ( sdf-1 ) , required for maintenance of endosteal niches . phlebotomy or lipopolysaccharide ( lps ) injection , two models of physiological stress , also increase osteoclastogenesis and trigger hspc egression . furthermore , the osteoclast inhibiting hormone calcitonin was reported to decrease hspc mobilization in response to lps injection . however , contradictory evidences demonstrate that inhibition of osteoclast activity by bisphosphonate does not impair hspc mobilization in response to granulocyte - colony stimulating factor ( g - csf ) treatment [ 1 , 10 ] , suggesting that osteoclasts may only intervene in certain types of hematopoietic stresses . mobilization of hspc is triggered by viral , bacterial , and plasmodium infection [ 1216 ] , as well as by phenylhydrazine- ( phz- ) induced anemia in mice [ 1719 ] , but the contribution of osteoclasts in these responses has not been yet characterized . blood stage malaria causes systemic inflammation and acute hemolytic anemia in mice , which are events that trigger stress hematopoiesis to generate phagocytic cells involved in parasite clearance and new erythrocytes to cope with anemia [ 2124 ] . in murine malaria models , stress hematopoiesis associates with egression of hspc from the bm to spleen , which becomes the major site of erythropoiesis [ 25 , 26 ] and myelopoiesis [ 27 , 28 ] this mobilization is indirectly involved in the development of splenomegaly [ 16 , 29 ] and extramedullary myelopoiesis , which are required in the resolution of acute parasitemia , through removal of parasitized red blood cells ( prbcs ) by splenic macrophages [ 3032 ] . we recently reported profound alterations in bone remodelling in mice with malarial or phz - induced hemolytic anemia , characterized by decreased bone formation , reduced osteoclastogenesis , and balance resulting in bone mass loss . knowing these alterations , the contribution of osteoclasts in hspc egression , stress erythropoiesis , and resolution of plasmodium chabaudi adami dk infection was investigated in calcitonin - treated mice . calcitonin is a peptide hormone known to inhibit osteoclasts bone resorption and osteoclast differentiation [ 3436 ] . our data indicate that calcitonin causes a significant drop in the basal activity of osteoclasts and partially interferes with the egression of hspc from bm to spleen in blood stage malaria . however , the development of splenic stress erythropoiesis is not affected by this treatment . unexpectedly , treatment with calcitonin exacerbates parasitemia in vivo and interferes with macrophage differentiation and proliferation in vivo and in vitro . all procedures in mice were approved by the animal care committee of the universit du qubec montral ( protocol 0210 - 677 - 0211 ) and according to relevant national and international guidelines . female balb / c mice ( charles river , canada ) aged 46 weeks were used in all experiments . mice were maintained in temperature controlled conditions ( 22 1c ) under a 12-hour light cycle ( 7:00 to 19:00 ) , having free access to osmosed water and rodent chow 5075 ( charles river , canada ) . mice were infected with the p. c. adami dk nonlethal strain , originally isolated from thamnomys rutilans and kindly provided by dr . david walliker , university of edinburgh ( congo - brazzaville , 1972 ) . to evaluate the contribution of osteoclasts in hspc mobilization during plasmodium infection , mice received 200 l salmon calcitonin ( 5 g / ml in pbs ) ( biotrend , switzerland ) during 5 consecutive days by the intraperitoneal route and were infected with 10 p. c. adami dk prbc in pbs by the intravenous route on the 3rd day of treatment . mice were euthanized on days 5 , 8 , and 10 after infection for analysis of hematopoietic parameters and cytokines in plasma by elisa ( biolegend , usa ) . to assess the effect of calcitonin - dependent osteoclasts inhibition on the erythropoietic responses to experimental hemolytic and nonhemolytic anemia , mice were injected intraperitoneally with 200 l of salmon calcitonin ( 5 g / ml in pbs ) ( biotrend , switzerland ) during 5 consecutive days . hemolytic anemia was induced in mice by peritoneal administration of phz ( sigma aldrich , canada ) at 40 mg / kg of body weight in 200 l of pbs , on the 3rd and 4th days of calcitonin treatment . to induce nonhemolytic anemia , the submandibular vein was incised with a 4 mm lancet ( medipoint , usa ) and 300 l of blood was withdrawn on the 3rd day of calcitonin treatment . mice received an equal volume of physiologic saline solution by the intraperitoneal route immediately after phlebotomy . in certain experiments with infected mice , 100 l did - loaded anionic liposomes ( formumax , usa ) were administered intravenously one day prior to calcitonin treatment . hemoglobin levels were monitored daily by diluting 2 l tail - vein blood in 500 l drabkin 's solution ( sigma aldrich , canada ) . following 15 min of incubation in the dark , 200 l of blood was transferred into 96-well plates ( costar , usa ) in duplicate and absorbance was measured at 540 nm in a microplate reader . values were converted to g / dl using a standard curve of rat hemoglobin ( sigma aldrich , canada ) prepared in drabkin 's solution . the percentages of reticulocytes in blood , 1 l of tail - vein blood was collected in 1 ml of pbs . the cell suspensions were then stained with anti - cd71-fitc antibody ( biolegend , usa ) and incubated at 4c for 30 min . data were acquired with an accuri c6 ( becton dickinson , usa ) and analyzed with the flowjo software ( tree star ) . in parallel , parasitemia was measured daily in methanol fixed blood smears stained with a 10% giemsa solution in pbs during 15 minutes . bone marrow single - cell suspensions were prepared by removal of tibia and femur epiphyses and flushing the marrow into 100 l of pbs by centrifugation of 300 g during 5 minutes . spleen single - cell suspensions were obtained by mechanically dissociating the spleen in a 60 mm petri dish containing 5 ml of pbs . recovered cells from the bm and the spleen were counted and resuspended at 10 cells / ml in rpmi 1640 , supplemented with 10% , penicillin ( 100 u / ml ) , and streptomycin ( 100 mg / ml ) ( invitrogen , usa ) . cells were distributed at 100 l / tube into 12 75 polypropylene tubes ( sarstedt , canada ) . for assessment of erythroid populations , cells were stained with 1 g of ter119-pe and anti - cd71-fitc ( clone ri7217 ) antibodies . for additional analysis , rbcs in spleen cell suspensions were lysed with the rbc lysing buffer hybri - max ( sigma aldrich , canada ) . hscs were stained with 10 l of the biotinylated lineage antibody cocktail ( cd3 , ly6/6c , b220 , cd11b , and ter119 ) and 1 g of streptavidin - apc , anti - sca1-fitc ( clone d7 ) , and anti - cd117-pe ( clone 28b ) . mature macrophages were stained with 1 g of anti - cd11b - fitc ( clone m1/70 ) and anti - f4/80-pe ( clone bm8 ) . dead cells were excluded from analysis with 7-amino - actinomycin d ( 7-aad ) ( biolegend , usa ) staining . analytical flow cytometry was performed on an accuri c6 ( becton dickinson , usa ) and data was analyzed using flojo software ( tree star ) . elisa for quantification of macrophage colony - stimulating factor ( m - csf , promokine , canada ) and c - terminal telopeptide ( ctx , mybiosource , usa ) were performed following the manufacturer 's instructions . briefly , 96-well elisa maxisorp plates ( nunc , usa ) were coated overnight with 60 l of capture antibody at 4c . wells were washed 4 times with washing buffer ( 0 , 05% tween 20 in pbs , 300 l ) and incubated with blocking buffer ( 1% bsa in pbs ) for 1 hour . following 4 washes , 60 l of samples and standards was added and incubated for 2 hours at room temperature , the plates were washed , and 60 l of detection antibody was added and incubated for 2 hours at room temperature . after 4 washes , 60 l of avidin - hrp was added and incubated for 30 min at room temperature , after which wells were washed 4 times . sixty l of tetramethylbenzidine ( tmb ) substrate solution ( sigma - aldrich , canada ) was added and incubated for 20 min at room temperature in the dark , and the reaction was stopped by adding 60 l 1 m h2so4 . single - cell suspensions from the spleen and bm were prepared at 1 10 and 2 10 cells / ml , respectively , in rpmi 1640 , supplemented with 10% , penicillin ( 100 u / ml ) , and streptomycin ( 100 mg / ml ) ( invitrogen , usa ) and 0.3 ml was added into 3 ml of methocult 03434 ( stemcell technologies , usa ) . the cell suspension was vortexed , allowed to stand for 10 min , and dispensed into 35 mm culture dishes ( stemcell technologies , usa ) using a 16-gauge blunt - end needle and a 3 ml syringe , 1.1 ml per dish in duplicate . the cultures were incubated at 37c , 5% co2 in air , and 95% humidity for 8 days after which colony - forming unit macrophages ( cfu - m ) were identified and counted . single - cell suspensions from bm were prepared at 1 10 cells / ml in rpmi 1640 supplemented with 10% fbs , penicillin ( 100 u / ml ) ( sigma , usa ) , streptomycin ( 100 mg / ml ) ( sigma , usa ) , 30% l929 cells supernatant ( atcc , usa ) , and 0 , 125 , 250 , and 500 ng / ml of salmon calcitonin ( biotrend , switzerland ) . after 6 days of incubation at 37c , 5% co2 in air , and 95% humidity , cells were harvested and macrophage differentiation was assessed by flow cytometry with cd11b - fitc ( biolegend , usa ) and f4/80-pe ( biolegend , usa ) staining . to evaluate cell proliferation , a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium inner salt / phenazine methosulfate ( mts / pms ) solution ( promega , usa ) after 2 h incubation at 37c , 5% co2 in air , and 95% humidity , absorbance was measured at 490 nm in a microplate reader . statistical analysis was performed with a one - way anova analysis of variance followed by tukey 's posttest for comparison of more than two groups and an unpaired t - test for comparison of two groups . p < 0.05 ; p < 0.01 ; p < 0.001 represent comparisons in respect to respective controls . in order to investigate the contribution for osteoclasts in malarial stress hematopoiesis with the p. c. adami dk infection model , balb / c mice were treated with calcitonin two days prior to infection and three days following infection . resolution of infection and subsequent stress erythropoiesis were monitored and compared in control and calcitonin - treated mice . for comparative purposes , as expected , significantly lower plasma concentrations of ctx were measured in calcitonin - treated mice on day 5 after infection , confirming inhibition of osteoclast activity ( figure 1(a ) ; p < 0.05 ) . parasitemia was exacerbated in calcitonin - treated mice throughout patent infection ( figure 1(b ) ; p < 0.01 ) , resulting in higher cumulative parasite burden ( figure 1(c ) ; p < 0.001 ) . consequent to higher parasitemia , hemoglobin levels were relatively lower in calcitonin - treated mice on days 11 and 12 after infection ( figure 1(d ) ; p < 0.01 and p < 0.05 , resp . ) . concurrent with enhanced anemia , the percentages of reticulocytes ( cd71 cells ) were higher in calcitonin - treated mice on day 14 after infection ( figure 1(e ) ) . the erythropoietic responses to anemia were also followed in phz - treated or phlebotomized mice ( figures 2(a ) and 2(b ) ) . in accordance with the responses to infection , comparable reticulocytosis occurred in control and calcitonin - treated mice under these two experimental conditions ( figures 2(c ) and 2(d ) ) . considering the crucial role of splenic stress hematopoiesis in the control of plasmodium infection [ 16 , 28 ] and anemia [ 21 , 25 , 26 , 38 ] , the effect of calcitonin on splenic hematopoietic parameters was investigated early ( day 5 after infection ) , at peak parasitemia ( day 8) , and following resolution of peak infection ( day 10 ) . as expected , spleen cellularity increased in mice with malaria ( figure 3(a ) ) , and this effect was apparent and comparable in control and calcitonin - treated mice until day 5 after infection ( figure 3(a ) ) . however , reduced numbers of splenocytes ( figure 3(a ) ; p < 0.001 ) and reduced splenic index ( supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/3909614 ; p < 0.01 ) were measured in calcitonin - treated mice at peak infection , indicating milder splenomegaly . parallel monitoring of the erythroid populations confirmed higher numbers of splenic cd71 ter119 cells in infected mice from day 5 after infection , which increased thereafter ( figure 3(b ) ; p < 0.001 ) ; this response was significantly attenuated in calcitonin - treated mice at the time of peak infection ( figure 3(b ) ; p < 0.01 ) . in respect to splenic lineage negative cells , their numbers increased significantly compared to uninfected controls only after peak infection . major differences were only apparent on day 8 after infection , reflected by significantly lower lineage negative cell numbers in calcitonin - treated infected mice ( figure 3(c ) ; p < 0.01 ) . in contrast , administration of calcitonin significantly reduced the number of splenic lineage negative ckit sca1 cells ( lsk ) at peak infection , as well as 10 days after infection ( figure 3(e ) ; p < 0.01 , 0.001 , resp . ) . the analysis of lymphoid populations in spleen revealed increased numbers of cd90 cells ( supplementary figure 2(a ) ; p < 0.001 ) and cd19 cells ( supplementary figure 2(b ) ; p < 0.001 ) at peak infection ; treatment with calcitonin resulted in lower cd19 cell numbers ( supplementary figure 2(b ) ; p < 0.05 ) . in respect to splenic myeloid cells , calcitonin did not affect the characteristic rise in cd11b cells ( supplementary figure 2(c ) ; p < 0.001 ) and fceria ckit cells at peak infection ( supplementary figure 2(d ) ; p < 0.01 ) . analysis of bm hematopoietic parameters confirmed the drops previously reported in bm cellularity of infected mice ( figure 4(a ) ) , and this effect was not modified by calcitonin . evaluation of the erythroid populations in the bm showed similar drops in cd71 ter119 cell numbers in the two groups of infected mice ( figure 4(b ) ) . compared to uninfected mice , the number of bm lineage negative cells significantly dropped on days 8 and 10 after infection in infected mice ( figure 4(c ) ) , becoming relatively higher in calcitonin - treated mice when compared to infected controls at peak infection ( figure 4(c ) ; p < 0.01 ) . the numbers of lsk cells also increased in the bm of infected mice and were significantly higher in calcitonin - treated mice on day 8 after infection ( figure 4(d ) , p < 0.01 ) . as reviewed by chua et al . , phagocytic macrophages are essential for clearance of prbcs , and dysregulation of their number or function negatively affects resolution of plasmodium infection . to determinate whether the increased parasitemia observed in calcitonin - treated mice was due to restricted production of macrophages , the numbers of f4 - 80 cells were analyzed in the bm and spleen . relative to uninfected mice , the infection did not provoke major effects on bm f4/80 cells , but their numbers were significantly higher in calcitonin - treated mice on day 10 after infection ( figure 5(a ) ; p < 0.05 ) . in contrast to the bm , the infection caused an important increase in splenic f4/80 cells , and this effect was significantly attenuated in calcitonin - treated mice on days 8 and 10 after infection ( figure 5(b ) ; p < 0.01 , 0.001 , resp . ) . analysis of progenitors colony forming unit - macrophage ( cfu - m ) did not reveal major modifications in the bm of infected mice ( figure 5(c ) ) , but lower numbers of cfu - m generated from the spleen of calcitonin - treated mice on day 8 after infection ( figure 5(d ) ; p < 0.05 ) . interestingly , serum m - csf levels remained comparable in infected and uninfected mice ( supplementary figure 3 ) . altogether , our results suggest altered production of macrophages in calcitonin - treated and infected mice . to discriminate the impact of calcitonin on preexisting versus de novo generated macrophages , did anionic liposomes were administered prior to infection , to specifically stain all phagocytic cells including macrophages . did ( diic185 ) is a viable lipophilic fluorescent dye weakly fluorescent in water , but highly fluorescent and photostable when incorporated into cell membranes , and allows discriminating mature macrophages ( did positive ) from de novo generated macrophages ( did negative ) ( supplementary figure 4 ) . the lipid composition of the anionic liposomes was originally designed to efficiently deplete phagocytic cells in vivo and in vitro when loaded with clodronate , and as nonphagocytic cells are not affected , this tool is efficient for targeting of macrophages [ 41 , 42 ] . compared to uninfected controls , in which the majority of bm macrophages were did , approximately 50% f4/80 did cells were found in infected mice on day 10 after infection , suggesting mobilization of mature macrophages from the bm and production of new ( did ) macrophages ( figure 5(e ) ; p < 0.01 ) . the numbers of splenic f4/80 did cells drastically increased on day 10 after infection , indicating recruitment of mature macrophages to the spleen after peak infection ( figure 5(f ) ; p < 0.001 ) . f4/80 did cells also increased during infection , but this response was half - fold in calcitonin - treated mice ( figure 5(f ) ; p < 0.01 ) , suggesting deficient de novo production of macrophages . calcitonin was originally documented to inhibit osteoclasts bone resorption activity and osteoclast differentiation [ 3436 ] . in order to investigate whether the lower macrophage numbers found in calcitonin - treated mice are concurrent with direct action of this hormone on myeloid precursors , the impact of calcitonin on differentiation of macrophage progenitors was evaluated in vitro . our data indicates reduced proliferation ( figure 6(a ) ; p < 0.01 ) and lower numbers of of f4/80 cells in bm cultures stimulated with m - csf ( figure 6(b ) ; p < 0.01 ) . herein , we evaluated the relative contribution for osteoclasts in the egression of hspc from the bm to the spleen during blood stage malaria , using the well - characterized p. c. adami dk infection model in balb / c mice . we previously compared bone remodelling markers in mice suffering from acute hemolytic anemia caused by p. c. adami infection or phz injection . these conditions are characterized by reduced bone mineralization and bone formation , as well as reduced numbers of osteoclasts and osteoclasts progenitors in the bm . considering that the levels of ctx , indicative of osteoclast - dependent bone resorption , remained comparable in infected , phz - treated , and naive mice , we concluded that the decreased bone mass density found in mice with hemolytic anemia was concurrent with imbalance favoring bone resorption , as has been reported for other hemolytic conditions [ 43 , 44 ] . herein , calcitonin was administered to mice three days prior to and during the first two days of infection to block bone resorption . interestingly , plasma ctx levels were comparable in naive and infected mice on day 5 after infection , suggesting no major stimulation of bone resorption in this murine malaria model . the fact that ctx levels were lower in calcitonin - treated mice confirmed an inhibitory action of this peptide hormone on steady state osteoclast activity . in our experimental malaria model , the number of hspc increased in a comparable manner in the bm of calcitonin - treated and control - infected mice on day 5 after infection , with no major effects in the spleen . however , at peak infection , calcitonin - treated mice had higher numbers of hspc in the bm and lower numbers of hspc in the spleen , also developing relatively milder splenomegaly . these results suggest that osteoclast - dependent mobilization of hspc to the spleen is partially responsible for splenomegaly in murine malaria . comparable recruitment of hpsc to the spleen has been reported in c57bl/6 mice infected with p. chabaudi as . in this malaria model , ifn- signalling and concurrent secretion of c - c motif ligand 2/7 ( ccl2/ccl7 ) chemokines by bm stromal cells seem required for hspc mobilization . interestingly , ifn- signalling indirectly stimulates the activity of osteoclasts , which may stimulate their effects in hspc mobilization . in opposition to reduced hspc mobilization appearance of circulating reticulocytes is considered an ultimate marker for functional erythropoiesis ( as reviewed in ) , and our data indicate comparable reticulocytosis in calcitonin - treated and control mice in response to malarial anemia , despite reduced mobilization of hspc and reduced numbers of erythroid progenitors in the spleen . accordingly , calcitonin did not affect reticulocytosis in mice recovering from anemia caused by bleeding or phz - treatment . calcitonin - treated mice developed higher parasitemia throughout the period of patent infection , resulting in enhanced anemia on days 11 and 12 after infection . in logic accordance with an exacerbated anemia , higher reticulocytosis occurred in these mice on day 14 after infection , which suggested that the drop in splenic erythroid progenitors on day 8 after infection did not have major effects on the recovery from anemia . in this context , stress burst forming unit - erythroid ( bfu - e ) with self - renewal properties have been reported , which efficiently generate ckit cd71 ter119 cells in response to erythropoietin ( epo ) , bone morphogenetic protein-4 ( bmp-4 ) , stem cell factor ( scf ) , and hypoxia [ 47 , 48 ] . as comparable numbers of ckit cd71 ter119 cells were measured in control and calcitonin - treated mice on day 8 after infection ( data not shown ) , we conclude that osteoclast - dependent hspc mobilization from the bm is not essential for splenic stress erythropoiesis in murine blood stage malaria . analysis of lymphocytic and granulocytic cell populations in the spleen at peak infection revealed no modifications in the numbers of t cells , mast cells , and myeloid cells in calcitonin - treated mice , although decreased numbers of b cells were noticed . the exacerbated parasitemia measured in calcitonin - treated mice may be concurrent with compromised parasite killing by macrophages . indeed , an important proportion of prbcs circulate in the blood stream and their elimination by spleen red pulp macrophages is pivotal for the control of infection [ 5052 ] . calcitonin - treated mice , which developed exacerbated parasitemia , had fewer numbers of macrophages in the spleen on days 8 and 10 after infection and , accordingly , reduced numbers of cfu - m generated from the spleen of these mice . b cells only intervene in the late resolution of blood stage malaria , and the reduction caused by calcitonin on cd19 cells numbers is not expected to affect the resolution of p. c. adami infection . in contrast , c - c chemokine receptor type 2- ( ccr2- ) dependent migration of monocytes from the bm to spleen and splenic myelopoiesis are essential for the control of p. chabaudi infection . administration of did - loaded liposomes prior to calcitonin treatment and infection efficiently labelled macrophages , allowing following their fate and distinguishing newly generated macrophages in vivo . in respect to noninfected controls , we evidenced comparable drops in the number of did macrophages in the bm and their concurrent increase in the spleen during infection . these results suggest that calcitonin does not alter the mobilization of monocytes / macrophages from the bm nor their recruitment to spleen . however , the fact that the numbers of splenic did macrophages were significantly lower in calcitonin - treated mice on day 10 after infection rather indicates impaired de novo generation of macrophages in mice treated with this hormone . the generally accepted paradigm proposes the recruitment of circulating monocytes to inflammatory site whereupon they differentiate into macrophages ( as reviewed in [ 24 , 55 , 56 ] ) . however , recent studies have reported local proliferation of resident macrophages in response to inflammation [ 57 , 58 ] and this process seems driven by m - csf . calcitonin did not alter m - csf levels in infected mice ; we hypothesize that it may interfere with m - csf - dependent signalling in certain bone marrow precursor cells and that impaired recruitment of hspc to the spleen may further compromise myelopoiesis . it is generally accepted that osteoclasts are the major cells responding to calcitonin ( as reviewed in ) . the hormone inhibits contraction and motility , as well as secretion of acid phosphatase [ 6163 ] . expression of the calcitonin receptor occurs during the differentiation of myeloid precursors into osteoclasts , requiring simultaneous action of m - csf and rankl [ 64 , 65 ] . as such , the inhibition caused by calcitonin on macrophage differentiation is puzzling and suggests that common bm precursors committing to the monocyte - macrophage differentiation may respond to this hormone . in summary , based on our preliminary study and our new data , we conclude that the inflammatory response and anemia caused by blood stage infection with p. c. adami parasites do not stimulate the activity of osteoclasts . inhibition of the basal activity of osteoclasts may be sufficient to partially block egression of hspc from the bm , but this egression seems not essential for the stress erythropoiesis in conditions of acute anemia . the intriguing effects of calcitonin on the differentiation and proliferation of macrophages remain to be characterized .

the anemia and inflammation concurrent with blood stage malaria trigger stress haematopoiesis and erythropoiesis . the activity of osteoclasts seems required for the mobilization of hematopoietic stem and progenitor cells ( hspc ) from the bone marrow to the periphery . knowing that balb / c mice with acute plasmodium chabaudi adami malaria have profound alterations in bone remodelling cells , we evaluated the extent to which osteoclasts influence their hematopoietic response to infection . for this , mice were treated with osteoclast inhibiting hormone calcitonin prior to parasite inoculation , and infection as well as hematological parameters was studied . in agreement with osteoclast - dependent hspc mobilization , administration of calcitonin led to milder splenomegaly , reduced numbers of hspc in the spleen , and their retention in the bone marrow . although c - terminal telopeptide ( ctx ) levels , indicative of bone resorption , were lower in calcitonin - treated infected mice , they remained comparable in naive and control infected mice . calcitonin - treated infected mice conveniently responded to anemia but generated less numbers of splenic macrophages and suffered from exacerbated infection ; interestingly , calcitonin also decreased the number of macrophages generated in vitro . globally , our results indicate that although osteoclast - dependent hsc mobilization from bone marrow to spleen is triggered in murine blood stage malaria , this activity is not essential for stress erythropoiesis .

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

quiescent hematopoietic stem cells ( hscs ) reside in the bone marrow ( bm ) near the endosteum , in specific microenvironments called endosteal niches [ 13 ] . low numbers of hscs leave the bm and enter the blood stream in steady state conditions [ 4 , 5 ] , thereafter establishing in extramedullary sites of hematopoiesis like the spleen and liver , or returning to the bm [ 6 , 7 ] . recent studies involve bone resorbing osteoclasts in the homeostasis and mobilization of hsc and hematopoietic progenitor cells ( hspc ) in conditions of stress , although this function remains controversial [ 811 ] . indeed , stimulation of osteoclasts activity by receptor activator of nuclear factor kappa - b ligand ( rankl ) increases hspc mobilization through secretion of cathepsin k , which cleaves c - kit - ligand and stromal cell - derived factor 1 ( sdf-1 ) , required for maintenance of endosteal niches . furthermore , the osteoclast inhibiting hormone calcitonin was reported to decrease hspc mobilization in response to lps injection . however , contradictory evidences demonstrate that inhibition of osteoclast activity by bisphosphonate does not impair hspc mobilization in response to granulocyte - colony stimulating factor ( g - csf ) treatment [ 1 , 10 ] , suggesting that osteoclasts may only intervene in certain types of hematopoietic stresses . mobilization of hspc is triggered by viral , bacterial , and plasmodium infection [ 1216 ] , as well as by phenylhydrazine- ( phz- ) induced anemia in mice [ 1719 ] , but the contribution of osteoclasts in these responses has not been yet characterized . blood stage malaria causes systemic inflammation and acute hemolytic anemia in mice , which are events that trigger stress hematopoiesis to generate phagocytic cells involved in parasite clearance and new erythrocytes to cope with anemia [ 2124 ] . in murine malaria models , stress hematopoiesis associates with egression of hspc from the bm to spleen , which becomes the major site of erythropoiesis [ 25 , 26 ] and myelopoiesis [ 27 , 28 ] this mobilization is indirectly involved in the development of splenomegaly [ 16 , 29 ] and extramedullary myelopoiesis , which are required in the resolution of acute parasitemia , through removal of parasitized red blood cells ( prbcs ) by splenic macrophages [ 3032 ] . we recently reported profound alterations in bone remodelling in mice with malarial or phz - induced hemolytic anemia , characterized by decreased bone formation , reduced osteoclastogenesis , and balance resulting in bone mass loss . knowing these alterations , the contribution of osteoclasts in hspc egression , stress erythropoiesis , and resolution of plasmodium chabaudi adami dk infection was investigated in calcitonin - treated mice . our data indicate that calcitonin causes a significant drop in the basal activity of osteoclasts and partially interferes with the egression of hspc from bm to spleen in blood stage malaria . however , the development of splenic stress erythropoiesis is not affected by this treatment . female balb / c mice ( charles river , canada ) aged 46 weeks were used in all experiments . to evaluate the contribution of osteoclasts in hspc mobilization during plasmodium infection , mice received 200 l salmon calcitonin ( 5 g / ml in pbs ) ( biotrend , switzerland ) during 5 consecutive days by the intraperitoneal route and were infected with 10 p. c. adami dk prbc in pbs by the intravenous route on the 3rd day of treatment . mice were euthanized on days 5 , 8 , and 10 after infection for analysis of hematopoietic parameters and cytokines in plasma by elisa ( biolegend , usa ) . to assess the effect of calcitonin - dependent osteoclasts inhibition on the erythropoietic responses to experimental hemolytic and nonhemolytic anemia , mice were injected intraperitoneally with 200 l of salmon calcitonin ( 5 g / ml in pbs ) ( biotrend , switzerland ) during 5 consecutive days . hemolytic anemia was induced in mice by peritoneal administration of phz ( sigma aldrich , canada ) at 40 mg / kg of body weight in 200 l of pbs , on the 3rd and 4th days of calcitonin treatment . in certain experiments with infected mice , 100 l did - loaded anionic liposomes ( formumax , usa ) were administered intravenously one day prior to calcitonin treatment . recovered cells from the bm and the spleen were counted and resuspended at 10 cells / ml in rpmi 1640 , supplemented with 10% , penicillin ( 100 u / ml ) , and streptomycin ( 100 mg / ml ) ( invitrogen , usa ) . elisa for quantification of macrophage colony - stimulating factor ( m - csf , promokine , canada ) and c - terminal telopeptide ( ctx , mybiosource , usa ) were performed following the manufacturer 's instructions . single - cell suspensions from the spleen and bm were prepared at 1 10 and 2 10 cells / ml , respectively , in rpmi 1640 , supplemented with 10% , penicillin ( 100 u / ml ) , and streptomycin ( 100 mg / ml ) ( invitrogen , usa ) and 0.3 ml was added into 3 ml of methocult 03434 ( stemcell technologies , usa ) . in order to investigate the contribution for osteoclasts in malarial stress hematopoiesis with the p. c. adami dk infection model , balb / c mice were treated with calcitonin two days prior to infection and three days following infection . resolution of infection and subsequent stress erythropoiesis were monitored and compared in control and calcitonin - treated mice . for comparative purposes , as expected , significantly lower plasma concentrations of ctx were measured in calcitonin - treated mice on day 5 after infection , confirming inhibition of osteoclast activity ( figure 1(a ) ; p < 0.05 ) . parasitemia was exacerbated in calcitonin - treated mice throughout patent infection ( figure 1(b ) ; p < 0.01 ) , resulting in higher cumulative parasite burden ( figure 1(c ) ; p < 0.001 ) . consequent to higher parasitemia , hemoglobin levels were relatively lower in calcitonin - treated mice on days 11 and 12 after infection ( figure 1(d ) ; p < 0.01 and p < 0.05 , resp . ) concurrent with enhanced anemia , the percentages of reticulocytes ( cd71 cells ) were higher in calcitonin - treated mice on day 14 after infection ( figure 1(e ) ) . in accordance with the responses to infection , comparable reticulocytosis occurred in control and calcitonin - treated mice under these two experimental conditions ( figures 2(c ) and 2(d ) ) . considering the crucial role of splenic stress hematopoiesis in the control of plasmodium infection [ 16 , 28 ] and anemia [ 21 , 25 , 26 , 38 ] , the effect of calcitonin on splenic hematopoietic parameters was investigated early ( day 5 after infection ) , at peak parasitemia ( day 8) , and following resolution of peak infection ( day 10 ) . as expected , spleen cellularity increased in mice with malaria ( figure 3(a ) ) , and this effect was apparent and comparable in control and calcitonin - treated mice until day 5 after infection ( figure 3(a ) ) . however , reduced numbers of splenocytes ( figure 3(a ) ; p < 0.001 ) and reduced splenic index ( supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/3909614 ; p < 0.01 ) were measured in calcitonin - treated mice at peak infection , indicating milder splenomegaly . parallel monitoring of the erythroid populations confirmed higher numbers of splenic cd71 ter119 cells in infected mice from day 5 after infection , which increased thereafter ( figure 3(b ) ; p < 0.001 ) ; this response was significantly attenuated in calcitonin - treated mice at the time of peak infection ( figure 3(b ) ; p < 0.01 ) . major differences were only apparent on day 8 after infection , reflected by significantly lower lineage negative cell numbers in calcitonin - treated infected mice ( figure 3(c ) ; p < 0.01 ) . in contrast , administration of calcitonin significantly reduced the number of splenic lineage negative ckit sca1 cells ( lsk ) at peak infection , as well as 10 days after infection ( figure 3(e ) ; p < 0.01 , 0.001 , resp . ) the analysis of lymphoid populations in spleen revealed increased numbers of cd90 cells ( supplementary figure 2(a ) ; p < 0.001 ) and cd19 cells ( supplementary figure 2(b ) ; p < 0.001 ) at peak infection ; treatment with calcitonin resulted in lower cd19 cell numbers ( supplementary figure 2(b ) ; p < 0.05 ) . in respect to splenic myeloid cells , calcitonin did not affect the characteristic rise in cd11b cells ( supplementary figure 2(c ) ; p < 0.001 ) and fceria ckit cells at peak infection ( supplementary figure 2(d ) ; p < 0.01 ) . compared to uninfected mice , the number of bm lineage negative cells significantly dropped on days 8 and 10 after infection in infected mice ( figure 4(c ) ) , becoming relatively higher in calcitonin - treated mice when compared to infected controls at peak infection ( figure 4(c ) ; p < 0.01 ) . the numbers of lsk cells also increased in the bm of infected mice and were significantly higher in calcitonin - treated mice on day 8 after infection ( figure 4(d ) , p < 0.01 ) . to determinate whether the increased parasitemia observed in calcitonin - treated mice was due to restricted production of macrophages , the numbers of f4 - 80 cells were analyzed in the bm and spleen . relative to uninfected mice , the infection did not provoke major effects on bm f4/80 cells , but their numbers were significantly higher in calcitonin - treated mice on day 10 after infection ( figure 5(a ) ; p < 0.05 ) . in contrast to the bm , the infection caused an important increase in splenic f4/80 cells , and this effect was significantly attenuated in calcitonin - treated mice on days 8 and 10 after infection ( figure 5(b ) ; p < 0.01 , 0.001 , resp . ) analysis of progenitors colony forming unit - macrophage ( cfu - m ) did not reveal major modifications in the bm of infected mice ( figure 5(c ) ) , but lower numbers of cfu - m generated from the spleen of calcitonin - treated mice on day 8 after infection ( figure 5(d ) ; p < 0.05 ) . interestingly , serum m - csf levels remained comparable in infected and uninfected mice ( supplementary figure 3 ) . altogether , our results suggest altered production of macrophages in calcitonin - treated and infected mice . to discriminate the impact of calcitonin on preexisting versus de novo generated macrophages , did anionic liposomes were administered prior to infection , to specifically stain all phagocytic cells including macrophages . the lipid composition of the anionic liposomes was originally designed to efficiently deplete phagocytic cells in vivo and in vitro when loaded with clodronate , and as nonphagocytic cells are not affected , this tool is efficient for targeting of macrophages [ 41 , 42 ] . compared to uninfected controls , in which the majority of bm macrophages were did , approximately 50% f4/80 did cells were found in infected mice on day 10 after infection , suggesting mobilization of mature macrophages from the bm and production of new ( did ) macrophages ( figure 5(e ) ; p < 0.01 ) . the numbers of splenic f4/80 did cells drastically increased on day 10 after infection , indicating recruitment of mature macrophages to the spleen after peak infection ( figure 5(f ) ; p < 0.001 ) . f4/80 did cells also increased during infection , but this response was half - fold in calcitonin - treated mice ( figure 5(f ) ; p < 0.01 ) , suggesting deficient de novo production of macrophages . in order to investigate whether the lower macrophage numbers found in calcitonin - treated mice are concurrent with direct action of this hormone on myeloid precursors , the impact of calcitonin on differentiation of macrophage progenitors was evaluated in vitro . herein , we evaluated the relative contribution for osteoclasts in the egression of hspc from the bm to the spleen during blood stage malaria , using the well - characterized p. c. adami dk infection model in balb / c mice . these conditions are characterized by reduced bone mineralization and bone formation , as well as reduced numbers of osteoclasts and osteoclasts progenitors in the bm . considering that the levels of ctx , indicative of osteoclast - dependent bone resorption , remained comparable in infected , phz - treated , and naive mice , we concluded that the decreased bone mass density found in mice with hemolytic anemia was concurrent with imbalance favoring bone resorption , as has been reported for other hemolytic conditions [ 43 , 44 ] . herein , calcitonin was administered to mice three days prior to and during the first two days of infection to block bone resorption . interestingly , plasma ctx levels were comparable in naive and infected mice on day 5 after infection , suggesting no major stimulation of bone resorption in this murine malaria model . the fact that ctx levels were lower in calcitonin - treated mice confirmed an inhibitory action of this peptide hormone on steady state osteoclast activity . in our experimental malaria model , the number of hspc increased in a comparable manner in the bm of calcitonin - treated and control - infected mice on day 5 after infection , with no major effects in the spleen . however , at peak infection , calcitonin - treated mice had higher numbers of hspc in the bm and lower numbers of hspc in the spleen , also developing relatively milder splenomegaly . these results suggest that osteoclast - dependent mobilization of hspc to the spleen is partially responsible for splenomegaly in murine malaria . in this malaria model , ifn- signalling and concurrent secretion of c - c motif ligand 2/7 ( ccl2/ccl7 ) chemokines by bm stromal cells seem required for hspc mobilization . interestingly , ifn- signalling indirectly stimulates the activity of osteoclasts , which may stimulate their effects in hspc mobilization . in opposition to reduced hspc mobilization appearance of circulating reticulocytes is considered an ultimate marker for functional erythropoiesis ( as reviewed in ) , and our data indicate comparable reticulocytosis in calcitonin - treated and control mice in response to malarial anemia , despite reduced mobilization of hspc and reduced numbers of erythroid progenitors in the spleen . in this context , stress burst forming unit - erythroid ( bfu - e ) with self - renewal properties have been reported , which efficiently generate ckit cd71 ter119 cells in response to erythropoietin ( epo ) , bone morphogenetic protein-4 ( bmp-4 ) , stem cell factor ( scf ) , and hypoxia [ 47 , 48 ] . as comparable numbers of ckit cd71 ter119 cells were measured in control and calcitonin - treated mice on day 8 after infection ( data not shown ) , we conclude that osteoclast - dependent hspc mobilization from the bm is not essential for splenic stress erythropoiesis in murine blood stage malaria . analysis of lymphocytic and granulocytic cell populations in the spleen at peak infection revealed no modifications in the numbers of t cells , mast cells , and myeloid cells in calcitonin - treated mice , although decreased numbers of b cells were noticed . the exacerbated parasitemia measured in calcitonin - treated mice may be concurrent with compromised parasite killing by macrophages . indeed , an important proportion of prbcs circulate in the blood stream and their elimination by spleen red pulp macrophages is pivotal for the control of infection [ 5052 ] . calcitonin - treated mice , which developed exacerbated parasitemia , had fewer numbers of macrophages in the spleen on days 8 and 10 after infection and , accordingly , reduced numbers of cfu - m generated from the spleen of these mice . b cells only intervene in the late resolution of blood stage malaria , and the reduction caused by calcitonin on cd19 cells numbers is not expected to affect the resolution of p. c. adami infection . in contrast , c - c chemokine receptor type 2- ( ccr2- ) dependent migration of monocytes from the bm to spleen and splenic myelopoiesis are essential for the control of p. chabaudi infection . administration of did - loaded liposomes prior to calcitonin treatment and infection efficiently labelled macrophages , allowing following their fate and distinguishing newly generated macrophages in vivo . in respect to noninfected controls , we evidenced comparable drops in the number of did macrophages in the bm and their concurrent increase in the spleen during infection . these results suggest that calcitonin does not alter the mobilization of monocytes / macrophages from the bm nor their recruitment to spleen . however , the fact that the numbers of splenic did macrophages were significantly lower in calcitonin - treated mice on day 10 after infection rather indicates impaired de novo generation of macrophages in mice treated with this hormone . calcitonin did not alter m - csf levels in infected mice ; we hypothesize that it may interfere with m - csf - dependent signalling in certain bone marrow precursor cells and that impaired recruitment of hspc to the spleen may further compromise myelopoiesis . in summary , based on our preliminary study and our new data , we conclude that the inflammatory response and anemia caused by blood stage infection with p. c. adami parasites do not stimulate the activity of osteoclasts . inhibition of the basal activity of osteoclasts may be sufficient to partially block egression of hspc from the bm , but this egression seems not essential for the stress erythropoiesis in conditions of acute anemia .

infant vaccination programmes have been highly successful in reducing severe disease and childhood deaths due to pertussis . despite these substantial gains , pertussis continues to circulate in all populations and most often affects very young , unimmunized or incompletely immunized infants , with a rising incidence in adolescents and adults [ 1 , 2 ] . family members have been implicated as important sources of pertussis for unprotected infants who are at highest risk of complications and death [ 14 ] . high coverage of infant pertussis vaccination has been longstanding in southeast asian countries ( ranging from 82% to 99% coverage in 2009 ) . although few data are available describing the epidemiology of pertussis disease in asian countries , the available evidence suggests that , as in other countries with high pertussis vaccine coverage in infants and young children , pertussis continues to circulate in adolescents and adults . in taiwan , a seroprevalence study found that adolescents and adults remain at risk of pertussis despite successful immunization programmes , and a surveillance study of patients with cough lasting more than 1 week estimated an incidence of up to 21% , with pertussis disease especially prevalent in adolescents and adults [ 6 , 7 ] . another seroprevalence study in singapore also showed pertussis to be prevalent in adults . although adults rarely die from pertussis infection , the medical costs and morbidity associated with pertussis in this age group can be considerable . possible complications of adult pertussis include secondary pneumonia , rib fractures and incontinence . in a hospital - based study of confirmed pertussis cases in taiwan the complication rate was 304% , of which pneumonia was most common ( 923% ) . an analysis of the 20112012 pertussis outbreak in england estimated an overall loss in quality of life of 0097 quality - adjusted life years ( qalys ) for pertussis patients , and 00365 qalys for coughing household contacts . findings from such epidemiological studies justify the need for pertussis vaccination in adolescents and young adults . we performed a cross - sectional study to determine the prevalence of serologically confirmed pertussis in adults in malaysia , taiwan and thailand with prolonged cough . pertussis vaccination schedule for these countries is shown in table 1 [ 1214 ] . table 1.pertussis vaccination schedule in malaysia , taiwan and thailandcountryprimary dosesbooster dosesmonth 2month 4month 6month 1846 years1112 yearsmalaysiadtap - hib - ipvdtap - hib - ipvdtap - hib - ipv*dtap - hib - ipvtaiwandtap - hib - ipvdtap - hib - ipvdtap - hib - ipvdtap - hib - ipvtdap - ipvthailanddtwp - hibdtwp - hibdtwp - hibdtwpdtwp(or dtap)(or dtap)(or dtap)(or dtap)(or dtap)tdapdark grey cells ( ) indicate optional vaccine . light grey cells ( ) indicate epi / nip vaccine.dtap , diphtheria , tetanus , pertussis ( acellular ) vaccine ; hib , haemophilus influenzae type b vaccine ; ipv , inactivated poliomyelitis vaccine ; dtwp , diphtheria , tetanus , pertussis ( whole cell ) vaccine ; dtap / tdap , diphtheria , tetanus , and pertussis ( acellular ) vaccine [ reduced antigen(s ) ] . epi , expanded programme on immunization ; nip , national immunization program.*month 5.after 5 years of age and before entering elementary school . pertussis vaccination schedule in malaysia , taiwan and thailand dark grey cells ( ) indicate optional vaccine . dtap , diphtheria , tetanus , pertussis ( acellular ) vaccine ; hib , haemophilus influenzae type b vaccine ; ipv , inactivated poliomyelitis vaccine ; dtwp , diphtheria , tetanus , pertussis ( whole cell ) vaccine ; dtap / tdap , diphtheria , tetanus , and pertussis ( acellular ) vaccine [ reduced antigen(s ) ] . this cross - sectional study was conducted between 20 june 2012 and 2 may 2013 according to the declaration of helsinki and good clinical practice guidelines . written informed consent or assent was obtained from subjects ( or parent / legally acceptable representative in taiwan and thailand where the age of consent is 20 years ) prior to enrolment . adult patients aged at least 19 years with prolonged cough were recruited from two centres in malaysia ( international medical university and university of malaya medical centre ) , three centres in thailand ( phramongkutklao hospital , srinagarind hospital and songklanagarind hospital ) and two centres in taiwan ( china medical university hospital and chang gung memorial hospital ) . these participating hospitals provide ambulatory primary healthcare facilities and treatment for outpatients besides accepting referral cases from other primary healthcare practitioners . all patients with prolonged cough ( present for 14 days ) were screened for eligibility . patients with known pulmonary disease causing chronic cough ( including pulmonary tuberculosis , chronic obstructive airways disease , bronchiectasis , asthma ) and patients with suspected or known immunodeficiency were excluded . patients treated with angiotensin - converting enzyme inhibitors within the previous 4 weeks prior to enrolment were also excluded from participation . demographic information ( age at enrolment , gender ) , medical history including vaccination history for pertussis , pneumococcal disease and influenza , and as well as information on clinical symptoms and treatments received were documented and a physical examination was performed . health - related quality of life [ euroqol-5d ( eq-5d ) ] and structured health economic questionnaires were administered by study staff to individuals at the time of enrolment . patients ' medical records were reviewed approximately 30 days after enrolment to re - validate the diagnosis and record the clinical outcome and results of additional medical procedures . a blood sample was collected from each patient for anti - pertussis toxin ( anti - pt ) level of igg antibodies testing . anti - pt was measured by elisa ( euroimmun , germany ) according to the manufacturer 's instructions at a laboratory designated by gsk vaccines with an assay cut - off for sensitivity of 10 iu / ml . sera were classified into four categories , based on manufacturer 's instructions , previous experience in the available literature [ 1517 ] and the absence of vaccination against pertussis in the last 12 months : a seronegative patient had an anti - pt igg level below the sensitivity limit of the assay ( < 10 iu / ml ) ; a seropositive patient had an anti - pt igg level 10 iu / ml ; an anti - pt igg 625 iu / ml was considered serological evidence of pertussis infection in the last 12 months ; and an anti - pt igg level 100 iu / ml was considered indicative of active or recent infection . health - related quality of life was assessed using the eq-5d questionnaire , which comprises five dimensions ( mobility , self - care , usual activities , pain / discomfort , anxiety / depression ) , with three levels of severity within each dimension . there are 245 possible health states , for which a mapping of utility scores can be conducted and the range of results is [ 0 ; 1 ] where 0 = death and 1 = perfect health . health economic impact such as absenteeism from work or school and associated income loss of the subjects or family members , healthcare resource utilization , and direct costs ( including medical costs and transportation costs ) were recorded using a standardized questionnaire . all costs were adjusted to 2012 us$ using the historical yearly mean exchange rate from www.oanda.com / currency / historical - rates/. statistical analyses were performed using sas v. 9.2 ( sas institute inc . , usa ) . previous studies have demonstrated a prevalence of pertussis of between 10% and 20% in adults with prolonged cough [ 19 , 20 ] . considering a 10% pertussis prevalence , 139 subjects would provide 5% precision on the estimate . considering a prevalence of pertussis of 18% assuming a drop - out rate of 10% , we therefore aimed to enrol 154 subjects in thailand and at least 78 subjects in malaysia and taiwan . the according - to - protocol ( atp ) cohort included all subjects with valid laboratory test results and excluded those subjects vaccinated with pertussis vaccines within 1 year before the blood sample . prevalence of pertussis infection with exact 95% confidence intervals ( cis ) was determined by the number of patients with laboratory evidence suggestive of pertussis infection in all subjects presenting with prolonged cough . in addition , clinical characteristics ( such as cough paroxysms , whoop , night cough , post - tussive vomiting , cyanosis , fever , etc . ) , contact with people with coughing disease before onset , comorbidities , antibiotic treatment and laboratory results of subsets of patients with and without serological evidence of pertussis , were assessed . the health economic impact in terms of absenteeism from work , healthcare resource utilization , and direct costs were described only for subjects with serologically confirmed pertussis . there were 337 patients enrolled in the study , of whom 25 ( 74% ) were excluded from the analysis leaving 312 subjects in the atp cohort ( fig . 1 ) . of the 312 subjects included in the analysis , 296 ( 949% ) had anti - pt igg levels below the pre - defined cut - off of 625 iu / ml , including 208 ( 6667% ) seronegative subjects ( anti - pt antibodies igg < 10 iu / ml ) . the other 16 of the 312 subjects ( 513% , 95% ci 296819 ) had serological evidence of pertussis infection in the last 12 months ( anti - pt 625 iu / ml ) : 6/76 ( 79% , 95% ci 2951640 ) in malaysia , 1/89 in taiwan ( 11% , 95% ci 003610 ) and 9/147 ( 61% , 95% ci 2841130 ) in thailand . four of these 16 subjects ( overall 13% , 4/312 , 95% ci 035325 ) had anti - pt antibodies 100 iu / ml indicative of active or recent infection . the demographic characteristics of enrolled patients with and without serological evidence of pertussis infection ( anti - pt 625 iu / ml ) are shown in table 2 . pertussis cases were distributed across all age groups , and were highest in age groups 5059 years ( 98% , 5/51 , 95% ci 3262141 ) and 1929 years ( 76% , 5/66 , 95% ci 2511680 ) ( figs . 2 , 3 ) . serological evidence of active pertussis ( anti - pt igg 100 iu / ml ) was most commonly observed in the 1929 years age group ( 30% , 2/66 , 95% ci 0371052 ) . the median duration of cough was not significantly different between patients with and without serological evidence of pertussis ( anti - pt igg 625 iu / ml ) : 43 days ( range 15120 ) vs. 31 days ( range 141682 ) , respectively ( p = 01 ) . duration of cough was > 90 days in 3/16 ( 188% ) of patients with serological evidence of pertussis compared to 40/296 ( 135% ) patients without ( table 3 ) . the most frequently reported duration of cough was longer in patients with serological evidence of pertussis ( 3160 days ) compared to those without ( 1430 days ) . a greater proportion of patients with serological evidence of pertussis ( 750% , 12/16 ) than those without ( 483% , 143/296 ) reported paroxysms ( p = 004 ) . breathlessness / chest pain was reported by 625% ( 10/16 ) of patients with serological evidence of pertussis vs. 358% ( 106/296 ) of patients without ( p = 003 ) . no other significant differences in clinical symptoms presence were observed between the two groups ( table 3 ) . the most commonly recorded comorbid conditions were categorized as other non - respiratory tract diseases ; however , there was no difference between those with / without serological evidence of pertussis in terms underlying medical conditions [ 1/16 ( 63% ) vs. 36/296 ( 122% ) , respectively , p = 070 , fisher 's exact test ] . 2.proportion of subjects in each age group with serological evidence of pertussis in the previous 12 months ( anti - pt igg 625 iu / ml ; according - to - protocol cohort ) . 3.distribution of anti - pt igg concentrations by ( a ) age and ( b ) cough duration ( according - to - protocol cohort ) . table 2.demographic characteristics of enrolled patients with and without evidence of pertussis in the last 12 months ( anti - pt igg 625 iu / ml)categoryanti - pt 655 iu / ml ( n = 16)anti - pt < 655 iu / ml ( n = 296)age ( years)mean ( sd)407 ( 133)431 ( 155)range21631983age group , years , n ( % ) 19295 ( 313)61 ( 206)30393 ( 188)86 ( 291)40492 ( 125)50 ( 169)50595 ( 313)46 ( 155)601 ( 63)53 ( 179)genderfemale , n ( % ) 8 ( 50)202 ( 682)male , n ( % ) 8 ( 50)94 ( 318)n , number of subjects ; n , number of subjects in a given category ; s.d . , standard deviation . table 3.clinical signs and symptoms of chronic cough by status of pertussis infection by anti - pt levels ( according - to - protocol cohort)clinical symptomswith serological evidence ( anti - pt 655 iu / ml ) ( n = 16)without serological evidence ( anti - pt < 655 iu / ml ) ( n = 296)p valueduration of cough , days , median ( range)43 ( 15120)31 ( 141682)cough duration , days , n ( % ) 14304 ( 250)142 ( 480)<00131607 ( 438)74 ( 250)61902 ( 125)40 ( 135)>903 ( 188)40 ( 135)fever since onset of cough , n ( % ) yes5 ( 313)74 ( 25)056presence of at least one of the following symptoms , n ( % ) yes13 ( 813)258 ( 872)045whoop2 ( 125)18 ( 61)027paroxysm12 ( 750)143 ( 483)004post - tussive vomiting6 ( 375)72 ( 243)024apnoea03 ( 10)100cyanosis00coughing up phlegm6 ( 375)180 ( 608)006sneezes6 ( 375)111 ( 375)100wheezes3 ( 188)34 ( 115)042cough at night11 ( 688)211 ( 713)078episodes of being unable to stop coughing7 ( 438)140 ( 473)078breathlessness / chest pain10 ( 625)106 ( 358)003n , number of subjects.fisher's exact test p value. test p value . proportion of subjects in each age group with serological evidence of pertussis in the previous 12 months ( anti - pt igg 625 iu / ml ; according - to - protocol cohort ) . distribution of anti - pt igg concentrations by ( a ) age and ( b ) cough duration ( according - to - protocol cohort ) . demographic characteristics of enrolled patients with and without evidence of pertussis in the last 12 months ( anti - pt igg 625 iu / ml ) n , number of subjects ; n , number of subjects in a given category ; s.d . , standard deviation . clinical signs and symptoms of chronic cough by status of pertussis infection by anti - pt levels ( according - to - protocol cohort ) n , number of subjects . chest x - ray was performed on 212/312 ( 679% ) patients ( seven with serological evidence of pertussis ) . the majority of chest x - rays [ 714% ( 5/7 ) in serologically confirmed and 863% ( 177/205 ) in patients with anti - pt igg < 625 polymerase chain reaction ( pcr ) was only performed in one patient without serological evidence of pertussis and failed to identify a pathogen . there were 94 ( 301% , 94/312 ) patients with suspected tuberculosis ; all of whom had negative examination results of sputum for acid - fast bacilli . one patient ( without serological evidence of pertussis ) had pleural tuberculosis confirmed by pleural biopsy ( post - enrolment and was included in study analyses ) . contact with a household or workplace member with known or suspected whooping cough was reported by 1/16 ( 63% ) patient with serological evidence of pertussis vs. 29/296 ( 98% ) of subjects without pertussis . contact with a household or workplace member with persistent cough was reported by 4/16 ( 250% ) pertussis patients and 109/296 ( 368% ) patients without pertussis , and contact with a household or workplace member who developed a cough subsequently was reported by 6/16 ( 375% ) and 106/296 ( 358% ) subjects , respectively . of the 16 patients with serological evidence of recent pertussis infection , three ( 188% ) were employed as teachers and one reported having contact with an infant aged < 1 year either at home or the workplace . of the 16 patients with serological evidence of pertussis , 10 ( 625% ) reported not receiving any previous dose of pertussis vaccine , with a further five ( 313% ) patients unable to recall their vaccination status . of the 296 patients without serological evidence of pertussis , 87 ( 294% ) reported not receiving any previous dose of pertussis vaccine while 114 ( 385% ) were unable to recall their vaccination status . the remaining 95 ( 321% ) patients reported previous pertussis vaccination history : 50 ( 526% ) received 45 doses , two ( 21% ) received 23 doses , and 43 ( 453% ) received one dose . however none of the patients ' vaccination histories could be verified . in patients with serological evidence of pertussis , the number of days absent from work ranged from 0 to 30 , with a median of 0 and a mean of 24 ( standard deviation 80 ) . one patient from malaysia reported lost income due to missing work , with an average daily income loss > us$97.5 . pertussis infection was associated with a median of 25 visits ( range 110 ) to healthcare professionals ( general practitioner , emergency room , or specialist ) . the median total direct medical cost of pertussis per episode in public hospitals ( including consultations with physicians and/or emergency room visits ) was us$13 ( range us$1316 ) or 40 myr ( range 4050 myr ) in malaysia ; us$83 ( or 2450 ntd ) in taiwan ( one patient ) and us$26 ( range us$12168 ) or 800 thb ( range 3605200 thb ) in thailand . the median time spent seeking medical care was 4 h ( range 02529 h ) . of subjects with serological evidence of pertussis infection , two patients ( 2/16 , 125% ) had problems with mobility , 6/16 ( 375% ) had problems in performing usual activities , 6/16 ( 375% ) suffered from pain / discomfort and 7/16 ( 438% ) suffered from anxiety / depression . we found serological evidence of pertussis infection in the last 12 months in 513% of adults presenting to hospitals with cough of at least 2 weeks ' duration in malaysia , taiwan and thailand . this is comparable with a prevalence of pertussis of 72% in taiwanese adults with cough duration 1 week tested by serology ( anti - pt ) or pcr , and 117% of adults with prolonged cough illness in the united states , denmark and korea tested for elevated anti - pt levels in non - outbreak settings ( reviewed in ) . in addition , two thirds of the subjects had no evidence of immunity against pertussis meaning they remain at risk of contracting infection . pertussis disease in adults is frequently difficult to diagnose because of the absence of classical clinical features such as the inspiratory whoop that are typically associated with disease in unvaccinated children . therefore , raising awareness in healthcare providers to consider pertussis in the differential diagnosis of cough illness in older individuals can help to improve the diagnostics . this may be particularly important as few clinical features were identified in our study that might facilitate differential diagnosis in this age group . bordetella pertussis is a fastidious organism that is difficult to culture , and while pcr improves pertussis detection rates compared to culture , these techniques are both expensive and not readily available in many asian laboratories . in addition , pcr is optimally sensitive during the first 3 weeks of cough , meaning patients in our study may have missed this window for detection . further difficulty in performing routine testing in asian countries is represented by the specialist training , experience and specific sample storage and transportation media required for the collection and processing of nasopharyngeal samples for pcr or culture . a single acute - phase serum sample , as used in our study , this method is also supported by kinetic studies showing that anti - pt antibodies decline rapidly in all age groups after infection . none of the patients with serological evidence of pertussis in the previous 12 months in our study received a diagnosis of pertussis . this suggests low awareness of pertussis in healthcare professionals and the potential for transmission of undetected pertussis to vulnerable infants . an increasing number of countries are adopting measures to reduce infant pertussis by vaccinating adults : this includes cocooning strategies and periodic booster doses at defined ages . of more recent interest is the potential to prevent infant pertussis through maternal immunization , with early evidence from the uk suggesting that high uptake and decreases in infant pertussis deaths are possible [ 24 , 25 ] . recent pertussis infection was associated with a reduced quality of life in our study , with over one third of subjects reporting pain / discomfort and problems conducting routine activities ( e.g. work , study , housework , family or leisure activities ) . moderately anxious. the median eq-5d score for subjects with recent pertussis infection was 072 suggesting that subjects with suspected pertussis perceived an impact on their quality of life . the direct medical costs associated with pertussis episodes in our study varied between countries and was likely related to individual healthcare systems . the 2012 average monthly wage was us$623 in malaysia , us$1546 in taiwan and us$359 in thailand . this means that pertussis - associated medical costs represented around 2% of the monthly wage in malaysia , 55% in taiwan and over 7% in thailand . patients recruited from secondary and tertiary hospitals could possibly have more severe conditions than those seen by primary healthcare physicians . this could result in either over- or under - estimation of the true pertussis burden . even though the patients were enrolled from primary healthcare centres within the hospitals , these patients may not be representative of patients seeking primary healthcare in general practitioners ' or family physicians ' clinics . however , both the pertussis positive and negative patients provided the information without the knowledge of the serological test results and thus were not likely to be biased by the diagnosis . in addition , pcr has not been widely used in laboratories of many asian countries for the diagnosis of pertussis . the diagnosis of pertussis in this study was based on serology , which could have resulted in an underestimation of the pertussis burden . furthermore , patients may not be fully aware of government subsidy levels for healthcare expenses , or they could be reluctant to share income information , resulting in an underestimation of the true health economic impact . in conclusion , approximately 5% of adult patients in malaysia , taiwan and thailand presenting to hospitals with cough of at least 2 weeks ' duration showed serological evidence of pertussis infection , while over two - thirds had no evidence of existing immunity . hence , pertussis should be considered in patients with prolonged or paroxysmal cough in the previous 12 months . pertussis infection posed a significant health economic burden and moderate impact on the health - related quality of life of the affected patients . increased awareness of pertussis by physicians and improved capacity of local laboratories is needed to improve early diagnostics and reduce the transmission of pertussis to children especially vulnerable young infants who are at the highest risk of severe disease and death .

summarysurveillance data on the burden of pertussis in asian adults are limited . this cross - sectional study evaluated the prevalence of serologically confirmed pertussis in adults with prolonged cough in malaysia , taiwan and thailand . adults ( 19 years ) with cough lasting for 14 days without other known underlying cause were enrolled from outpatient clinics of seven public and/or private hospitals . single blood samples for anti - pertussis toxin antibodies ( anti - pt igg ) were analysed and economic impact and health - related quality of life ( eq-5d ) questionnaires assessed . sixteen ( 513% ) of the 312 chronically coughing adults had serological evidence of pertussis infection within the previous 12 months ( anti - pt igg titre 625 iu / ml ) . three of them were teachers . longer duration of cough , paroxysms ( 75% seroconfirmed , 48% non - seroconfirmed ) and breathlessness / chest pain ( 63% seroconfirmed , 36% non - seroconfirmed ) were associated with pertussis ( p < 004 ) . of the seroconfirmed patients , the median total direct medical cost per pertussis episode in public hospitals ( including physician consultations and/or emergency room visits ) was us$13 in malaysia , us$83 in taiwan ( n = 1 ) and us$26 in thailand . the overall median eq-5d index score of cases was 072 ( range 042100 ) . pertussis should be considered in the aetiology of adults with a prolonged or paroxysmal cough , and vaccination programmes considered .

INTRODUCTION METHODS RESULTS DISCUSSION

although few data are available describing the epidemiology of pertussis disease in asian countries , the available evidence suggests that , as in other countries with high pertussis vaccine coverage in infants and young children , pertussis continues to circulate in adolescents and adults . in taiwan , a seroprevalence study found that adolescents and adults remain at risk of pertussis despite successful immunization programmes , and a surveillance study of patients with cough lasting more than 1 week estimated an incidence of up to 21% , with pertussis disease especially prevalent in adolescents and adults [ 6 , 7 ] . although adults rarely die from pertussis infection , the medical costs and morbidity associated with pertussis in this age group can be considerable . in a hospital - based study of confirmed pertussis cases in taiwan the complication rate was 304% , of which pneumonia was most common ( 923% ) . an analysis of the 20112012 pertussis outbreak in england estimated an overall loss in quality of life of 0097 quality - adjusted life years ( qalys ) for pertussis patients , and 00365 qalys for coughing household contacts . we performed a cross - sectional study to determine the prevalence of serologically confirmed pertussis in adults in malaysia , taiwan and thailand with prolonged cough . table 1.pertussis vaccination schedule in malaysia , taiwan and thailandcountryprimary dosesbooster dosesmonth 2month 4month 6month 1846 years1112 yearsmalaysiadtap - hib - ipvdtap - hib - ipvdtap - hib - ipv*dtap - hib - ipvtaiwandtap - hib - ipvdtap - hib - ipvdtap - hib - ipvdtap - hib - ipvtdap - ipvthailanddtwp - hibdtwp - hibdtwp - hibdtwpdtwp(or dtap)(or dtap)(or dtap)(or dtap)(or dtap)tdapdark grey cells ( ) indicate optional vaccine . pertussis vaccination schedule in malaysia , taiwan and thailand dark grey cells ( ) indicate optional vaccine . dtap , diphtheria , tetanus , pertussis ( acellular ) vaccine ; hib , haemophilus influenzae type b vaccine ; ipv , inactivated poliomyelitis vaccine ; dtwp , diphtheria , tetanus , pertussis ( whole cell ) vaccine ; dtap / tdap , diphtheria , tetanus , and pertussis ( acellular ) vaccine [ reduced antigen(s ) ] . this cross - sectional study was conducted between 20 june 2012 and 2 may 2013 according to the declaration of helsinki and good clinical practice guidelines . written informed consent or assent was obtained from subjects ( or parent / legally acceptable representative in taiwan and thailand where the age of consent is 20 years ) prior to enrolment . adult patients aged at least 19 years with prolonged cough were recruited from two centres in malaysia ( international medical university and university of malaya medical centre ) , three centres in thailand ( phramongkutklao hospital , srinagarind hospital and songklanagarind hospital ) and two centres in taiwan ( china medical university hospital and chang gung memorial hospital ) . all patients with prolonged cough ( present for 14 days ) were screened for eligibility . patients with known pulmonary disease causing chronic cough ( including pulmonary tuberculosis , chronic obstructive airways disease , bronchiectasis , asthma ) and patients with suspected or known immunodeficiency were excluded . health - related quality of life [ euroqol-5d ( eq-5d ) ] and structured health economic questionnaires were administered by study staff to individuals at the time of enrolment . a blood sample was collected from each patient for anti - pertussis toxin ( anti - pt ) level of igg antibodies testing . anti - pt was measured by elisa ( euroimmun , germany ) according to the manufacturer 's instructions at a laboratory designated by gsk vaccines with an assay cut - off for sensitivity of 10 iu / ml . sera were classified into four categories , based on manufacturer 's instructions , previous experience in the available literature [ 1517 ] and the absence of vaccination against pertussis in the last 12 months : a seronegative patient had an anti - pt igg level below the sensitivity limit of the assay ( < 10 iu / ml ) ; a seropositive patient had an anti - pt igg level 10 iu / ml ; an anti - pt igg 625 iu / ml was considered serological evidence of pertussis infection in the last 12 months ; and an anti - pt igg level 100 iu / ml was considered indicative of active or recent infection . health - related quality of life was assessed using the eq-5d questionnaire , which comprises five dimensions ( mobility , self - care , usual activities , pain / discomfort , anxiety / depression ) , with three levels of severity within each dimension . health economic impact such as absenteeism from work or school and associated income loss of the subjects or family members , healthcare resource utilization , and direct costs ( including medical costs and transportation costs ) were recorded using a standardized questionnaire . previous studies have demonstrated a prevalence of pertussis of between 10% and 20% in adults with prolonged cough [ 19 , 20 ] . considering a prevalence of pertussis of 18% assuming a drop - out rate of 10% , we therefore aimed to enrol 154 subjects in thailand and at least 78 subjects in malaysia and taiwan . prevalence of pertussis infection with exact 95% confidence intervals ( cis ) was determined by the number of patients with laboratory evidence suggestive of pertussis infection in all subjects presenting with prolonged cough . , contact with people with coughing disease before onset , comorbidities , antibiotic treatment and laboratory results of subsets of patients with and without serological evidence of pertussis , were assessed . the health economic impact in terms of absenteeism from work , healthcare resource utilization , and direct costs were described only for subjects with serologically confirmed pertussis . there were 337 patients enrolled in the study , of whom 25 ( 74% ) were excluded from the analysis leaving 312 subjects in the atp cohort ( fig . of the 312 subjects included in the analysis , 296 ( 949% ) had anti - pt igg levels below the pre - defined cut - off of 625 iu / ml , including 208 ( 6667% ) seronegative subjects ( anti - pt antibodies igg < 10 iu / ml ) . the other 16 of the 312 subjects ( 513% , 95% ci 296819 ) had serological evidence of pertussis infection in the last 12 months ( anti - pt 625 iu / ml ) : 6/76 ( 79% , 95% ci 2951640 ) in malaysia , 1/89 in taiwan ( 11% , 95% ci 003610 ) and 9/147 ( 61% , 95% ci 2841130 ) in thailand . four of these 16 subjects ( overall 13% , 4/312 , 95% ci 035325 ) had anti - pt antibodies 100 iu / ml indicative of active or recent infection . the demographic characteristics of enrolled patients with and without serological evidence of pertussis infection ( anti - pt 625 iu / ml ) are shown in table 2 . pertussis cases were distributed across all age groups , and were highest in age groups 5059 years ( 98% , 5/51 , 95% ci 3262141 ) and 1929 years ( 76% , 5/66 , 95% ci 2511680 ) ( figs . serological evidence of active pertussis ( anti - pt igg 100 iu / ml ) was most commonly observed in the 1929 years age group ( 30% , 2/66 , 95% ci 0371052 ) . the median duration of cough was not significantly different between patients with and without serological evidence of pertussis ( anti - pt igg 625 iu / ml ) : 43 days ( range 15120 ) vs. 31 days ( range 141682 ) , respectively ( p = 01 ) . duration of cough was > 90 days in 3/16 ( 188% ) of patients with serological evidence of pertussis compared to 40/296 ( 135% ) patients without ( table 3 ) . the most frequently reported duration of cough was longer in patients with serological evidence of pertussis ( 3160 days ) compared to those without ( 1430 days ) . a greater proportion of patients with serological evidence of pertussis ( 750% , 12/16 ) than those without ( 483% , 143/296 ) reported paroxysms ( p = 004 ) . breathlessness / chest pain was reported by 625% ( 10/16 ) of patients with serological evidence of pertussis vs. 358% ( 106/296 ) of patients without ( p = 003 ) . the most commonly recorded comorbid conditions were categorized as other non - respiratory tract diseases ; however , there was no difference between those with / without serological evidence of pertussis in terms underlying medical conditions [ 1/16 ( 63% ) vs. 36/296 ( 122% ) , respectively , p = 070 , fisher 's exact test ] . 2.proportion of subjects in each age group with serological evidence of pertussis in the previous 12 months ( anti - pt igg 625 iu / ml ; according - to - protocol cohort ) . 3.distribution of anti - pt igg concentrations by ( a ) age and ( b ) cough duration ( according - to - protocol cohort ) . table 2.demographic characteristics of enrolled patients with and without evidence of pertussis in the last 12 months ( anti - pt igg 625 iu / ml)categoryanti - pt 655 iu / ml ( n = 16)anti - pt < 655 iu / ml ( n = 296)age ( years)mean ( sd)407 ( 133)431 ( 155)range21631983age group , years , n ( % ) 19295 ( 313)61 ( 206)30393 ( 188)86 ( 291)40492 ( 125)50 ( 169)50595 ( 313)46 ( 155)601 ( 63)53 ( 179)genderfemale , n ( % ) 8 ( 50)202 ( 682)male , n ( % ) 8 ( 50)94 ( 318)n , number of subjects ; n , number of subjects in a given category ; s.d . table 3.clinical signs and symptoms of chronic cough by status of pertussis infection by anti - pt levels ( according - to - protocol cohort)clinical symptomswith serological evidence ( anti - pt 655 iu / ml ) ( n = 16)without serological evidence ( anti - pt < 655 iu / ml ) ( n = 296)p valueduration of cough , days , median ( range)43 ( 15120)31 ( 141682)cough duration , days , n ( % ) 14304 ( 250)142 ( 480)<00131607 ( 438)74 ( 250)61902 ( 125)40 ( 135)>903 ( 188)40 ( 135)fever since onset of cough , n ( % ) yes5 ( 313)74 ( 25)056presence of at least one of the following symptoms , n ( % ) yes13 ( 813)258 ( 872)045whoop2 ( 125)18 ( 61)027paroxysm12 ( 750)143 ( 483)004post - tussive vomiting6 ( 375)72 ( 243)024apnoea03 ( 10)100cyanosis00coughing up phlegm6 ( 375)180 ( 608)006sneezes6 ( 375)111 ( 375)100wheezes3 ( 188)34 ( 115)042cough at night11 ( 688)211 ( 713)078episodes of being unable to stop coughing7 ( 438)140 ( 473)078breathlessness / chest pain10 ( 625)106 ( 358)003n , number of subjects.fisher's exact test p value. proportion of subjects in each age group with serological evidence of pertussis in the previous 12 months ( anti - pt igg 625 iu / ml ; according - to - protocol cohort ) . distribution of anti - pt igg concentrations by ( a ) age and ( b ) cough duration ( according - to - protocol cohort ) . demographic characteristics of enrolled patients with and without evidence of pertussis in the last 12 months ( anti - pt igg 625 iu / ml ) n , number of subjects ; n , number of subjects in a given category ; s.d . clinical signs and symptoms of chronic cough by status of pertussis infection by anti - pt levels ( according - to - protocol cohort ) n , number of subjects . chest x - ray was performed on 212/312 ( 679% ) patients ( seven with serological evidence of pertussis ) . the majority of chest x - rays [ 714% ( 5/7 ) in serologically confirmed and 863% ( 177/205 ) in patients with anti - pt igg < 625 polymerase chain reaction ( pcr ) was only performed in one patient without serological evidence of pertussis and failed to identify a pathogen . one patient ( without serological evidence of pertussis ) had pleural tuberculosis confirmed by pleural biopsy ( post - enrolment and was included in study analyses ) . contact with a household or workplace member with known or suspected whooping cough was reported by 1/16 ( 63% ) patient with serological evidence of pertussis vs. 29/296 ( 98% ) of subjects without pertussis . contact with a household or workplace member with persistent cough was reported by 4/16 ( 250% ) pertussis patients and 109/296 ( 368% ) patients without pertussis , and contact with a household or workplace member who developed a cough subsequently was reported by 6/16 ( 375% ) and 106/296 ( 358% ) subjects , respectively . of the 16 patients with serological evidence of recent pertussis infection , three ( 188% ) were employed as teachers and one reported having contact with an infant aged < 1 year either at home or the workplace . of the 16 patients with serological evidence of pertussis , 10 ( 625% ) reported not receiving any previous dose of pertussis vaccine , with a further five ( 313% ) patients unable to recall their vaccination status . of the 296 patients without serological evidence of pertussis , 87 ( 294% ) reported not receiving any previous dose of pertussis vaccine while 114 ( 385% ) were unable to recall their vaccination status . in patients with serological evidence of pertussis , the number of days absent from work ranged from 0 to 30 , with a median of 0 and a mean of 24 ( standard deviation 80 ) . pertussis infection was associated with a median of 25 visits ( range 110 ) to healthcare professionals ( general practitioner , emergency room , or specialist ) . the median total direct medical cost of pertussis per episode in public hospitals ( including consultations with physicians and/or emergency room visits ) was us$13 ( range us$1316 ) or 40 myr ( range 4050 myr ) in malaysia ; us$83 ( or 2450 ntd ) in taiwan ( one patient ) and us$26 ( range us$12168 ) or 800 thb ( range 3605200 thb ) in thailand . of subjects with serological evidence of pertussis infection , two patients ( 2/16 , 125% ) had problems with mobility , 6/16 ( 375% ) had problems in performing usual activities , 6/16 ( 375% ) suffered from pain / discomfort and 7/16 ( 438% ) suffered from anxiety / depression . we found serological evidence of pertussis infection in the last 12 months in 513% of adults presenting to hospitals with cough of at least 2 weeks ' duration in malaysia , taiwan and thailand . this is comparable with a prevalence of pertussis of 72% in taiwanese adults with cough duration 1 week tested by serology ( anti - pt ) or pcr , and 117% of adults with prolonged cough illness in the united states , denmark and korea tested for elevated anti - pt levels in non - outbreak settings ( reviewed in ) . in addition , two thirds of the subjects had no evidence of immunity against pertussis meaning they remain at risk of contracting infection . pertussis disease in adults is frequently difficult to diagnose because of the absence of classical clinical features such as the inspiratory whoop that are typically associated with disease in unvaccinated children . therefore , raising awareness in healthcare providers to consider pertussis in the differential diagnosis of cough illness in older individuals can help to improve the diagnostics . in addition , pcr is optimally sensitive during the first 3 weeks of cough , meaning patients in our study may have missed this window for detection . further difficulty in performing routine testing in asian countries is represented by the specialist training , experience and specific sample storage and transportation media required for the collection and processing of nasopharyngeal samples for pcr or culture . a single acute - phase serum sample , as used in our study , this method is also supported by kinetic studies showing that anti - pt antibodies decline rapidly in all age groups after infection . none of the patients with serological evidence of pertussis in the previous 12 months in our study received a diagnosis of pertussis . recent pertussis infection was associated with a reduced quality of life in our study , with over one third of subjects reporting pain / discomfort and problems conducting routine activities ( e.g. the median eq-5d score for subjects with recent pertussis infection was 072 suggesting that subjects with suspected pertussis perceived an impact on their quality of life . the direct medical costs associated with pertussis episodes in our study varied between countries and was likely related to individual healthcare systems . the 2012 average monthly wage was us$623 in malaysia , us$1546 in taiwan and us$359 in thailand . this means that pertussis - associated medical costs represented around 2% of the monthly wage in malaysia , 55% in taiwan and over 7% in thailand . even though the patients were enrolled from primary healthcare centres within the hospitals , these patients may not be representative of patients seeking primary healthcare in general practitioners ' or family physicians ' clinics . the diagnosis of pertussis in this study was based on serology , which could have resulted in an underestimation of the pertussis burden . furthermore , patients may not be fully aware of government subsidy levels for healthcare expenses , or they could be reluctant to share income information , resulting in an underestimation of the true health economic impact . in conclusion , approximately 5% of adult patients in malaysia , taiwan and thailand presenting to hospitals with cough of at least 2 weeks ' duration showed serological evidence of pertussis infection , while over two - thirds had no evidence of existing immunity . hence , pertussis should be considered in patients with prolonged or paroxysmal cough in the previous 12 months . pertussis infection posed a significant health economic burden and moderate impact on the health - related quality of life of the affected patients .

autistic spectrum disorder ( asd ) represents a group of poorly understood disorders with complex etiologies , confounded by substantial clinical and genetic heterogeneity . males are affected four times as often as females ( chakrabarti and fombonne , 2005 ) with a population prevalence of 0.60.7% ( szatmari et al . autism is broadly defined as a neurodevelopmental syndrome with markedly abnormal or impaired development in social interaction and communication , and involving repetitive or restricted patterns of behavior ( dsm - iv ) . asd also includes atypical autism , asperger 's syndrome , rett syndrome , overactive disorder , and pervasive developmental disorders , all of which are etiologically related to autism ( reichenberg et al . , 2006 ) . although the neurobiology of asd is not completely understood , one key neurobiological feature of asd is early brain overgrowth , which is thought to reflect defects in neural patterning and wiring ( courchesne et al . , 2007 ) . while the etiology of asd remains unknown , twin and family studies have supported a significant genetic contribution to the disorder . the concordance rates for autism and asd in monozygotic ( mz ) twins are 70 and 90% , respectively , compared to the dizygotic ( dz ) concordance rates of 5 and 10% ( sebat et al . , 2007 ) . rett syndrome is a rare form of asd that arises from mutations in the methyl - cpg - binding protein-2 ( mecp2 ) gene ( monteggia and kavalali , 2009 ) . however , asd displays considerable heterogeneity and the recent large collaborative genome scan by the autism genome project ( agp ) of nearly 1200 sibling pairs with asd identified several regions of interest , yet failed to identify one region with genome - wide significance ( szatmari et al . , 2007 ) . the genetic contribution to asd etiology is thus likely to involve subtle alterations including rare single nucleotide polymorphisms ( snp ) or changes in copy - number variation ( cnv ) . cnv represents a gain ( duplication ) or loss ( deletion or inversion ) in a chromosomal region greater than 1 kb in size . with approximately 12% of the human genome varying in copy number ( carter , 2007 ) , a recent surge of research has revealed associations between cnvs and autism , including both duplications and deletions ( berg et al . , 2007 ; sebat et al . , 2007 ; weiss et al . , 2008 ; xu et al . , 2008 ; cho et al . , 2009 ; glessner et al . , 2009 ; in addition to genetic variation , several other factors have been identified from epidemiological studies , which increase the risk for asd diagnosis , including obstetric complications , such as pre - eclampsia ( mann et al . , 2010 ) , premature birth or low birth weight ( eaton et al . , 2001 ) , and advanced paternal age ( apa ; reichenberg et al . , 2006 ) . apa is a risk factor identified by epidemiology that is potentially modifiable , and thus represents a rare target for asd research . advanced paternal age is associated with a broad range of disease outcomes in children , ranging from an increased risk for neural tube defects and cleft palate ( mcintosh et al . , 1995 ) , intellectual impairments ( malaspina et al . , 2005 ; saha et al . , 2009 ) , epilepsy ( vestergaard et al . , 2005 ) , and bipolar disorder ( frans et al . , 2008 ) . the most robust and replicated studies in this field have demonstrated a link between apa and elevated risk for the neurodevelopmental disorders autism ( for example , reichenberg et al . , 2006 ; durkin et al . , 2008 ; tsuchiya et al . , 2008 ; lundstrom et al . , 2010 ; shelton et al . , 2010 ) and schizophrenia ( malaspina et al . , 2001 ; brown et al . , 2002 ; dalman and allebeck , 2002 ; byrne et al . , 2003 ; el - saadi et al . , 2004 ; tsuchiya et al . , one study assessed a cohort of 132,271 individuals and found that children of men aged 40 years or older had a nearly six - fold increased risk of asd compared to the children of men aged 30 years or younger ( reichenberg et al . , 2006 ) . this finding has been replicated in a large , population - based danish study ( larsson et al . , 2005 ) . in recent years several large us studies ( some of which may include overlapping cases ) have also reported an association between apa and increased risk of asd ( croen et al . , 2007 ; durkin et al . , 2008 ; grether et al . , 2009 ; shelton et al . , advanced paternal age represents an opportunity to investigate gene and environment interactions with the potential to help understand how de novo mutation events occur and contribute to disorders such as asd ( malaspina , 2001 ; sebat et al . , 2007 ) . however , the molecular mechanisms underlying the effects of apa on disease outcomes in children remain essentially unknown . a large number of factors influences human development and could covary with paternal age , one of which is maternal age ( saha et al . , 2009 ) . most attention to date has focused on the effects of aging on the integrity of germ cells , because mammalian germ cell development differs between males and females . a greater number of germ line cell divisions occur in the sperm relative to the egg and the disparity between the quantity of male and female divisions increases with age . with respect to humans , only 24 cell divisions are required in the development of an oocyte and 22 of these occur before birth . in contrast , spermatogonia undergo an estimated 30 cell divisions before puberty and then divide every 16 days thereafter . therefore , the sperm of a 40-year - old man has undergone over four times as many replications than that of a 20-year - old . each time these cells divide , the replication of the genome increases susceptibility to copy error mutations ( crow , 2000 ) . apa - related copy error is likely to result in rare mutations in individual sperm . this suggests that such copy error could contribute to changes in structural variation , such as cnvs . therefore , one challenge lies in the clarification of an association between apa and an increase in cnvs using a suitable model system . dysregulation of epigenetic processes during spermatogenesis in older men also has the potential to contribute to the association between apa and neurocognitive disorders ( perrin et al . , 2007 ) . changes in chromatin packaging and integrity ( zubkova and robaire , 2006 ) and hypermethylation in ribosomal dna ( oakes et al . , 2003 ) were evident in spermatozoa of older rats when compared to younger rats . further , it is possible that age - related alterations in gene imprinting mediate apa effects on the next generation . only one of the alleles is transcribed , the other allele is represented in a parent - of - origin dependent manner ( surani , 1998 ; reik and walter , 2001 ) . within the genome , the majority of imprinted genes occur in large clusters controlled by imprinting centers ( koerner and barlow , 2010 ) . in some instances , imprinted gene expression can be tissue - specific and within the brain it may occur in a region or cell - specific manner ( davies et al . changes to the expression of imprinted genes may alter brain development ( gregg et al . , 2010b ) and have been connected with neurocognitive disease ( davies et al . , 2004 ; isles et al . perrin et al . ( 2007 ) suggests that apa could increase the likelihood of epimutations or mutations within imprinted genes or imprinting centers . the next challenge is , therefore , to develop an animal model to test the hypothesis that apa can directly alter epigenetic processes . there is some indirect evidence from clinical research to support the hypothesis that these apa - related mechanisms may be implicated in asd . genetic studies indicate that cnvs are significantly more common in asd ( sebat et al . , 2007 ; christian et al . , 2008 ; marshall et al . , 2008 ; merikangas et al . , 2009 ; weiss and arking , 2009 ; pinto et al . , 2010 ) . rett syndrome , an x chromosome - linked asd , has been associated with mutations in mecp2 gene encoding a protein with key functions in the regulation of epigenetic patterning ( nagarajan et al . , 2008 ; feinberg , 2010 ; gonzales and lasalle , 2010 ) . furthermore , loss of methylation at the maternally imprinted insulin - like growth factor ii ( igf2 ) gene was associated with apa in cord blood cells of chinese subjects ( dai et al . , 2007 ) . interestingly , the levels of this growth hormone were elevated in blood of boys with asd or autism and this may have contributed to the larger head circumference and body mass index seen in these boys ( mills et al . , 2007 ) . moreover , global methylation profiling of lymphoblastoid cell lines from monozygotic twins discordant for autism showed altered methylation in promoters for several candidate genes previously linked to autism ( nguyen et al . , 2010 ) . the molecular mechanisms described above may not be mutually exclusive but they may interact with each other to increase the risk of developing asd ( schanen , 2006 ) . thus , the one commonly recurrent cytogenetic aberration associated with asd entails duplications of chromosome 15q1113 , which occur in up to 5% of patients with asd ( cook et al . the affected region includes paternally and maternally imprinted genes and maternally derived duplications confer an 85% increase in the risk of developing asd ( cook et al . , 1997 ) . genome - wide expression profiling using lymphoblasts from asd patients with duplications in this region identified over 100 consistently differentially expressed transcripts , but most of this arose from genes outside the duplication ( baron et al . these observations emphasize the notion that the autism phenotype is an incompletely penetrant , multigenic trait . although clinical research is clearly important , testing causative and mechanistic hypothesis is difficult in humans because multiple genetic and environmental factors are involved in the etiology of asd . animal models that focus on behavioral dimensions associated with underlying biological mechanisms are valuable for this purpose . however , many core behavioral symptoms of asd include aspects that may be impossible to model in mice . for example , theory of mind , which is the ability to empathize with the feelings and intentions of others , as well as language deficits are not easily evaluated in the mouse . therefore , asd may be particularly difficult to model in rodents ( crawley , 2007 ) . in contrast , social behavior can be effectively assessed in mouse models , because mice are known to engage in high levels of social interaction and social communication ( gheusi et al . , 1994 ) . the first of these used targeted disruption or mutation of genes that may be involved in asd susceptibility , such as the fmr1 gene that is associated with fragile x syndrome ( mcnaughton et al . , 2008 ) . the drawback of this approach is that the targeted allele in the mouse may be functionally distinct from the allele present in the asd population ( moy et al . , 2006 ) . a second approach has been to generate models based on defects in neurotransmission , such as neonatal serotonin depletion , or alterations in parts of the serotonin signaling pathway ( moy and nadler , 2008 ) . the third major approach to modeling autism in the mouse has been based on known epidemiological risk factors such as apa . this is an emerging area of animal modeling that correlates naturalistically with disease etiology in humans . the first rodent study investigating the consequences of apa on adult behavior assessed the offspring of 2.523 month - old male wistar rats on measures of spontaneous activity and learning capacity in a conditioned avoidance task ( auroux , 1983 ) . this study revealed a significant decrease in learning capacity in male and female offspring with increasing paternal age . this learning deficit was seen in male offspring of sires that were 6 months of age or older , but only became apparent in female offspring when the sires were 18 months old . while the auroux ( 1983 ) study showed a cognitive defect resulting from apa in the rat , inbred mouse strains are preferred for this type of research . the use of inbred strains minimizes genetic variability in order to optimize the dissection of potential genetic and epigenetic mechanisms underlying apa . additionally , the genome of the inbred mouse provides the opportunity for genetic manipulation to test specific hypotheses . when generating new models of complex psychiatric disease , it is difficult to decide the most appropriate conditions for the model . the longevity of laboratory mice varies substantially by strain and laboratory . while male c57bl/6j mice have an average life span of 28.5 months ( massie et al . , 1991 ) ; non - representative animals that live for extended periods influence this measure . the jackson laboratory ( http://jaxmice.jax.org/strain/000664.html ) notes that the median age span for c57 is approximately 20 months . if a naturalistic model is preferred , then one would select sires for apa models as close to the age at which there is a known apa effect on psychiatric illness in humans . for autism and schizophrenia , this is 4050 years in the human ( malaspina et al . , 2001 ; reichenberg et al . , 2006 ) , which is equivalent to 1215 months in the mouse ( or 1620 months in the rat ) . to date , there are three published models investigating the behavioral effects of apa in the mouse . the first study investigated the effects of apa on spontaneous activity and passive avoidance learning in adult offspring from a hybrid ( c57bl/6jico x cba / jico ) mouse strain ( garcia - palomares et al . , 2009 ) . these authors demonstrated a learning deficit in the offspring of sires aged 25 and 30 months , compared to the offspring of sires 3 months old , and a decrease in motor activity . however , this reduced activity was only found in the offspring of extremely old sires ( the 30-month - old group ) . only 5 sires out of 20 survived at this age and as such , may have contributed less variation than from the other age groups ( all comprising 20 sires ) . thus , the behavioral phenotype in this model could be a result of a combination apa and unusual paternal longevity . = apa did not alter this behavior . a second mouse model of apa utilized the inbred c57bl/6j strain to examine apa effects on social and exploratory behaviors in the offspring of 2 month - old and 10 month - old sires ( smith et al . , 2009 ) . they reported less engagement in social activity and reduced exploratory behavior for the offspring of older sires . however , locomotion was not altered in this model , as seen in the garcia - palomares et al . ( 2009 ) study , and no other behavioral domains were examined . the third apa mouse model developed by our group uses 4 month - old ( control ) and 1218 month - old ( apa ) sires of the inbred c57bl/6j strain ( foldi et al . , 2010 ) . apa sire ages were selected because they best match the at - risk relative age in humans . sires were mated with 4 month - old females and adult offspring were examined on a comprehensive battery of behavioral tests , designed to assess the broad domains of anxiety , exploration , locomotion , learned helplessness , sensorimotor gating , and working and spatial memories . following behavioral phenotyping , the brains of these mice were imaged ex vivo on a 16.4 t mri scanner . the behavioral phenotype of apa mice in our study included a significant increase in anxiety - related behavior and exploration and an altered coping strategy to an aversive environment ( the forced swim test ) . ( 2009 ) study , we showed no alterations in locomotion or learning , although different paradigms were used to assess learning in both studies . ( 2009 ) study , social behavior in apa mice was normal and exploration was increased , not decreased . the trajectory of cortical development was also altered for male apa mice , in that as neonates , apa males had significantly thinner cortices than did control males . this appeared to reverse by adulthood in that the cortical volumes of male apa mice were now significantly larger than control male mice . this volumetric increase was specific to the rostral portion of the cortex ( i.e. , rostral to the lateral ventricles ; foldi et al . the altered pattern of cortical growth in apa mice is of particular relevance to the association between apa and autism . the cortices of apa neonates were thinner than those of controls , indicating that apa altered the trajectory of cortical development in this model . the cerebral cortex in the brain of a neonatal mouse is similar to that of a late second trimester or early third trimester human fetus ( clancy et al . , 2001 ) . this pattern of cortical development may be similar to indices of brain growth in children with autism . although head circumference is only a proxy marker of brain size , head circumference in children who develop autism is slightly smaller at birth but the growth in circumference accelerates abnormally 621 months after birth ( courchesne et al . , 2003 ; dementieva et al . , 2005 neuroimaging studies have also suggested that autism is associated with rapid and excessive brain growth in the first years of life , resulting in increased cortical volume at 24 years of age ( courchesne et al . , 2001 ; sparks et al . , 2002 furthermore , cerebral overgrowth in autism is greatest in the frontal and temporal cortices ( carper and courchesne , 2005 ) , which is consistent with the neuroanatomical findings described in apa mice by foldi et al . , our apa mouse model seems to recapitulate part of the structural neurobiology of autism . in addition , the anatomical changes were observed only in male apa offspring , with no alterations shown for female mice . considering the marked excess of autism in males this further supports the relevance of our model to the disorder . while there remains a lack of consistency in the behavioral phenotypes of these three apa - related mouse models , there are important differences in how they are constituted . importantly , the age of the apa sires used for each study varied ( see table 1 ) . it is known that behavioral phenotypes are markedly affected by housing and testing conditions , even when significant precautions are taken to equate test apparatus , protocols , and other features of animal husbandry ( crabbe et al . so perhaps it is unsurprising that the reported apa phenotypes differ so significantly , considering inconsistencies in both animal husbandry and testing techniques . what is also obvious from comparing phenotypes is that apa in the mouse is not associated with rare outliers , but results in shifts in various behavioral outcomes that are significant at the population level . one would presume that if we were dealing with a model of rare , dominant de novo genetic events , that this would result in qualitative alterations in phenotype , rather than quantitative population - level effects . alternately it may be that because these population - level effects are in such non - selective behaviors , that they are the result of a cumulative effect of different rare mutations or cnvs that impact on the same behaviors . the instability of the phenotype may also simply reflect the broad associations with disease outcomes in human populations . it is possible that offspring behavioral phenotypes are altered by non - genetic transmission of paternal characteristics , such as paternal behavior . one study has explored the strength of paternal offspring open - field behavior correlations , based on the length of sire exposure ( lse ) prenatally ( alter et al . , 2009 ) . this study demonstrated stronger correlations when sires were housed with pregnant females ( dams ) for longer durations . lse could impact offspring development via maternal stress , such as by altering maternal hormone levels ( lupo di prisco et al . , 1978 ) or other variation in male phenotypic qualities ( drickamer et al . we have recently shown that prenatal lse alone does not influence behavioral outcomes in offspring , by comparing the two predominant methods for breeding laboratory mice . the offspring of both pair - housed and time - mated breeding dyads had equivalent behavioral phenotypes ( foldi et al . , 2011 ) . however , the effects of preconception and prenatal paternal behavior may be highlighted when variables such as paternal drug exposure are included . for example , paternal cocaine abuse in mice prior to coitus has been shown to decrease attention and working memory in offspring ( he et al . , 2006 ) . whether the reported apa rodent phenotypes are affected by the postnatal presence or absence of the sire remains unknown , since these four studies removed the sires prior to birth . , 1980 ; frick et al . , 2000 ) , it is plausible that sire behavior would contribute to offspring phenotypes . the hypothesized mechanism of apa involves rare genetic events , affecting only a small percentage of offspring . perhaps the field needs to move into a model where rare mutations can be detected in very large populations , for example in drosophila ( burne et al . , 2011 ) . additionally , it may be that a reliable phenotype only becomes apparent in the offspring of extremely old male mice , as was seen in the garcia - palomares ( 2009 ) study . thus , the next generation of apa mouse models should utilize an apa dose design ( e.g. , 12 , 18 , and 24 months . this will ascertain firstly , whether increased apa in the mouse produces an increased phenotypic difference and secondly , whether apa dose can explain the disparity within the three published behavioral phenotypes . regardless , the apa mouse model will allow the exploration of within - mouse correlations between behavioral , neuroanatomical , and genomic outcomes . in many instances it will be possible to correlate brain structure versus behavior in the same animal , and behavioral and brain structural outcomes versus genome - wide assessment of methylation and/or copy - number variants in the same animal . in some respects , the apa model , which is based on age - related mutagenesis , represents a naturalistic forward genetics platform . traditionally such platforms use chemicals to induce mutations in sperm stem cells and then use selective breeding on phenotypes of interest to map genotype phenotype relationships ( kile and hilton , 2005 ; caspary and anderson , 2006 ) . the offspring of older fathers have an increased risk of various adverse health outcomes , including autism and schizophrenia . while the molecular mechanisms underpinning these associations are unclear , potential candidates include cnvs and epigenetics . these mechanisms are not mutually exclusive factors but could combine and interact to produce an altered neurocognitive phenotype . while there are only four published rodent models of apa , the phenotypes differ substantially between these models . this is an emerging area of research and future animal studies will need to refine the behavioral and structural phenotype to further explore genetic and epigenetic mechanisms that may underlie these changes . apa models might not only provide clues to upstream mechanisms of action in asd , but could serve as screening platforms for treatments . the urgency for the research community to understand the effects of apa are becoming increasingly important given current trends toward delaying parenting ( bray et al . , 2006 ; toriello and meck , 2008 ) . 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 .

offspring of older fathers have an increased risk of various adverse health outcomes , including autism and schizophrenia . with respect to biological mechanisms for this association , there are many more germline cell divisions in the life history of a sperm relative to that of an oocyte . this leads to more opportunities for copy error mutations in germ cells from older fathers . evidence also suggests that epigenetic patterning in the sperm from older men is altered . rodent models provide an experimental platform to examine the association between paternal age and brain development . several rodent models of advanced paternal age ( apa ) have been published with relevance to intermediate phenotypes related to autism . all four published apa models vary in key features creating a lack of consistency with respect to behavioral phenotypes . a consideration of common phenotypes that emerge from these apa - related mouse models may be informative in the exploration of the molecular and neurobiological correlates of apa .

Autistic Spectrum Disorder Advanced Paternal Age Biological Mechanisms Underpinning APA and Risk of Autism Animal Models Animal Models of APA Conclusion Conflict of Interest Statement

autistic spectrum disorder ( asd ) represents a group of poorly understood disorders with complex etiologies , confounded by substantial clinical and genetic heterogeneity . autism is broadly defined as a neurodevelopmental syndrome with markedly abnormal or impaired development in social interaction and communication , and involving repetitive or restricted patterns of behavior ( dsm - iv ) . asd also includes atypical autism , asperger 's syndrome , rett syndrome , overactive disorder , and pervasive developmental disorders , all of which are etiologically related to autism ( reichenberg et al . , 2006 ) . while the etiology of asd remains unknown , twin and family studies have supported a significant genetic contribution to the disorder . the concordance rates for autism and asd in monozygotic ( mz ) twins are 70 and 90% , respectively , compared to the dizygotic ( dz ) concordance rates of 5 and 10% ( sebat et al . rett syndrome is a rare form of asd that arises from mutations in the methyl - cpg - binding protein-2 ( mecp2 ) gene ( monteggia and kavalali , 2009 ) . the genetic contribution to asd etiology is thus likely to involve subtle alterations including rare single nucleotide polymorphisms ( snp ) or changes in copy - number variation ( cnv ) . cnv represents a gain ( duplication ) or loss ( deletion or inversion ) in a chromosomal region greater than 1 kb in size . with approximately 12% of the human genome varying in copy number ( carter , 2007 ) , a recent surge of research has revealed associations between cnvs and autism , including both duplications and deletions ( berg et al . , 2007 ; sebat et al . , 2007 ; weiss et al . , 2008 ; xu et al . , 2008 ; cho et al . , 2009 ; in addition to genetic variation , several other factors have been identified from epidemiological studies , which increase the risk for asd diagnosis , including obstetric complications , such as pre - eclampsia ( mann et al . , 2010 ) , premature birth or low birth weight ( eaton et al . , 2001 ) , and advanced paternal age ( apa ; reichenberg et al . , 2006 ) . apa is a risk factor identified by epidemiology that is potentially modifiable , and thus represents a rare target for asd research . advanced paternal age is associated with a broad range of disease outcomes in children , ranging from an increased risk for neural tube defects and cleft palate ( mcintosh et al . , 1995 ) , intellectual impairments ( malaspina et al . , 2005 ; saha et al . , 2009 ) , epilepsy ( vestergaard et al . , 2005 ) , and bipolar disorder ( frans et al . the most robust and replicated studies in this field have demonstrated a link between apa and elevated risk for the neurodevelopmental disorders autism ( for example , reichenberg et al . , 2008 ; tsuchiya et al . , 2010 ) and schizophrenia ( malaspina et al . , 2002 ; dalman and allebeck , 2002 ; byrne et al . , 2003 ; el - saadi et al . , 2004 ; tsuchiya et al . , one study assessed a cohort of 132,271 individuals and found that children of men aged 40 years or older had a nearly six - fold increased risk of asd compared to the children of men aged 30 years or younger ( reichenberg et al . , 2006 ) . , 2005 ) . in recent years several large us studies ( some of which may include overlapping cases ) have also reported an association between apa and increased risk of asd ( croen et al . , 2007 ; durkin et al . , 2008 ; grether et al . , 2009 ; shelton et al . , advanced paternal age represents an opportunity to investigate gene and environment interactions with the potential to help understand how de novo mutation events occur and contribute to disorders such as asd ( malaspina , 2001 ; sebat et al . however , the molecular mechanisms underlying the effects of apa on disease outcomes in children remain essentially unknown . a large number of factors influences human development and could covary with paternal age , one of which is maternal age ( saha et al . most attention to date has focused on the effects of aging on the integrity of germ cells , because mammalian germ cell development differs between males and females . a greater number of germ line cell divisions occur in the sperm relative to the egg and the disparity between the quantity of male and female divisions increases with age . with respect to humans , only 24 cell divisions are required in the development of an oocyte and 22 of these occur before birth . in contrast , spermatogonia undergo an estimated 30 cell divisions before puberty and then divide every 16 days thereafter . therefore , the sperm of a 40-year - old man has undergone over four times as many replications than that of a 20-year - old . each time these cells divide , the replication of the genome increases susceptibility to copy error mutations ( crow , 2000 ) . apa - related copy error is likely to result in rare mutations in individual sperm . this suggests that such copy error could contribute to changes in structural variation , such as cnvs . therefore , one challenge lies in the clarification of an association between apa and an increase in cnvs using a suitable model system . dysregulation of epigenetic processes during spermatogenesis in older men also has the potential to contribute to the association between apa and neurocognitive disorders ( perrin et al . , 2007 ) . changes in chromatin packaging and integrity ( zubkova and robaire , 2006 ) and hypermethylation in ribosomal dna ( oakes et al . , 2003 ) were evident in spermatozoa of older rats when compared to younger rats . further , it is possible that age - related alterations in gene imprinting mediate apa effects on the next generation . only one of the alleles is transcribed , the other allele is represented in a parent - of - origin dependent manner ( surani , 1998 ; reik and walter , 2001 ) . in some instances , imprinted gene expression can be tissue - specific and within the brain it may occur in a region or cell - specific manner ( davies et al . changes to the expression of imprinted genes may alter brain development ( gregg et al . , 2010b ) and have been connected with neurocognitive disease ( davies et al . , 2004 ; isles et al . ( 2007 ) suggests that apa could increase the likelihood of epimutations or mutations within imprinted genes or imprinting centers . the next challenge is , therefore , to develop an animal model to test the hypothesis that apa can directly alter epigenetic processes . there is some indirect evidence from clinical research to support the hypothesis that these apa - related mechanisms may be implicated in asd . genetic studies indicate that cnvs are significantly more common in asd ( sebat et al . , 2008 ; merikangas et al . rett syndrome , an x chromosome - linked asd , has been associated with mutations in mecp2 gene encoding a protein with key functions in the regulation of epigenetic patterning ( nagarajan et al . furthermore , loss of methylation at the maternally imprinted insulin - like growth factor ii ( igf2 ) gene was associated with apa in cord blood cells of chinese subjects ( dai et al . , 2007 ) . interestingly , the levels of this growth hormone were elevated in blood of boys with asd or autism and this may have contributed to the larger head circumference and body mass index seen in these boys ( mills et al . , 2007 ) . moreover , global methylation profiling of lymphoblastoid cell lines from monozygotic twins discordant for autism showed altered methylation in promoters for several candidate genes previously linked to autism ( nguyen et al . , 2010 ) . the molecular mechanisms described above may not be mutually exclusive but they may interact with each other to increase the risk of developing asd ( schanen , 2006 ) . the affected region includes paternally and maternally imprinted genes and maternally derived duplications confer an 85% increase in the risk of developing asd ( cook et al . , 1997 ) . although clinical research is clearly important , testing causative and mechanistic hypothesis is difficult in humans because multiple genetic and environmental factors are involved in the etiology of asd . animal models that focus on behavioral dimensions associated with underlying biological mechanisms are valuable for this purpose . however , many core behavioral symptoms of asd include aspects that may be impossible to model in mice . for example , theory of mind , which is the ability to empathize with the feelings and intentions of others , as well as language deficits are not easily evaluated in the mouse . therefore , asd may be particularly difficult to model in rodents ( crawley , 2007 ) . in contrast , social behavior can be effectively assessed in mouse models , because mice are known to engage in high levels of social interaction and social communication ( gheusi et al . the first of these used targeted disruption or mutation of genes that may be involved in asd susceptibility , such as the fmr1 gene that is associated with fragile x syndrome ( mcnaughton et al . , 2008 ) . the drawback of this approach is that the targeted allele in the mouse may be functionally distinct from the allele present in the asd population ( moy et al . a second approach has been to generate models based on defects in neurotransmission , such as neonatal serotonin depletion , or alterations in parts of the serotonin signaling pathway ( moy and nadler , 2008 ) . the third major approach to modeling autism in the mouse has been based on known epidemiological risk factors such as apa . the first rodent study investigating the consequences of apa on adult behavior assessed the offspring of 2.523 month - old male wistar rats on measures of spontaneous activity and learning capacity in a conditioned avoidance task ( auroux , 1983 ) . this study revealed a significant decrease in learning capacity in male and female offspring with increasing paternal age . this learning deficit was seen in male offspring of sires that were 6 months of age or older , but only became apparent in female offspring when the sires were 18 months old . while the auroux ( 1983 ) study showed a cognitive defect resulting from apa in the rat , inbred mouse strains are preferred for this type of research . additionally , the genome of the inbred mouse provides the opportunity for genetic manipulation to test specific hypotheses . when generating new models of complex psychiatric disease , it is difficult to decide the most appropriate conditions for the model . the longevity of laboratory mice varies substantially by strain and laboratory . while male c57bl/6j mice have an average life span of 28.5 months ( massie et al . , 1991 ) ; non - representative animals that live for extended periods influence this measure . if a naturalistic model is preferred , then one would select sires for apa models as close to the age at which there is a known apa effect on psychiatric illness in humans . for autism and schizophrenia , this is 4050 years in the human ( malaspina et al . , 2006 ) , which is equivalent to 1215 months in the mouse ( or 1620 months in the rat ) . to date , there are three published models investigating the behavioral effects of apa in the mouse . the first study investigated the effects of apa on spontaneous activity and passive avoidance learning in adult offspring from a hybrid ( c57bl/6jico x cba / jico ) mouse strain ( garcia - palomares et al . , 2009 ) . these authors demonstrated a learning deficit in the offspring of sires aged 25 and 30 months , compared to the offspring of sires 3 months old , and a decrease in motor activity . however , this reduced activity was only found in the offspring of extremely old sires ( the 30-month - old group ) . only 5 sires out of 20 survived at this age and as such , may have contributed less variation than from the other age groups ( all comprising 20 sires ) . thus , the behavioral phenotype in this model could be a result of a combination apa and unusual paternal longevity . = apa did not alter this behavior . a second mouse model of apa utilized the inbred c57bl/6j strain to examine apa effects on social and exploratory behaviors in the offspring of 2 month - old and 10 month - old sires ( smith et al . , 2009 ) . they reported less engagement in social activity and reduced exploratory behavior for the offspring of older sires . however , locomotion was not altered in this model , as seen in the garcia - palomares et al . the third apa mouse model developed by our group uses 4 month - old ( control ) and 1218 month - old ( apa ) sires of the inbred c57bl/6j strain ( foldi et al . , 2010 ) . apa sire ages were selected because they best match the at - risk relative age in humans . the behavioral phenotype of apa mice in our study included a significant increase in anxiety - related behavior and exploration and an altered coping strategy to an aversive environment ( the forced swim test ) . ( 2009 ) study , we showed no alterations in locomotion or learning , although different paradigms were used to assess learning in both studies . the trajectory of cortical development was also altered for male apa mice , in that as neonates , apa males had significantly thinner cortices than did control males . this appeared to reverse by adulthood in that the cortical volumes of male apa mice were now significantly larger than control male mice . this volumetric increase was specific to the rostral portion of the cortex ( i.e. , rostral to the lateral ventricles ; foldi et al . the altered pattern of cortical growth in apa mice is of particular relevance to the association between apa and autism . the cortices of apa neonates were thinner than those of controls , indicating that apa altered the trajectory of cortical development in this model . the cerebral cortex in the brain of a neonatal mouse is similar to that of a late second trimester or early third trimester human fetus ( clancy et al . this pattern of cortical development may be similar to indices of brain growth in children with autism . , 2005 neuroimaging studies have also suggested that autism is associated with rapid and excessive brain growth in the first years of life , resulting in increased cortical volume at 24 years of age ( courchesne et al . , 2001 ; sparks et al . , 2002 furthermore , cerebral overgrowth in autism is greatest in the frontal and temporal cortices ( carper and courchesne , 2005 ) , which is consistent with the neuroanatomical findings described in apa mice by foldi et al . , our apa mouse model seems to recapitulate part of the structural neurobiology of autism . considering the marked excess of autism in males this further supports the relevance of our model to the disorder . while there remains a lack of consistency in the behavioral phenotypes of these three apa - related mouse models , there are important differences in how they are constituted . importantly , the age of the apa sires used for each study varied ( see table 1 ) . it is known that behavioral phenotypes are markedly affected by housing and testing conditions , even when significant precautions are taken to equate test apparatus , protocols , and other features of animal husbandry ( crabbe et al . so perhaps it is unsurprising that the reported apa phenotypes differ so significantly , considering inconsistencies in both animal husbandry and testing techniques . what is also obvious from comparing phenotypes is that apa in the mouse is not associated with rare outliers , but results in shifts in various behavioral outcomes that are significant at the population level . one would presume that if we were dealing with a model of rare , dominant de novo genetic events , that this would result in qualitative alterations in phenotype , rather than quantitative population - level effects . alternately it may be that because these population - level effects are in such non - selective behaviors , that they are the result of a cumulative effect of different rare mutations or cnvs that impact on the same behaviors . the instability of the phenotype may also simply reflect the broad associations with disease outcomes in human populations . it is possible that offspring behavioral phenotypes are altered by non - genetic transmission of paternal characteristics , such as paternal behavior . one study has explored the strength of paternal offspring open - field behavior correlations , based on the length of sire exposure ( lse ) prenatally ( alter et al . this study demonstrated stronger correlations when sires were housed with pregnant females ( dams ) for longer durations . lse could impact offspring development via maternal stress , such as by altering maternal hormone levels ( lupo di prisco et al . the offspring of both pair - housed and time - mated breeding dyads had equivalent behavioral phenotypes ( foldi et al . however , the effects of preconception and prenatal paternal behavior may be highlighted when variables such as paternal drug exposure are included . whether the reported apa rodent phenotypes are affected by the postnatal presence or absence of the sire remains unknown , since these four studies removed the sires prior to birth . , 1980 ; frick et al . , 2000 ) , it is plausible that sire behavior would contribute to offspring phenotypes . the hypothesized mechanism of apa involves rare genetic events , affecting only a small percentage of offspring . additionally , it may be that a reliable phenotype only becomes apparent in the offspring of extremely old male mice , as was seen in the garcia - palomares ( 2009 ) study . thus , the next generation of apa mouse models should utilize an apa dose design ( e.g. , 12 , 18 , and 24 months . this will ascertain firstly , whether increased apa in the mouse produces an increased phenotypic difference and secondly , whether apa dose can explain the disparity within the three published behavioral phenotypes . regardless , the apa mouse model will allow the exploration of within - mouse correlations between behavioral , neuroanatomical , and genomic outcomes . in many instances it will be possible to correlate brain structure versus behavior in the same animal , and behavioral and brain structural outcomes versus genome - wide assessment of methylation and/or copy - number variants in the same animal . in some respects , the apa model , which is based on age - related mutagenesis , represents a naturalistic forward genetics platform . traditionally such platforms use chemicals to induce mutations in sperm stem cells and then use selective breeding on phenotypes of interest to map genotype phenotype relationships ( kile and hilton , 2005 ; caspary and anderson , 2006 ) . the offspring of older fathers have an increased risk of various adverse health outcomes , including autism and schizophrenia . while the molecular mechanisms underpinning these associations are unclear , potential candidates include cnvs and epigenetics . these mechanisms are not mutually exclusive factors but could combine and interact to produce an altered neurocognitive phenotype . while there are only four published rodent models of apa , the phenotypes differ substantially between these models . apa models might not only provide clues to upstream mechanisms of action in asd , but could serve as screening platforms for treatments . the urgency for the research community to understand the effects of apa are becoming increasingly important given current trends toward delaying parenting ( bray et al . 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 .

hepatocellular carcinoma ( hcc ) is one of the most common cancers and chronic infection with hepatitis b virus ( hbv ) is the most common cause of hcc worldwide . the mainland of china , an area endemic for hbv genotypes b and c , accounts for one - third of global hbv - infected subjects and half of global hcc cases . according to the differences in cancer - inducing capacities of hbv genotypes b and c , the endemicity of hbv genotypes in the mainland of china , and the incidence of hcc in a cohort study carried out in taiwan ( china ) where genotype b is endemic , we estimated that 30% of male and 10% of female hbv - infected subjects in the mainland of china will develop hcc before 75 years old . thus , at least 20 million hbv - caused hcc cases will be diagnosed in the mainland of china within next 50 years , even though , postnatal hbv infection has been greatly diminished by hbv vaccination since 1992 and other effective prophylaxis . hcc is a highly fatal malignancy , with a 5-year survival rate of 10% for patients without surgical treatment and 30% for those who received curative surgery . the occurrence of hbv - related hcc in chronic hbv carriers can be greatly reduced via active prophylaxis using anti - hbv treatments . it is , therefore , a great challenge to identify the hbv - infected subjects who are more likely to develop hcc and need the specific prophylaxis in advance . epidemiological studies have shown that increasing age , male gender , hbv genotype c ( vs. genotype b ) , high levels of circulating hbv dna ( > 10 copies / ml ) , hepatitis b e antigen ( hbeag ) expression , and certain hbv mutations are significantly associated with an increased risk of hcc in hbv - infected subjects . we and others have reported that hbv mutations c1653 t , t1753v , a1762t / g1764a , t1674c / g , and c1766t / t1768a in the enhancer ii / basal core promoter ( enhii / bcp ) region ; g1899a , c2002 t , a2159 g , a2189c , and g2203a / t in the precore / core region ; as well as t53c , pres2 start codon mutation , pres1 deletion , c2964a , a2962 g , c3116 t , and c7a in the pres region of hbv genome are significantly associated with an increased risk of hcc . hbv reverse transcriptase lacks proofreading activity , resulting in viral nucleotide substitution mutations during viral replication . moreover , inflammatory factors promote hbv mutations , possibly through inducing expression of cytidine deaminases . chronic inflammatory microenvironment can facilitate the selection of hcc - related hbv mutations by host innate and adaptive antiviral immunity . however , the effect of hbv mutations on hcc development might be influenced by their interactions with host genetic susceptibility . phosphatase and tensin homolog ( pten ) , which is located at chromosome 10q23.3 , spans 105 kb and includes 9 exons and 8 introns . it involves in a variety of biological processes including regulation of cell growth , proliferation , migration , apoptosis , and cell cycle control . pten protein is a dual phosphatase that acts at both serine - threonine and tyrosine sites . although pten mutation is a rare event in hcc , the signaling affected by pten , namely pi3k / akt / mammalian target of rapamycin , is aberrantly regulated in half of hcc cases . pten may affect the development of hcc via modulating hbv x protein - mediated signaling . single nucleotide polymorphisms ( snps ) of pten have been linked to multiple histotypes of cancers including nasopharyngeal carcinoma . however , the effects of pten snps on hcc risk and their interactions with hbv mutations on hcc risk have not been reported . we hypothesized that pten genetic polymorphisms may play a role in chronic hbv infection , hbv - related inflammation , and hepatocarcinogenesis , alone or possibly through interacting with hbv mutations . in this large epidemiological study , we investigated the effects of several pten polymorphisms and their interactions with important hbv mutations on the risk of hcc in chronic hbv - infected subjects . this study should be helpful in identifying hbv - infected subjects who are more likely to develop hcc and need active prophylaxis . healthy controls and hbv - infected subjects with or without hcc enrolled in this study have been previously described . in brief , 1012 healthy controls , 316 asymptomatic hepatitis b surface antigen ( hbsag ) carriers , 316 chronic hepatitis b ( chb ) patients , 358 liver cirrhosis patients , and 1021 hcc patients were recruited from our community - based epidemiological study in yangpu district of shanghai and our collaborative hospitals in shanghai , shandong province , jiangsu province , and chongqing between september 2009 and september 2011 . we also enrolled 302 natural clearance subjects from our epidemiological survey in yangpu district during this period . subjects who were positive for antibodies against hepatitis c virus ( hcv ) , hepatitis delta virus ( hdv ) , and/or human immunodeficiency virus ( hiv ) were excluded . the study was approved by the human research ethics committee of the second military medical university , and the protocol was conformed to the ethical guidelines of the 1975 declaration of helsinki . briefly , 5 ml fasting blood samples were obtained with a vacuum blood collection tube without anticoagulant during recruitment before any treatment . the serum was separated by centrifugation at 4c and stored in a sterile tube at 80c within 6 h of sample collection . hbv serological markers , including hbeag , antibody to hbe , hbsag , antibody to hbs were examined by architect - i2000 ( abbott laboratories , north chicago , il , usa ) . liver function tests including alanine aminotransferase ( alt ) were conducted by serum chemistry autoanalyzer ( model 7600 , hitachi , tokyo , japan ) using commercial reagents ( wako , japan ) ; alpha - fetoprotein ( afp ) was tested by bayer acs-180 ( bayer , germany ) using commercial reagents ( bayer ) . antibody to hdv was examined using commercial kits ( wantai bio - pharm , beijing , china ) . serum viral load was measured in the lightcycler ( roche , mannheim , germany ) , using the quantitative hbv polymerase chain reaction ( pcr ) fluorogence diagnostic kit ( pg biotechnology , shenzhen , china ) . the enhii / bcp / pc region and pres region of hbv genome were amplified by nested - pcr as previously described . the amplicons were directly sequenced in both forward and reverse directions using abi prism bigdye sequencing kits and an abi 3730 genetic analyzer ( applied biosystems , foster city , ca , usa ) . the hbv sequences were aligned and analyzed by mega 5.0 ( http://www.megasoftware.net/ ) and bioedit 7.0 software packages ( ibis therapeutics carlsbad , ca , usa ) . the mutations of hbv genotype b ( b2 ) and genotype c ( c2 ) were defined , respectively . hcc - related hbv mutations in the enhii / bcp / precore region and the pres region of the hbv genome were evaluated , as previously characterized . phosphatase and tensin homolog snps in the promoter , intronic , intron / exon boundary , exonic and 3utr regions were initially screened using the international hapmap project tag snp information in the han chinese population ( http://hapmap.ncbi.nlm.nih.gov/ ) . the snps in the intron ( rs1234213 , rs1234220 , rs2299939 , rs532678 , rs1234219 , rs12572106 , and rs2299941 ) , at intron / exon boundaries ( rs1903858 ) , and in the 3utr ( rs701848 ) were selected based on the hapmap screening and previous literatures regarding the associations of pten snps with cancers or inflammation - related diseases . we excluded snps if they were monomorphic or had low frequencies in our samples ( rs12572106 , rs1234219 , and rs701848 ) or their surrounding sequences were not suitable for designing the minor groove binder ( mgb ) probes ( rs532678 , rs2299941 , and rs1903858 ) . three intronic snps rs1234220 ( t > c , in the intron 3 ) , rs2299939 ( g > t , in the intron 5 ) , and rs1234213 ( c > t , in the intron 7 ) were finally selected in this study . genomic dna was extracted from 200 l peripheral blood using a qiaamp blood kit ( qiagen , hilden , germany ) according to the manufacturer 's instruction . snps were genotyped using fluorescent probe real - time quantitative pcr in a lightcycler 480 ( roche , basel , switzerland ) . primers and mgb probes [ table 1 ] were designed and synthesized by genecore biotechnologies ( shanghai , china ) . mol / l of primers and probes , 14 ng/l purified templates in premix ex tag reaction system ( takara , dalian , china ) . the conditions for pcr were : initial denaturalizing at 95c for 10 s , followed by 45 cycles of 95c for 10 s and 60c for 30 s , then 40c for 1 s. for quality control purpose , one blank control ( ddh2o ) was included in each 384-well plate . each snp was tested for deviation from the hardy - weinberg equilibrium ( hwe ) in healthy controls using the exact test . student 's t - test and test were used to compare continuous variables and categorical variables , respectively . unconditional logistic regression model was applied to test the effects of pten snps on hcc risk and other hbv - related characteristics , and to calculate odds ratios ( ors ) and their 95% confidence intervals ( cis ) . multivariate logistic regression analysis was applied to evaluate the effect of multiplicative interactions of snps with hbv mutations on hcc development , adjusting for age and gender . all statistical tests were two - sided , and a p < 0.05 was considered statistically significant . all analyses were performed by spss 16.0 for windows ( spss inc . , chicago , il , usa ) . healthy controls and hbv - infected subjects with or without hcc enrolled in this study have been previously described . in brief , 1012 healthy controls , 316 asymptomatic hepatitis b surface antigen ( hbsag ) carriers , 316 chronic hepatitis b ( chb ) patients , 358 liver cirrhosis patients , and 1021 hcc patients were recruited from our community - based epidemiological study in yangpu district of shanghai and our collaborative hospitals in shanghai , shandong province , jiangsu province , and chongqing between september 2009 and september 2011 . we also enrolled 302 natural clearance subjects from our epidemiological survey in yangpu district during this period . subjects who were positive for antibodies against hepatitis c virus ( hcv ) , hepatitis delta virus ( hdv ) , and/or human immunodeficiency virus ( hiv ) were excluded . the study was approved by the human research ethics committee of the second military medical university , and the protocol was conformed to the ethical guidelines of the 1975 declaration of helsinki . , 5 ml fasting blood samples were obtained with a vacuum blood collection tube without anticoagulant during recruitment before any treatment . the serum was separated by centrifugation at 4c and stored in a sterile tube at 80c within 6 h of sample collection . hbv serological markers , including hbeag , antibody to hbe , hbsag , antibody to hbs were examined by architect - i2000 ( abbott laboratories , north chicago , il , usa ) . liver function tests including alanine aminotransferase ( alt ) were conducted by serum chemistry autoanalyzer ( model 7600 , hitachi , tokyo , japan ) using commercial reagents ( wako , japan ) ; alpha - fetoprotein ( afp ) was tested by bayer acs-180 ( bayer , germany ) using commercial reagents ( bayer ) . antibody to hdv was examined using commercial kits ( wantai bio - pharm , beijing , china ) . serum viral load was measured in the lightcycler ( roche , mannheim , germany ) , using the quantitative hbv polymerase chain reaction ( pcr ) fluorogence diagnostic kit ( pg biotechnology , shenzhen , china ) . the enhii / bcp / pc region and pres region of hbv genome were amplified by nested - pcr as previously described . the amplicons were directly sequenced in both forward and reverse directions using abi prism bigdye sequencing kits and an abi 3730 genetic analyzer ( applied biosystems , foster city , ca , usa ) . the hbv sequences were aligned and analyzed by mega 5.0 ( http://www.megasoftware.net/ ) and bioedit 7.0 software packages ( ibis therapeutics carlsbad , ca , usa ) . the mutations of hbv genotype b ( b2 ) and genotype c ( c2 ) were defined , respectively . hcc - related hbv mutations in the enhii / bcp / precore region and the pres region of the hbv genome were evaluated , as previously characterized . phosphatase and tensin homolog snps in the promoter , intronic , intron / exon boundary , exonic and 3utr regions were initially screened using the international hapmap project tag snp information in the han chinese population ( http://hapmap.ncbi.nlm.nih.gov/ ) . the snps in the intron ( rs1234213 , rs1234220 , rs2299939 , rs532678 , rs1234219 , rs12572106 , and rs2299941 ) , at intron / exon boundaries ( rs1903858 ) , and in the 3utr ( rs701848 ) were selected based on the hapmap screening and previous literatures regarding the associations of pten snps with cancers or inflammation - related diseases . we excluded snps if they were monomorphic or had low frequencies in our samples ( rs12572106 , rs1234219 , and rs701848 ) or their surrounding sequences were not suitable for designing the minor groove binder ( mgb ) probes ( rs532678 , rs2299941 , and rs1903858 ) . three intronic snps rs1234220 ( t > c , in the intron 3 ) , rs2299939 ( g > t , in the intron 5 ) , and rs1234213 ( c > t , in the intron 7 ) were finally selected in this study . genomic dna was extracted from 200 l peripheral blood using a qiaamp blood kit ( qiagen , hilden , germany ) according to the manufacturer 's instruction . snps were genotyped using fluorescent probe real - time quantitative pcr in a lightcycler 480 ( roche , basel , switzerland ) . primers and mgb probes [ table 1 ] were designed and synthesized by genecore biotechnologies ( shanghai , china ) . each reaction mixture contained 0.2 mol / l of primers and probes , 14 ng/l purified templates in premix ex tag reaction system ( takara , dalian , china ) . the conditions for pcr were : initial denaturalizing at 95c for 10 s , followed by 45 cycles of 95c for 10 s and 60c for 30 s , then 40c for 1 s. for quality control purpose , one blank control ( ddh2o ) was included in each 384-well plate . each snp was tested for deviation from the hardy - weinberg equilibrium ( hwe ) in healthy controls using the exact test . student 's t - test and test were used to compare continuous variables and categorical variables , respectively . unconditional logistic regression model was applied to test the effects of pten snps on hcc risk and other hbv - related characteristics , and to calculate odds ratios ( ors ) and their 95% confidence intervals ( cis ) . multivariate logistic regression analysis was applied to evaluate the effect of multiplicative interactions of snps with hbv mutations on hcc development , adjusting for age and gender . all statistical tests were two - sided , and a p < 0.05 was considered statistically significant . all analyses were performed by spss 16.0 for windows ( spss inc . , chicago , il , usa ) . briefly , the group of hcc patients had a higher proportion of males compared to other groups . healthy controls and hbv natural clearance subjects were relatively older than hbv - infected patients including hcc patients . there was a larger portion of hbeag negativity in hcc patients compared to hbv - infected patients without hcc . baseline characteristics of the study population * hbv - infected subjects with hcc versus healthy controls ; hbv - infected subjects with hcc versus hbv - infected subjects without hcc ; all hbv - infected subjects including hcc versus hbv natural clearances ; hbv - infected subjects without hcc versus healthy controls . for multiple comparisons , p value was corrected by the bonferroni correction ( p = 0.010 ) . alt : alanine aminotransferase ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; hbeag : hepatitis b e antigen ; hcc : hepatocellular carcinoma ; hbv : hepatitis b virus ; lc : liver cirrhosis ; nd : no data ; sd : standard deviation . all three snps were conformed to hwe in healthy controls ( p > 0.05 for all ) . we first evaluated the associations of pten snps with the risk of hcc [ table 3 ] . compared with healthy controls , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( adjusted or [ aor ] = 1.37 , 95% ci = 1.081.73 and aor = 1.35 , 95% ci = 1.071.69 , respectively ) , after adjusting for age and gender . when compared to hbv - infected patients without hcc , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 1.28 , 95% ci = 1.021.61 and aor = 1.27 , 95% ci = 1.011.57 , respectively ) , whereas rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with reduced risks of hcc ( aor = 0.75 , 95% ci = 0.620.92 , and aor = 0.79 , 95% ci = 0.650.96 , respectively ) . compared with all subjects without hcc , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 1.30 , 95% ci = 1.081.56 and aor = 1.29 , 95% ci = 1.071.54 , respectively ) , while rs2299939 gt genotype was significantly associated with a reduced risk of hcc ( aor = 0.84 , 95% ci = 0.710.99 ) . however , after stratifying the participants by gender , the significant results were only evident in males , but not in females [ table 3 ] . association of pten polymorphisms with the risk of hcc aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; hwe : hardy - weinberg equilibrium ; pten : phosphatase and tensin homolog ; snp : single nucleotide polymorphism . as hbv genotypes and hbeag status are potential confounders in elucidating the associations of pten snps with the risk of hcc , we performed further stratified analyses . after stratifying by hbv genotypes , we found that rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 2.02 , 95% ci = 1.143.59 and aor = 2.02 , 95% ci = 1.143.59 , respectively ) in genotype b hbv subjects , compared with genotype b hbv - infected patients without hcc ; however , these effects did not exist in genotype c hbv - infected subjects [ table 4 ] . as compared with hbv - infected patients without hcc , rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with reduced risks of hcc ( aor = 0.68 , 95% ci = 0.530.87 and aor = 0.72 , 95% ci = 0.570.92 , respectively ) in hbeag - negative hbv - infected subjects . however , these effects were not evident in hbeag - positive ones . association of pten polymorphisms with the risk of hcc stratified by hbv genotypes aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; non - hcc : asc + chb + lc ; pten : phosphatase and tensin homolog ; snp : single nucleotide polymorphism ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; lc : liver cirrhosis . we then investigated the associations of pten snps with hbv - related characteristics in hcc - free hbv - infected subjects . as shown in table 5 , none of the three pten snps were significantly associated with hbv persistence compared to the hbsag seroclearance subjects or healthy controls . rs2299939 t ( gt+tt ) allele was significantly associated with high alt level ( aor = 1.26 , 95% ci = 1.011.57 ) ; rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with high viral load ( 10 copies / ml ) ( aor = 1.33 , 95% ci = 1.061.67 and aor = 1.29 , 95% ci = 1.031.60 , respectively ) after adjusting for age and gender . interestingly , rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with high alt level ( aor = 1.35 , 95% ci = 1.041.75 and aor = 1.36 , 95% ci = 1.061.74 , respectively ) and high viral load ( 10 copies / ml ) ( aor = 1.38 , 95% ci = 1.071.78 and aor = 1.29 , 95% ci = 1.011.66 , respectively ) in males . associations of pten polymorphisms with hbv - related characteristics in hcc - free hbv - infected subjects * hcc - free hbv - infected subjects : ascs , chb patients , and lc patients . aor : adjusted odds ratio ( adjusted for age and gender in the total subjects ; adjusted for age after stratification by gender ) ; pten : phosphatase and tensin homolog ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; snp : single nucleotide polymorphism ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; alt : alanine aminotransferase ; ci : confidence interval ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; lc : liver cirrhosis . the associations of the pten snps with the hcc - related hbv mutations that were described in our previous studies were assessed using the data of the hbv - infected patients including those with hcc . it was found that the variant genotype ( tc ) of rs1234220 was significantly associated with increased frequencies of hbv mutations a1652 g ( aor = 4.16 , 95% ci = 1.6410.55 ) , c1673 t ( aor = 2.40 , 95% ci = 1.025.66 ) , and c1730 g ( aor = 2.34 , 95% ci = 1.025.39 ) in genotype b hbv - infected subjects . we tested the interactions of pten snps with several important hcc - related hbv mutations on hcc risk . there were significant interactions between pten polymorphisms and a3054 t or c3116 t , important hbv mutations in the pres region [ table 6 ] . although the variant genotype of rs2299939 was significantly associated with a reduced risk of hcc , the interaction of rs2299939 variant genotypes with a3054 t was significantly associated with an increased risk of hcc . hbv c3116 t mutation was a significant risk factor of hcc ; however , its interaction with rs2299939 variant genotypes was significantly associated with a reduced risk of hcc . we also found that the interaction of rs1234213 variant genotypes with hbv c3116 t mutation significantly increased the risk of hcc in the hbv - infected subjects . after stratifying by gender , it was found that the interaction between the polymorphism of rs2299939 gt+tt and c3116 t significantly reduced the risk of hcc in male hbv - infected subjects ( aor = 0.27 , 95% ci = 0.130.57 ) . similarly , the interaction between rs1234213 ct+tt and c3116 t also significantly increased hcc risk in male hbv - infected subjects ( aor = 3.61 , 95% ci = 1.548.42 ) after stratifying by gender . interactions of pten polymorphisms with hbv mutations on hcc risk aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; hcc : asc + chb + lc ; pten : phosphatase and tensin homolog ; lc : liver cirrhosis ; snp : single nucleotide polymorphism ; or : odds ratio . the three pten snps were in linkage disequilibrium with each other in our study population . the estimated haplotype frequencies of hcc patients ( n = 1016 ) and healthy controls ( n = 1004 ) indicated that only one haplotype of rs1234220(c)-rs2299939(g)-rs1234213(t ) was significantly more frequent in hcc patients compared to healthy controls after adjusting for age and gender ( aor = 1.32 , 95% ci = 1.061.63 , p = 0.012 ) . the frequencies of other estimated haplotypes did not differ significantly between the two groups . briefly , the group of hcc patients had a higher proportion of males compared to other groups . healthy controls and hbv natural clearance subjects were relatively older than hbv - infected patients including hcc patients . there was a larger portion of hbeag negativity in hcc patients compared to hbv - infected patients without hcc . baseline characteristics of the study population * hbv - infected subjects with hcc versus healthy controls ; hbv - infected subjects with hcc versus hbv - infected subjects without hcc ; all hbv - infected subjects including hcc versus hbv natural clearances ; hbv - infected subjects without hcc versus healthy controls . for multiple comparisons , p value was corrected by the bonferroni correction ( p = 0.010 ) . alt : alanine aminotransferase ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; hbeag : hepatitis b e antigen ; hcc : hepatocellular carcinoma ; hbv : hepatitis b virus ; lc : liver cirrhosis ; nd : no data ; sd : standard deviation . all three snps were conformed to hwe in healthy controls ( p > 0.05 for all ) . we first evaluated the associations of pten snps with the risk of hcc [ table 3 ] . compared with healthy controls , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( adjusted or [ aor ] = 1.37 , 95% ci = 1.081.73 and aor = 1.35 , 95% ci = 1.071.69 , respectively ) , after adjusting for age and gender . when compared to hbv - infected patients without hcc , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 1.28 , 95% ci = 1.021.61 and aor = 1.27 , 95% ci = 1.011.57 , respectively ) , whereas rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with reduced risks of hcc ( aor = 0.75 , 95% ci = 0.620.92 , and aor = 0.79 , 95% ci = 0.650.96 , respectively ) . compared with all subjects without hcc , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 1.30 , 95% ci = 1.081.56 and aor = 1.29 , 95% ci = 1.071.54 , respectively ) , while rs2299939 gt genotype was significantly associated with a reduced risk of hcc ( aor = 0.84 , 95% ci = 0.710.99 ) . however , after stratifying the participants by gender , the significant results were only evident in males , but not in females [ table 3 ] . association of pten polymorphisms with the risk of hcc aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; hwe : hardy - weinberg equilibrium ; pten : phosphatase and tensin homolog ; snp : single nucleotide polymorphism . as hbv genotypes and hbeag status are potential confounders in elucidating the associations of pten snps with the risk of hcc , we performed further stratified analyses . after stratifying by hbv genotypes , we found that rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 2.02 , 95% ci = 1.143.59 and aor = 2.02 , 95% ci = 1.143.59 , respectively ) in genotype b hbv subjects , compared with genotype b hbv - infected patients without hcc ; however , these effects did not exist in genotype c hbv - infected subjects [ table 4 ] . as compared with hbv - infected patients without hcc , rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with reduced risks of hcc ( aor = 0.68 , 95% ci = 0.530.87 and aor = 0.72 , 95% ci = 0.570.92 , respectively ) in hbeag - negative hbv - infected subjects . association of pten polymorphisms with the risk of hcc stratified by hbv genotypes aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; non - hcc : asc + chb + lc ; pten : phosphatase and tensin homolog ; snp : single nucleotide polymorphism ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; lc : liver cirrhosis . we then investigated the associations of pten snps with hbv - related characteristics in hcc - free hbv - infected subjects . as shown in table 5 , none of the three pten snps were significantly associated with hbv persistence compared to the hbsag seroclearance subjects or healthy controls . rs2299939 t ( gt+tt ) allele was significantly associated with high alt level ( aor = 1.26 , 95% ci = 1.011.57 ) ; rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with high viral load ( 10 copies / ml ) ( aor = 1.33 , 95% ci = 1.061.67 and aor = 1.29 , 95% ci = 1.031.60 , respectively ) after adjusting for age and gender . interestingly , rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with high alt level ( aor = 1.35 , 95% ci = 1.041.75 and aor = 1.36 , 95% ci = 1.061.74 , respectively ) and high viral load ( 10 copies / ml ) ( aor = 1.38 , 95% ci = 1.071.78 and aor = 1.29 , 95% ci = 1.011.66 , respectively ) in males . associations of pten polymorphisms with hbv - related characteristics in hcc - free hbv - infected subjects * hcc - free hbv - infected subjects : ascs , chb patients , and lc patients . aor : adjusted odds ratio ( adjusted for age and gender in the total subjects ; adjusted for age after stratification by gender ) ; pten : phosphatase and tensin homolog ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; snp : single nucleotide polymorphism ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; alt : alanine aminotransferase ; ci : confidence interval ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; lc : liver cirrhosis . the associations of the pten snps with the hcc - related hbv mutations that were described in our previous studies were assessed using the data of the hbv - infected patients including those with hcc . it was found that the variant genotype ( tc ) of rs1234220 was significantly associated with increased frequencies of hbv mutations a1652 g ( aor = 4.16 , 95% ci = 1.6410.55 ) , c1673 t ( aor = 2.40 , 95% ci = 1.025.66 ) , and c1730 g ( aor = 2.34 , 95% ci = 1.025.39 ) in genotype b hbv - infected subjects . we tested the interactions of pten snps with several important hcc - related hbv mutations on hcc risk . there were significant interactions between pten polymorphisms and a3054 t or c3116 t , important hbv mutations in the pres region [ table 6 ] . although the variant genotype of rs2299939 was significantly associated with a reduced risk of hcc , the interaction of rs2299939 variant genotypes with a3054 t was significantly associated with an increased risk of hcc . hbv c3116 t mutation was a significant risk factor of hcc ; however , its interaction with rs2299939 variant genotypes was significantly associated with a reduced risk of hcc . we also found that the interaction of rs1234213 variant genotypes with hbv c3116 t mutation significantly increased the risk of hcc in the hbv - infected subjects . after stratifying by gender , it was found that the interaction between the polymorphism of rs2299939 gt+tt and c3116 t significantly reduced the risk of hcc in male hbv - infected subjects ( aor = 0.27 , 95% ci = 0.130.57 ) . similarly , the interaction between rs1234213 ct+tt and c3116 t also significantly increased hcc risk in male hbv - infected subjects ( aor = 3.61 , 95% ci = 1.548.42 ) after stratifying by gender . interactions of pten polymorphisms with hbv mutations on hcc risk aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; hcc : asc + chb + lc ; pten : phosphatase and tensin homolog ; lc : liver cirrhosis ; snp : single nucleotide polymorphism ; or : odds ratio . the three pten snps were in linkage disequilibrium with each other in our study population . the estimated haplotype frequencies of hcc patients ( n = 1016 ) and healthy controls ( n = 1004 ) indicated that only one haplotype of rs1234220(c)-rs2299939(g)-rs1234213(t ) was significantly more frequent in hcc patients compared to healthy controls after adjusting for age and gender ( aor = 1.32 , 95% ci = 1.061.63 , p = 0.012 ) . the frequencies of other estimated haplotypes did not differ significantly between the two groups . in the present study , we found that the variant genotypes or c allele of rs1234220 were significantly associated with increased risks of hcc compared to healthy controls and hcc - free hbv - infected subjects , whereas the variant genotypes of rs2299939 were significantly associated with a reduced risk of hcc compared to hcc - free hbv - infected subjects [ table 3 ] . these pten snps have not been found as susceptible genetic loci of cancers especially hcc in genome - wide association studies carried out in chinese . we also found that three important interactions of genetic factors ( snps ) with environmental factors ( hbv mutations ) significantly affected the risks of hcc [ table 6 ] . interestingly , the associations of the variant genotypes of rs1234220 and rs2299939 with the risk of hcc were solely evident in males but not in females . hbv - hcc occurs more frequently in males than in females , with a male - to - female ratio of 3:1 . these findings should be of clinical and/or public health importance in determining the hbv - infected males who are more likely to develop hcc and need specific prophylaxis . the mechanisms by which the two pten snps affected the risk of hcc remain to be determined . as pten is a well - known tumor suppressor gene , the variant genotypes or c allele of rs1234220 may predispose hbv - infected subjects to a reduced function of pten in their liver . the variant genotypes of rs2299939 were reversely associated with hcc risk in hbeag - negative hbv - infected subjects ; however , they were significantly associated with high viral load ( > 10 copies / ml ) and alt abnormality ( 40 u / ml ) [ table 5 ] . our data were in contradiction with the observations in previous prospective studies that high viral load and hepatic inflammation predicted the occurrence of hcc in hbv - infected subjects . our study is of cross - sectional case - control design that can only indicate the statistical association between genetic polymorphisms and diseases . the results of our study indicate that rs2299939 variant genotypes may predispose the hbv - infected subjects to occur acute - on - chb . it has been demonstrated that reduced pten expression level is correlated with tumor progression , high afp levels , and poor prognosis of hcc patients . however , somatic mutation frequencies of pten were rarely detected in hcc , suggesting other mechanisms such as promoter methylation , decreased transcription / translation by noncoding rna ( ncrna ) , and increased protein degradation / phosphorylation may be responsible for pten down - regulation . in this study , we found that 2 of the 3 intronic snps of pten affected the risk of hcc , and the effects were only evident in males . although introns do not code for proteins , they may regulate gene expression , or generate ncrnas . there should be inherent as yet unknown links between androgen / androgen receptor and pten signaling in hbv - induced hepatocarcinogenesis . the intronic snps may function in hepatocarcinogenesis via affecting gene expression as putative enhancers , altering alternative splicing , and/or generating ncrnas upon hbv infection . our stratification analysis indicated that rs1234220 ct genotype and c ( ct+cc ) allele significantly increased the risk of hcc only in hbv genotype b subjects , not in genotype c hbv - infected subjects [ table 4 ] . we also found that rs1234220 variant genotype was significantly associated with increased frequencies of hbv mutations a1652 g , c1673 t , and c1730 g in genotype b hbv - infected subjects . it is reasonable to speculate that rs1234220 variant genotypes increase hcc risk , possibly via facilitating the generation of a1652 g , c1673 t , and c1730 g , the three hcc - risk hbv mutations in genotype b hbv - infected subjects . in our previous studies , we found that the genetic polymorphisms of a group of the key immune and/or proinflammatory molecules facilitated the generation of hcc - risk mutations , possibly because these immune / proinflammatory molecules predispose the host immunity to select these hcc - risk mutations , especially in genotype c hbv - infected subjects . to our surprise , the immune selection milieu predisposed by the pten snps might be special in genotype b hbv - infected subjects who are more likely to develop hcc . as pten has an anti - inflammatory activity , rs1234220 variant genotypes may be associated with reduced expression or activity of pten , thus facilitating the formation of active inflammation essential for the immune selection of hcc - risk mutation . compared to hbv genotype c , genotype b is more apt to cause active inflammation and the occurrence at the young without cirrhosis background and recurrence of hcc . thus , the mechanisms by which the hcc - risk hbv mutations are selected might be different between hbv genotype b and genotype c. interestingly , the associations of the pten polymorphisms with the risk of hcc were significantly affected by the hbv mutations . the interactions of rs2299939 polymorphism with a3054 t mutation or rs1234213 polymorphism with c3116 t mutation significantly increased the risk of hcc in male hbv - infected subjects ; whereas the interaction of rs2299939 polymorphism with c3116 t mutation significantly decreased the risk of hcc [ table 6 ] . t and c3116 t on hcc susceptibility can be moderated by host genetic susceptibility such as pten polymorphisms . nevertheless , these interactions should be helpful in identifying hbv - infected subjects who are more likely to develop hcc . to the best of our knowledge , this is the first study investigating the effects of pten genetic polymorphisms on the generation of hcc - related hbv mutations , and their interactions on the risk of hcc in hbv - infected subjects . moreover , our epidemiological study with a relatively large sample size provided convincing data to support the role of pten polymorphisms in determining individual 's genetic susceptibility to hcc . however , several limitations should be addressed . first , other important information such as environment risk factor , alcohol consumption , and family history of hcc were not collected , and we could not adjust for those covariates . second , the current study is cross - sectional in nature , and our findings need to be validated in prospective cohort studies . in conclusion , the variant genotypes of rs1234220 are significantly associated with increased risks of hcc , whereas the variant genotypes of rs2299939 are significantly associated with a reduced risk of hcc in male hbv - infected subjects . the effect of rs1234220 variant genotypes on hcc risk is only observed in genotype b hbv - infected subjects , possibly via facilitating the immune selection of a1652 g , c1673 t , and c1730 g , the three hcc - risk mutations in genotype b hbv - infected subjects . the interactions of rs2299939 polymorphism with a3054 t mutation or rs1234213 polymorphism with c3116 t mutation significantly increased the risk of hcc in male hbv - infected subjects ; whereas the interaction of rs2299939 polymorphism with c3116 t mutation significantly decreased the risk of hcc . these findings should be of clinical and/or public health importance in determining the hbv - infected males who are more likely to develop hcc and , therefore , need specific prophylaxis . the effects of the interactions of genetic predisposition ( pten snps ) with environmental factors ( hbv mutations ) on hcc occurrence and progression should be validated in large prospective cohort studies .

background : chronic hepatitis b virus ( hbv ) infection is the major cause of hepatocellular carcinoma ( hcc ) . some hbv mutants and dysregulation of phosphatase and tensin homolog ( pten ) may promote the development of hcc synergistically . we aimed to test the effects of pten genetic polymorphisms and their interactions with important hbv mutations on the development of hcc in hbv - infected subjects.methods:quantitative polymerase chain reaction was applied to genotype pten polymorphisms ( rs1234220 , rs2299939 , rs1234213 ) in 1012 healthy controls , 302 natural clearance subjects , and 2011 chronic hbv - infected subjects including 1021 hcc patients . hbv mutations were determined by sequencing . the associations of pten polymorphisms and their interactions with hbv mutations with hcc risk were assessed using multivariate logistic regression analysis.results:rs1234220 c allele was significantly associated with hcc risk compared to healthy controls ( adjusted odds ratio [ aor ] = 1.35 , 95% confidence interval [ ci ] = 1.071.69 ) and hcc - free hbv - infected subjects ( aor = 1.27 , 95% ci = 1.011.57 ) . rs1234220 c allele was significantly associated with increased frequencies of hcc - risk a1652 g , c1673 t , and c1730 g mutations in genotype b hbv - infected subjects . rs2299939 gt genotype was inversely associated with hcc risk in hbv - infected patients ( aor = 0.75 , 95% ci = 0.620.92 ) . the interaction of rs2299939 variant genotypes ( gt+tt ) with a3054 t mutation significantly increased hcc risk ( aor = 2.41 , 95% ci = 1.085.35 ) ; whereas its interaction with c3116 t mutation significantly reduced hcc risk ( aor = 0.34 , 95% ci = 0.180.66 ) . these significant effects were only evident in males after stratification.conclusions:pten polymorphisms and their interactions with hbv mutations may contribute to hepatocarcinogenesis in males . the host - virus interactions are important in identifying hbv - infected subjects who are more likely to develop hcc .

I M Study population Serological hepatitis B virus parameter examination, hepatitis B virus genotyping, and viral mutation analysis Polymorphism selection and genotyping Statistical analysis R Characteristics of study participants Associations of phosphatase and tensin homolog single nucleotide polymorphisms with the risk of hepatocellular carcinoma Associations of phosphatase and tensin homolog single nucleotide polymorphisms with the risks of other hepatitis B virus-related characteristics Interactions of phosphatase and tensin homolog single nucleotide polymorphisms with hepatitis B virus mutations and their associations with hepatocellular carcinoma risk Haplotype analysis D

hepatocellular carcinoma ( hcc ) is one of the most common cancers and chronic infection with hepatitis b virus ( hbv ) is the most common cause of hcc worldwide . epidemiological studies have shown that increasing age , male gender , hbv genotype c ( vs. genotype b ) , high levels of circulating hbv dna ( > 10 copies / ml ) , hepatitis b e antigen ( hbeag ) expression , and certain hbv mutations are significantly associated with an increased risk of hcc in hbv - infected subjects . in this large epidemiological study , we investigated the effects of several pten polymorphisms and their interactions with important hbv mutations on the risk of hcc in chronic hbv - infected subjects . this study should be helpful in identifying hbv - infected subjects who are more likely to develop hcc and need active prophylaxis . unconditional logistic regression model was applied to test the effects of pten snps on hcc risk and other hbv - related characteristics , and to calculate odds ratios ( ors ) and their 95% confidence intervals ( cis ) . unconditional logistic regression model was applied to test the effects of pten snps on hcc risk and other hbv - related characteristics , and to calculate odds ratios ( ors ) and their 95% confidence intervals ( cis ) . compared with healthy controls , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( adjusted or [ aor ] = 1.37 , 95% ci = 1.081.73 and aor = 1.35 , 95% ci = 1.071.69 , respectively ) , after adjusting for age and gender . when compared to hbv - infected patients without hcc , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 1.28 , 95% ci = 1.021.61 and aor = 1.27 , 95% ci = 1.011.57 , respectively ) , whereas rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with reduced risks of hcc ( aor = 0.75 , 95% ci = 0.620.92 , and aor = 0.79 , 95% ci = 0.650.96 , respectively ) . compared with all subjects without hcc , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 1.30 , 95% ci = 1.081.56 and aor = 1.29 , 95% ci = 1.071.54 , respectively ) , while rs2299939 gt genotype was significantly associated with a reduced risk of hcc ( aor = 0.84 , 95% ci = 0.710.99 ) . association of pten polymorphisms with the risk of hcc aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; hwe : hardy - weinberg equilibrium ; pten : phosphatase and tensin homolog ; snp : single nucleotide polymorphism . after stratifying by hbv genotypes , we found that rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 2.02 , 95% ci = 1.143.59 and aor = 2.02 , 95% ci = 1.143.59 , respectively ) in genotype b hbv subjects , compared with genotype b hbv - infected patients without hcc ; however , these effects did not exist in genotype c hbv - infected subjects [ table 4 ] . as compared with hbv - infected patients without hcc , rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with reduced risks of hcc ( aor = 0.68 , 95% ci = 0.530.87 and aor = 0.72 , 95% ci = 0.570.92 , respectively ) in hbeag - negative hbv - infected subjects . association of pten polymorphisms with the risk of hcc stratified by hbv genotypes aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; non - hcc : asc + chb + lc ; pten : phosphatase and tensin homolog ; snp : single nucleotide polymorphism ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; lc : liver cirrhosis . rs2299939 t ( gt+tt ) allele was significantly associated with high alt level ( aor = 1.26 , 95% ci = 1.011.57 ) ; rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with high viral load ( 10 copies / ml ) ( aor = 1.33 , 95% ci = 1.061.67 and aor = 1.29 , 95% ci = 1.031.60 , respectively ) after adjusting for age and gender . interestingly , rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with high alt level ( aor = 1.35 , 95% ci = 1.041.75 and aor = 1.36 , 95% ci = 1.061.74 , respectively ) and high viral load ( 10 copies / ml ) ( aor = 1.38 , 95% ci = 1.071.78 and aor = 1.29 , 95% ci = 1.011.66 , respectively ) in males . aor : adjusted odds ratio ( adjusted for age and gender in the total subjects ; adjusted for age after stratification by gender ) ; pten : phosphatase and tensin homolog ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; snp : single nucleotide polymorphism ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; alt : alanine aminotransferase ; ci : confidence interval ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; lc : liver cirrhosis . it was found that the variant genotype ( tc ) of rs1234220 was significantly associated with increased frequencies of hbv mutations a1652 g ( aor = 4.16 , 95% ci = 1.6410.55 ) , c1673 t ( aor = 2.40 , 95% ci = 1.025.66 ) , and c1730 g ( aor = 2.34 , 95% ci = 1.025.39 ) in genotype b hbv - infected subjects . although the variant genotype of rs2299939 was significantly associated with a reduced risk of hcc , the interaction of rs2299939 variant genotypes with a3054 t was significantly associated with an increased risk of hcc . we also found that the interaction of rs1234213 variant genotypes with hbv c3116 t mutation significantly increased the risk of hcc in the hbv - infected subjects . after stratifying by gender , it was found that the interaction between the polymorphism of rs2299939 gt+tt and c3116 t significantly reduced the risk of hcc in male hbv - infected subjects ( aor = 0.27 , 95% ci = 0.130.57 ) . similarly , the interaction between rs1234213 ct+tt and c3116 t also significantly increased hcc risk in male hbv - infected subjects ( aor = 3.61 , 95% ci = 1.548.42 ) after stratifying by gender . interactions of pten polymorphisms with hbv mutations on hcc risk aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; hcc : asc + chb + lc ; pten : phosphatase and tensin homolog ; lc : liver cirrhosis ; snp : single nucleotide polymorphism ; or : odds ratio . the estimated haplotype frequencies of hcc patients ( n = 1016 ) and healthy controls ( n = 1004 ) indicated that only one haplotype of rs1234220(c)-rs2299939(g)-rs1234213(t ) was significantly more frequent in hcc patients compared to healthy controls after adjusting for age and gender ( aor = 1.32 , 95% ci = 1.061.63 , p = 0.012 ) . compared with healthy controls , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( adjusted or [ aor ] = 1.37 , 95% ci = 1.081.73 and aor = 1.35 , 95% ci = 1.071.69 , respectively ) , after adjusting for age and gender . when compared to hbv - infected patients without hcc , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 1.28 , 95% ci = 1.021.61 and aor = 1.27 , 95% ci = 1.011.57 , respectively ) , whereas rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with reduced risks of hcc ( aor = 0.75 , 95% ci = 0.620.92 , and aor = 0.79 , 95% ci = 0.650.96 , respectively ) . compared with all subjects without hcc , rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 1.30 , 95% ci = 1.081.56 and aor = 1.29 , 95% ci = 1.071.54 , respectively ) , while rs2299939 gt genotype was significantly associated with a reduced risk of hcc ( aor = 0.84 , 95% ci = 0.710.99 ) . association of pten polymorphisms with the risk of hcc aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; hwe : hardy - weinberg equilibrium ; pten : phosphatase and tensin homolog ; snp : single nucleotide polymorphism . after stratifying by hbv genotypes , we found that rs1234220 ct genotype and c ( ct+cc ) allele were significantly associated with increased risks of hcc ( aor = 2.02 , 95% ci = 1.143.59 and aor = 2.02 , 95% ci = 1.143.59 , respectively ) in genotype b hbv subjects , compared with genotype b hbv - infected patients without hcc ; however , these effects did not exist in genotype c hbv - infected subjects [ table 4 ] . as compared with hbv - infected patients without hcc , rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with reduced risks of hcc ( aor = 0.68 , 95% ci = 0.530.87 and aor = 0.72 , 95% ci = 0.570.92 , respectively ) in hbeag - negative hbv - infected subjects . association of pten polymorphisms with the risk of hcc stratified by hbv genotypes aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; non - hcc : asc + chb + lc ; pten : phosphatase and tensin homolog ; snp : single nucleotide polymorphism ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; lc : liver cirrhosis . rs2299939 t ( gt+tt ) allele was significantly associated with high alt level ( aor = 1.26 , 95% ci = 1.011.57 ) ; rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with high viral load ( 10 copies / ml ) ( aor = 1.33 , 95% ci = 1.061.67 and aor = 1.29 , 95% ci = 1.031.60 , respectively ) after adjusting for age and gender . interestingly , rs2299939 gt genotype and t ( gt+tt ) allele were significantly associated with high alt level ( aor = 1.35 , 95% ci = 1.041.75 and aor = 1.36 , 95% ci = 1.061.74 , respectively ) and high viral load ( 10 copies / ml ) ( aor = 1.38 , 95% ci = 1.071.78 and aor = 1.29 , 95% ci = 1.011.66 , respectively ) in males . aor : adjusted odds ratio ( adjusted for age and gender in the total subjects ; adjusted for age after stratification by gender ) ; pten : phosphatase and tensin homolog ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; snp : single nucleotide polymorphism ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; alt : alanine aminotransferase ; ci : confidence interval ; asc : asymptomatic hepatitis b surface antigen carrier ; chb : chronic hepatitis b ; lc : liver cirrhosis . it was found that the variant genotype ( tc ) of rs1234220 was significantly associated with increased frequencies of hbv mutations a1652 g ( aor = 4.16 , 95% ci = 1.6410.55 ) , c1673 t ( aor = 2.40 , 95% ci = 1.025.66 ) , and c1730 g ( aor = 2.34 , 95% ci = 1.025.39 ) in genotype b hbv - infected subjects . although the variant genotype of rs2299939 was significantly associated with a reduced risk of hcc , the interaction of rs2299939 variant genotypes with a3054 t was significantly associated with an increased risk of hcc . we also found that the interaction of rs1234213 variant genotypes with hbv c3116 t mutation significantly increased the risk of hcc in the hbv - infected subjects . after stratifying by gender , it was found that the interaction between the polymorphism of rs2299939 gt+tt and c3116 t significantly reduced the risk of hcc in male hbv - infected subjects ( aor = 0.27 , 95% ci = 0.130.57 ) . similarly , the interaction between rs1234213 ct+tt and c3116 t also significantly increased hcc risk in male hbv - infected subjects ( aor = 3.61 , 95% ci = 1.548.42 ) after stratifying by gender . interactions of pten polymorphisms with hbv mutations on hcc risk aor : adjusted odds ratio ( adjusted for age and gender ) ; ci : confidence interval ; hbv : hepatitis b virus ; hcc : hepatocellular carcinoma ; hcc : asc + chb + lc ; pten : phosphatase and tensin homolog ; lc : liver cirrhosis ; snp : single nucleotide polymorphism ; or : odds ratio . the estimated haplotype frequencies of hcc patients ( n = 1016 ) and healthy controls ( n = 1004 ) indicated that only one haplotype of rs1234220(c)-rs2299939(g)-rs1234213(t ) was significantly more frequent in hcc patients compared to healthy controls after adjusting for age and gender ( aor = 1.32 , 95% ci = 1.061.63 , p = 0.012 ) . in the present study , we found that the variant genotypes or c allele of rs1234220 were significantly associated with increased risks of hcc compared to healthy controls and hcc - free hbv - infected subjects , whereas the variant genotypes of rs2299939 were significantly associated with a reduced risk of hcc compared to hcc - free hbv - infected subjects [ table 3 ] . we also found that rs1234220 variant genotype was significantly associated with increased frequencies of hbv mutations a1652 g , c1673 t , and c1730 g in genotype b hbv - infected subjects . it is reasonable to speculate that rs1234220 variant genotypes increase hcc risk , possibly via facilitating the generation of a1652 g , c1673 t , and c1730 g , the three hcc - risk hbv mutations in genotype b hbv - infected subjects . to our surprise , the immune selection milieu predisposed by the pten snps might be special in genotype b hbv - infected subjects who are more likely to develop hcc . the interactions of rs2299939 polymorphism with a3054 t mutation or rs1234213 polymorphism with c3116 t mutation significantly increased the risk of hcc in male hbv - infected subjects ; whereas the interaction of rs2299939 polymorphism with c3116 t mutation significantly decreased the risk of hcc [ table 6 ] . nevertheless , these interactions should be helpful in identifying hbv - infected subjects who are more likely to develop hcc . to the best of our knowledge , this is the first study investigating the effects of pten genetic polymorphisms on the generation of hcc - related hbv mutations , and their interactions on the risk of hcc in hbv - infected subjects . the effect of rs1234220 variant genotypes on hcc risk is only observed in genotype b hbv - infected subjects , possibly via facilitating the immune selection of a1652 g , c1673 t , and c1730 g , the three hcc - risk mutations in genotype b hbv - infected subjects . the interactions of rs2299939 polymorphism with a3054 t mutation or rs1234213 polymorphism with c3116 t mutation significantly increased the risk of hcc in male hbv - infected subjects ; whereas the interaction of rs2299939 polymorphism with c3116 t mutation significantly decreased the risk of hcc .

bone scaffolds , which provide the benefit of avoiding unwanted immunological responses and eliminate the risk of acquiring infectious diseases from autografts and allografts , are widely used by orthopedic surgeons when repairing different types of bone defects . the traditional methods of manufacturing scaffolds mainly focus on reshaping the structure of specific types of materials and give the scaffold some biomedical properties via processes such as leaching or soaking . these methods can make scaffolds with higher porosity , but their size , shape , and interconnectivity are not easy to control , which may limit the prognosis in many aspects ; thus , getting satisfactory outcomes when treating bone defects using bone scaffolds is still very challenging . the development of three - dimensional ( 3d ) printing technology has dramatically changed scaffold designs in regenerative medicine . scaffold microstructures are able to regulate cell behaviors such as proliferation , differentiation , and apoptosis . with the recent rapid development of 3d printing technology , not only can we print 3d scaffolds with controllable inner microstructures but also we can have scaffolds composed with components in the extracellular matrix to deliver biomaterials . platelets represent a type of specific source of growth factors and cytokines that are involved in wound healing and tissue repair . many platelet - derived factors play important roles in cell proliferation and differentiation including platelet - derived growth factor ( pdgf ) , transforming growth factor ( tgf)-1 , and insulin - like growth factors-1 . in this study , we designed a 3d printed scaffold with gelatin and platelets , examined the proliferation of preosteoblasts in the scaffolds using a cell counting kit-8 ( cck-8 ) assay and the growth factor release at various time points . some of the pdgfs that were measured include tgf-1 and vascular endothelial growth factor ( vegf ) , which play significant roles in wound healing and tissue regeneration . blood from healthy donors was collected into 3.8% ( mass fraction ) sodium citrate - containing tubes ( lankang company , jinan , china ) . blood samples were centrifuged at 1500 g for 6 min , after which the samples were divided into three layers : a bottom layer composed of red blood cells , an intermediate layer composed of white blood cells , and a top player composed of plasma . all of the plasma layer and 3 mm of the intermediate layer were then centrifuged for another 6 min at 1000 g to obtain a two - part plasma : the upper three - quarters consisted of platelet - poor plasma and the lower quarter consisted of platelet - rich plasma ( prp ) . the scaffolds possess an internal porous structure designed in a computer aided design environment using the software rhino 5.0 ( robert mcneel & associates , usa ) . the internal pores were orderly arranged regular dodecahedrons within the scaffolds with = 1500 m . the titanium skeletons that formed the internal porous structure and the external appearance of scaffolds were 100 m in diameter . the 3d porous titanium was printed based on laser sintering technology ( concept laser mlab , germany ) in two sizes : 5 mm diameter and 3 mm thickness for cultivating cells in 96-well plate and 8 mm diameter and 3 mm thickness for implanting in vivo , respectively . three groups of scaffolds were prepared including clear scaffolds ( blank group ) , scaffolds with gelatin ( control group ) , and scaffolds with gelatin and platelets ( treated group ) . to prepare the scaffold with gelatin , 1 g nacl was added to 5 ml of gelatin solution ( 5% ) , and then the solution was perfused into a 3d printed scaffold and lyophilized well . the nacl was absterged using distilled water , and then freeze - dried for the second time . the sterilization process was treatment with ethylene oxide , and the scaffolds were prepared with gelatin . the treated groups were prepared by adding 20 l of prp to the scaffolds with gelatin then freeze - dried . the structure of the clear scaffold and scaffolds with gelatin and platelets was imaged as shown in figure 1 via scanning electron microscopy . all scaffolds were molded at 3 mm height and 5 mm diameter , which fit the wells of the 96-well microplate ( 3559 , 96wl , corning , usa ) . ( a ) the internal pores of the blank group ( scaffolds with gelatin ) , diameters ranging from 150 to 220 m . bar = 20 m . ( b ) the internal pores of the treated group ( scaffolds with gelatin and platelet ) , diameters ranging from 70 to 200 m . bar = 20 m . preosteoblasts ( mc3t3-e1 , osteoblast cell line ) were bought from the chinese academy of medical sciences . the cells were cultured in an alpha - minimum essential medium ( -mem ) supplemented with 5% fetal bovine serum , 2 mmol / l glutamine , and 100 g / ml penicillin streptomycin and incubated at 37c in a humidified atmosphere with 5% co2 . first , 5000 cells were seeded on a 75 cm culture flask with 8 ml medium . after the cells adhered to the flasks , the medium was removed and replaced by a new 8 ml medium . the medium was replaced every 3 days until the cells were filled in the flasks . then , 1 ml 0.25% trypsin was added to the flask , which was lightly joggled . the flask was cultivated in an incubator for 23 min and 6 ml of a new medium was added to stop digestion . the whole mixture medium was removed to a 15 ml centrifuge tube so that it could be centrifuged for 5 min at 1000 g . the supernatant was dumped , and 5 ml of a new medium was added followed by mechanical isolation . we set four groups including a control group and three types of scaffold groups in a 96-well microplate . three types of scaffolds with 200 l -mem were placed into the wells of the treated groups . each well in the 96-well microplate then received 100 l of cell suspension ( cell concentration : 2 10 cells / ml ) . the 96-well microplate was then incubated in a co2 incubator until we measured the cell viability and cytotoxicity . the gelatin scaffolds group and the gelatin and platelet scaffolds group were compared for release levels of vegf and tgf- after 3 , 6 , 9 , 12 , 15 , 18 , and 21 days via enzyme - linked immunosorbent assay ( elisa ) analysis . the levels of vegf and tgf- release were also measured via elisa after the scaffolds were stored at 25c for 3 months . samples were treated at room temperature , and the concentrations of vegf and tgf- were measured using company kits ( r&d systems , minneapolis , usa ) . two wells in each group were measured simultaneously and averaged , and then 10 l of cck-8 was added to each well of the 96-well microplate . the absorbance was measured at 450 nm with a microplate reader ( thermo scientific , multiskan go , usa ) . the time points chosen to evaluate the cytotoxicity were 1 , 3 , 5 , and 7 days . two wells in each group were measured simultaneously and averaged , then 10 l of cck-8 was added to each well of the 96-well microplate . the absorbance at 450 nm was measured with a microplate reader ( thermo scientific ) . chicago , il , usa ) by student 's single - sample t - test . blood from healthy donors was collected into 3.8% ( mass fraction ) sodium citrate - containing tubes ( lankang company , jinan , china ) . blood samples were centrifuged at 1500 g for 6 min , after which the samples were divided into three layers : a bottom layer composed of red blood cells , an intermediate layer composed of white blood cells , and a top player composed of plasma . all of the plasma layer and 3 mm of the intermediate layer were then centrifuged for another 6 min at 1000 g to obtain a two - part plasma : the upper three - quarters consisted of platelet - poor plasma and the lower quarter consisted of platelet - rich plasma ( prp ) . the scaffolds possess an internal porous structure designed in a computer aided design environment using the software rhino 5.0 ( robert mcneel & associates , usa ) . the internal pores were orderly arranged regular dodecahedrons within the scaffolds with = 1500 m . the titanium skeletons that formed the internal porous structure and the external appearance of scaffolds were 100 m in diameter . the 3d porous titanium was printed based on laser sintering technology ( concept laser mlab , germany ) in two sizes : 5 mm diameter and 3 mm thickness for cultivating cells in 96-well plate and 8 mm diameter and 3 mm thickness for implanting in vivo , respectively . three groups of scaffolds were prepared including clear scaffolds ( blank group ) , scaffolds with gelatin ( control group ) , and scaffolds with gelatin and platelets ( treated group ) . to prepare the scaffold with gelatin , 1 g nacl was added to 5 ml of gelatin solution ( 5% ) , and then the solution was perfused into a 3d printed scaffold and lyophilized well . the nacl was absterged using distilled water , and then freeze - dried for the second time . the sterilization process was treatment with ethylene oxide , and the scaffolds were prepared with gelatin . the treated groups were prepared by adding 20 l of prp to the scaffolds with gelatin then freeze - dried . the structure of the clear scaffold and scaffolds with gelatin and platelets was imaged as shown in figure 1 via scanning electron microscopy . all scaffolds were molded at 3 mm height and 5 mm diameter , which fit the wells of the 96-well microplate ( 3559 , 96wl , corning , usa ) . ( a ) the internal pores of the blank group ( scaffolds with gelatin ) , diameters ranging from 150 to 220 m . bar = 20 m . ( b ) the internal pores of the treated group ( scaffolds with gelatin and platelet ) , diameters ranging from 70 to 200 m . preosteoblasts ( mc3t3-e1 , osteoblast cell line ) were bought from the chinese academy of medical sciences . the cells were cultured in an alpha - minimum essential medium ( -mem ) supplemented with 5% fetal bovine serum , 2 mmol / l glutamine , and 100 g / ml penicillin streptomycin and incubated at 37c in a humidified atmosphere with 5% co2 . first , 5000 cells were seeded on a 75 cm culture flask with 8 ml medium . after the cells adhered to the flasks , the medium was removed and replaced by a new 8 ml medium . the medium was replaced every 3 days until the cells were filled in the flasks . then , 1 ml 0.25% trypsin was added to the flask , which was lightly joggled . the flask was cultivated in an incubator for 23 min and 6 ml of a new medium was added to stop digestion . the whole mixture medium was removed to a 15 ml centrifuge tube so that it could be centrifuged for 5 min at 1000 g . the supernatant was dumped , and 5 ml of a new medium was added followed by mechanical isolation . we set four groups including a control group and three types of scaffold groups in a 96-well microplate . three types of scaffolds with 200 l -mem were placed into the wells of the treated groups . each well in the 96-well microplate then received 100 l of cell suspension ( cell concentration : 2 10 cells / ml ) . the 96-well microplate was then incubated in a co2 incubator until we measured the cell viability and cytotoxicity . the gelatin scaffolds group and the gelatin and platelet scaffolds group were compared for release levels of vegf and tgf- after 3 , 6 , 9 , 12 , 15 , 18 , and 21 days via enzyme - linked immunosorbent assay ( elisa ) analysis . the levels of vegf and tgf- release were also measured via elisa after the scaffolds were stored at 25c for 3 months . samples were treated at room temperature , and the concentrations of vegf and tgf- were measured using company kits ( r&d systems , minneapolis , usa ) . two wells in each group were measured simultaneously and averaged , and then 10 l of cck-8 was added to each well of the 96-well microplate . the absorbance was measured at 450 nm with a microplate reader ( thermo scientific , multiskan go , usa ) . the time points chosen to evaluate the cytotoxicity were 1 , 3 , 5 , and 7 days . two wells in each group were measured simultaneously and averaged , then 10 l of cck-8 was added to each well of the 96-well microplate . the absorbance at 450 nm was measured with a microplate reader ( thermo scientific ) . chicago , il , usa ) by student 's single - sample t - test . as shown in figure 1 , the gelatin microscaffolds were successfully created in pores of the 3d printed porous titanium . we calculated that 100 gelatin pores existed in microscaffolds and found that their diameter was in the range of 100300 m . after freeze - drying the prp , the microscaffold could also stay in the titanium pores because of the expansibility of gelatin when absorbing water . the scaffold with platelets became smaller than before because the platelets adhered to the gelatin . elisa was used to measure the vegf and tgf-1 released over 21 days , with results as shown in figure 2 . it could be determined that there was a triggered release after the first 3 days and the growth factors released would be low and plain at later days , which presented a better sustained release function . all the treated groups showed significantly higher release of vegf and tgf-1 than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . data are shown as mean standard deviation , n = 3 . * p < 0.05 versus control . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . after the scaffolds were stored at 25c for 3 months , vegf and tgf-1 were measured for another 21 days , with results as shown in figure 3 . the releasing curve not only seemed irregular compared to fresh scaffolds but also proved that the release function was sustained after 3 months of storage at room temperature . at 3 , 9 , 12 , 15 , 18 , and 21 days time points , treated group exhibited significantly higher than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . * p < 0.05 versus control group . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . we chose three wells in each group randomly three times and measured the optical density at 450 nm to determine the number of viable cells [ figure 4 ] . after 21 days of culture , the cck-8 cell count of the control and treated groups was significantly higher than that of the blank group ( p = 0.019 and 0.024 , respectively ) . however , there was no significant difference ( p = 0.364 ) between the control group and the treated group after 21 days of culture . cell proliferation assay of the mc3t3-e1 using cell counting kit-8 assay after a 21-day culture . * p < 0.05 versus the blank group . the number of the living cells was determined by the cck-8 assay to detect cytotoxicity [ figure 5 ] . at the 3 days time point of culturing , the value of the cck-8 cell count of the control and treated groups was obviously higher than that of the blank group ( p < 0.05 ) . at 5 day of culture , the value of the cck-8 cell count of the control group remained above the level of the blank group ( p < 0.05 ) , with the treated group almost equal to the blank group . however , at 7 d of culture , cck-8 cell count of the control and treated groups was lower than that of the blank group ( p < 0.05 ) . as shown in figure 1 , the gelatin microscaffolds were successfully created in pores of the 3d printed porous titanium . we calculated that 100 gelatin pores existed in microscaffolds and found that their diameter was in the range of 100300 m . after freeze - drying the prp , the microscaffold could also stay in the titanium pores because of the expansibility of gelatin when absorbing water . the scaffold with platelets became smaller than before because the platelets adhered to the gelatin . elisa was used to measure the vegf and tgf-1 released over 21 days , with results as shown in figure 2 . it could be determined that there was a triggered release after the first 3 days and the growth factors released would be low and plain at later days , which presented a better sustained release function . all the treated groups showed significantly higher release of vegf and tgf-1 than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . data are shown as mean standard deviation , n = 3 . * p < 0.05 versus control . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . after the scaffolds were stored at 25c for 3 months , vegf and tgf-1 were measured for another 21 days , with results as shown in figure 3 . the releasing curve not only seemed irregular compared to fresh scaffolds but also proved that the release function was sustained after 3 months of storage at room temperature . at 3 , 9 , 12 , 15 , 18 , and 21 days time points , treated group exhibited significantly higher than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . * p < 0.05 versus control group . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . we chose three wells in each group randomly three times and measured the optical density at 450 nm to determine the number of viable cells [ figure 4 ] . after 21 days of culture , the cck-8 cell count of the control and treated groups was significantly higher than that of the blank group ( p = 0.019 and 0.024 , respectively ) . however , there was no significant difference ( p = 0.364 ) between the control group and the treated group after 21 days of culture . cell proliferation assay of the mc3t3-e1 using cell counting kit-8 assay after a 21-day culture . * p < 0.05 versus the blank group . the number of the living cells was determined by the cck-8 assay to detect cytotoxicity [ figure 5 ] . at the 3 days time point of culturing , the value of the cck-8 cell count of the control and treated groups was obviously higher than that of the blank group ( p < 0.05 ) . at 5 day of culture , the value of the cck-8 cell count of the control group remained above the level of the blank group ( p < 0.05 ) , with the treated group almost equal to the blank group . however , at 7 d of culture , cck-8 cell count of the control and treated groups was lower than that of the blank group ( p < 0.05 ) . as modern medicine advances , many varied methodologies are being explored to treat severe bone diseases . however , curing bone defects it remains very challenging , not only because of the complex stratified architecture of bone itself but also due to changes in the microenvironment including cytokines and blood supplies . our work is a combination of 3d printing technology that gives the scaffold an accurate structure , and biological treatments with platelets to enhance the osteogenesis effect , thus helping patients with bone defects obtain a better prognosis . the 3d printed scaffold appears gray , bright , and clean . using a scanning electron microscope the pores are equally distributed and connected , with a diameter of 1500 m , and are visible within the scaffold . the micropore structure is clearly visible at 200 magnification , with diameters in the range of 100300 m . it has been reported that when the pore diameter of a porous structure is 1540 m , fibrous tissue would grow into the interior of the material . when the diameter of pores is 40100 m when the diameter of the pores exceeds 150 m , the bone tissue can infiltrate completely into the pore structure of the scaffold , potentially improving the osteogenesis effect . the 3d geometric structure and interconnected pores of our scaffold offer a quite large internal surface area and a 3d structure , in which preosteoblasts are able to absorb more large molecules , promote cell adhesion , proliferation , and osteogenic capabilities . during the design process , we treated the scaffold with platelets for the sake of utilizing its cytokines secretion to enhance the regeneration of the defected area . platelets attract considerable attention for their bioactivation of scaffolds due to their simplicity , safety , and cost - effectiveness of autologous blood preparation . we tested the vegf and tgf-1 release for 21 days for different times at which the platelets were made immediately and stored for 3 months at 25c [ figures 2 and 3 ] to address whether the control group and treated group had different effects on cytokines control - release . from these results , it was indicated that the scaffolds with platelets adhered on the microscaffold could release growth factors effectively and be restored for a long time via this special preparation method . in the study the cells proliferated further and numerous cells were measured at day 21 via cck-8 assay , the cell count of the control group and treated groups was significantly higher than that of the blank group . however , there was no significant difference between the control group and the treated group . this result demonstrated that the gelatin and platelets in the 3d porous structure of the scaffold could also promote cytokine secretion and the infiltration of nutrients and metabolites in the medium , which enhanced the 3d printed scaffolds biocompatibility . therefore , this kind of scaffold may be used as a bone graft substitute material . the method we used to evaluate the viability was cck-8 assay , which calculated the relative cell proliferation rate based on 450 nm light absorption indirectly , to assess the degree of cytotoxicity . a higher cell proliferation rate was associated with the lower toxicity of different types of scaffolds . in this study , detection based on cytotoxicity test standards in vitro showed that preosteoblast proliferation increased rapidly in the early stage ( days 15 ) and then the velocity decreased ; we can see the results in figure 5 . in three groups with scaffolds and one group without a scaffold , preosteoblasts maintained remarkably good proliferation in the experimental group . however , the preosteoblasts proliferation seems poor in the treated group compared to the other two groups with scaffolds at day 7 because the cells became confluent on the well surface . this finding is in agreement with previous studies that prp at low concentration in a culture medium has positive effects on cell growth , while high concentrations of prp suppress viability and proliferation . therefore , the results indicate that the scaffolds we designed are not toxic to preosteoblast cells and may be safe to implant in animals . in conclusion , the results of this study confirm that our 3d printed scaffolds provided good tissue compatibility and improved the adhesion and proliferation of preosteoblasts . furthermore , the platelets we treated also showed a terrific sustained release effect for vegf and tgf-1 , even after being stored for 3 months . therefore , a 3d printed scaffold with gelatin and platelets is a suitable implant for cell proliferation and may be a good bone graft substitute material for bone defects . in the present study , 3d scaffolds were only observed in vitro for 3 months ; therefore , a longer observation period and in vivo experiments will be performed in future studies . this research was supported by a grant from the youth foundation of chinese academy of medical sciences ( no . this research was supported by a grant from the youth foundation of chinese academy of medical sciences ( no .

background : three - dimensional ( 3d ) printing technology holds great promise for treating diseases or injuries that affect human bones with enhanced performance over traditional techniques . different patterns of design can lead to various mechanical properties and biocompatibility to various degrees . however , there is still a long way to go before we can fully take advantage of 3d printing technologies.methods:this study tailored 3d printed scaffolds with gelatin and platelets to maximize bone regeneration . the scaffolds were designed with special internal porous structures that can allow bone tissue and large molecules to infiltrate better into the scaffolds . they were then treated with gelatin and platelets via thermo - crosslinking and freeze - drying , respectively . vascular endothelial growth factor ( vegf ) and transforming growth factor ( tgf)-1 were measured at different time points after the scaffolds had been made . cell proliferation and cytotoxicity were determined via cell counting kit-8 ( cck-8 ) assay.results:there was a massive boost in the level of vegf and tgf-1 released by the scaffolds with gelatin and platelets compared to that of scaffolds with only gelatin . after 21 days of culture , the cck-8 cell counts of the control group and treated group were significantly higher than that of the blank group ( p < 0.05 ) . the cytotoxicity test also indicated the safety of the scaffolds.conclusions:our experiments confirmed that the 3d printed scaffolds we had designed could provide a sustained - release effect for growth factors and improve the proliferation of preosteoblasts with little cytotoxicity in vitro . they may hold promise as bone graft substitute materials in the future .

I M Preparation of platelets Preparation of scaffolds Cell culture and culture medium Growth factors release test Cell counting kit-8 scaffold proliferation and cytotoxicity test Statistical analysis R Characteristics of the complex scaffolds Vascular endothelial growth factor and transforming growth factor-1 sustained-release Cell proliferation Scaffold cytotoxicity test D Financial support and sponsorship Conflicts of interest

these methods can make scaffolds with higher porosity , but their size , shape , and interconnectivity are not easy to control , which may limit the prognosis in many aspects ; thus , getting satisfactory outcomes when treating bone defects using bone scaffolds is still very challenging . the development of three - dimensional ( 3d ) printing technology has dramatically changed scaffold designs in regenerative medicine . with the recent rapid development of 3d printing technology , not only can we print 3d scaffolds with controllable inner microstructures but also we can have scaffolds composed with components in the extracellular matrix to deliver biomaterials . platelets represent a type of specific source of growth factors and cytokines that are involved in wound healing and tissue repair . many platelet - derived factors play important roles in cell proliferation and differentiation including platelet - derived growth factor ( pdgf ) , transforming growth factor ( tgf)-1 , and insulin - like growth factors-1 . in this study , we designed a 3d printed scaffold with gelatin and platelets , examined the proliferation of preosteoblasts in the scaffolds using a cell counting kit-8 ( cck-8 ) assay and the growth factor release at various time points . some of the pdgfs that were measured include tgf-1 and vascular endothelial growth factor ( vegf ) , which play significant roles in wound healing and tissue regeneration . all of the plasma layer and 3 mm of the intermediate layer were then centrifuged for another 6 min at 1000 g to obtain a two - part plasma : the upper three - quarters consisted of platelet - poor plasma and the lower quarter consisted of platelet - rich plasma ( prp ) . the scaffolds possess an internal porous structure designed in a computer aided design environment using the software rhino 5.0 ( robert mcneel & associates , usa ) . the internal pores were orderly arranged regular dodecahedrons within the scaffolds with = 1500 m . the titanium skeletons that formed the internal porous structure and the external appearance of scaffolds were 100 m in diameter . the 3d porous titanium was printed based on laser sintering technology ( concept laser mlab , germany ) in two sizes : 5 mm diameter and 3 mm thickness for cultivating cells in 96-well plate and 8 mm diameter and 3 mm thickness for implanting in vivo , respectively . three groups of scaffolds were prepared including clear scaffolds ( blank group ) , scaffolds with gelatin ( control group ) , and scaffolds with gelatin and platelets ( treated group ) . to prepare the scaffold with gelatin , 1 g nacl was added to 5 ml of gelatin solution ( 5% ) , and then the solution was perfused into a 3d printed scaffold and lyophilized well . the sterilization process was treatment with ethylene oxide , and the scaffolds were prepared with gelatin . the treated groups were prepared by adding 20 l of prp to the scaffolds with gelatin then freeze - dried . the structure of the clear scaffold and scaffolds with gelatin and platelets was imaged as shown in figure 1 via scanning electron microscopy . all scaffolds were molded at 3 mm height and 5 mm diameter , which fit the wells of the 96-well microplate ( 3559 , 96wl , corning , usa ) . ( a ) the internal pores of the blank group ( scaffolds with gelatin ) , diameters ranging from 150 to 220 m . ( b ) the internal pores of the treated group ( scaffolds with gelatin and platelet ) , diameters ranging from 70 to 200 m . after the cells adhered to the flasks , the medium was removed and replaced by a new 8 ml medium . we set four groups including a control group and three types of scaffold groups in a 96-well microplate . three types of scaffolds with 200 l -mem were placed into the wells of the treated groups . the gelatin scaffolds group and the gelatin and platelet scaffolds group were compared for release levels of vegf and tgf- after 3 , 6 , 9 , 12 , 15 , 18 , and 21 days via enzyme - linked immunosorbent assay ( elisa ) analysis . the levels of vegf and tgf- release were also measured via elisa after the scaffolds were stored at 25c for 3 months . samples were treated at room temperature , and the concentrations of vegf and tgf- were measured using company kits ( r&d systems , minneapolis , usa ) . two wells in each group were measured simultaneously and averaged , and then 10 l of cck-8 was added to each well of the 96-well microplate . the time points chosen to evaluate the cytotoxicity were 1 , 3 , 5 , and 7 days . two wells in each group were measured simultaneously and averaged , then 10 l of cck-8 was added to each well of the 96-well microplate . all of the plasma layer and 3 mm of the intermediate layer were then centrifuged for another 6 min at 1000 g to obtain a two - part plasma : the upper three - quarters consisted of platelet - poor plasma and the lower quarter consisted of platelet - rich plasma ( prp ) . the scaffolds possess an internal porous structure designed in a computer aided design environment using the software rhino 5.0 ( robert mcneel & associates , usa ) . the internal pores were orderly arranged regular dodecahedrons within the scaffolds with = 1500 m . the titanium skeletons that formed the internal porous structure and the external appearance of scaffolds were 100 m in diameter . the 3d porous titanium was printed based on laser sintering technology ( concept laser mlab , germany ) in two sizes : 5 mm diameter and 3 mm thickness for cultivating cells in 96-well plate and 8 mm diameter and 3 mm thickness for implanting in vivo , respectively . three groups of scaffolds were prepared including clear scaffolds ( blank group ) , scaffolds with gelatin ( control group ) , and scaffolds with gelatin and platelets ( treated group ) . to prepare the scaffold with gelatin , 1 g nacl was added to 5 ml of gelatin solution ( 5% ) , and then the solution was perfused into a 3d printed scaffold and lyophilized well . the sterilization process was treatment with ethylene oxide , and the scaffolds were prepared with gelatin . the treated groups were prepared by adding 20 l of prp to the scaffolds with gelatin then freeze - dried . the structure of the clear scaffold and scaffolds with gelatin and platelets was imaged as shown in figure 1 via scanning electron microscopy . all scaffolds were molded at 3 mm height and 5 mm diameter , which fit the wells of the 96-well microplate ( 3559 , 96wl , corning , usa ) . ( a ) the internal pores of the blank group ( scaffolds with gelatin ) , diameters ranging from 150 to 220 m . ( b ) the internal pores of the treated group ( scaffolds with gelatin and platelet ) , diameters ranging from 70 to 200 m . after the cells adhered to the flasks , the medium was removed and replaced by a new 8 ml medium . we set four groups including a control group and three types of scaffold groups in a 96-well microplate . three types of scaffolds with 200 l -mem were placed into the wells of the treated groups . the gelatin scaffolds group and the gelatin and platelet scaffolds group were compared for release levels of vegf and tgf- after 3 , 6 , 9 , 12 , 15 , 18 , and 21 days via enzyme - linked immunosorbent assay ( elisa ) analysis . the levels of vegf and tgf- release were also measured via elisa after the scaffolds were stored at 25c for 3 months . samples were treated at room temperature , and the concentrations of vegf and tgf- were measured using company kits ( r&d systems , minneapolis , usa ) . two wells in each group were measured simultaneously and averaged , and then 10 l of cck-8 was added to each well of the 96-well microplate . the time points chosen to evaluate the cytotoxicity were 1 , 3 , 5 , and 7 days . two wells in each group were measured simultaneously and averaged , then 10 l of cck-8 was added to each well of the 96-well microplate . as shown in figure 1 , the gelatin microscaffolds were successfully created in pores of the 3d printed porous titanium . we calculated that 100 gelatin pores existed in microscaffolds and found that their diameter was in the range of 100300 m . after freeze - drying the prp , the microscaffold could also stay in the titanium pores because of the expansibility of gelatin when absorbing water . elisa was used to measure the vegf and tgf-1 released over 21 days , with results as shown in figure 2 . it could be determined that there was a triggered release after the first 3 days and the growth factors released would be low and plain at later days , which presented a better sustained release function . all the treated groups showed significantly higher release of vegf and tgf-1 than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . * p < 0.05 versus control . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . after the scaffolds were stored at 25c for 3 months , vegf and tgf-1 were measured for another 21 days , with results as shown in figure 3 . the releasing curve not only seemed irregular compared to fresh scaffolds but also proved that the release function was sustained after 3 months of storage at room temperature . at 3 , 9 , 12 , 15 , 18 , and 21 days time points , treated group exhibited significantly higher than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . * p < 0.05 versus control group . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . after 21 days of culture , the cck-8 cell count of the control and treated groups was significantly higher than that of the blank group ( p = 0.019 and 0.024 , respectively ) . however , there was no significant difference ( p = 0.364 ) between the control group and the treated group after 21 days of culture . cell proliferation assay of the mc3t3-e1 using cell counting kit-8 assay after a 21-day culture . * p < 0.05 versus the blank group . the number of the living cells was determined by the cck-8 assay to detect cytotoxicity [ figure 5 ] . at the 3 days time point of culturing , the value of the cck-8 cell count of the control and treated groups was obviously higher than that of the blank group ( p < 0.05 ) . at 5 day of culture , the value of the cck-8 cell count of the control group remained above the level of the blank group ( p < 0.05 ) , with the treated group almost equal to the blank group . however , at 7 d of culture , cck-8 cell count of the control and treated groups was lower than that of the blank group ( p < 0.05 ) . as shown in figure 1 , the gelatin microscaffolds were successfully created in pores of the 3d printed porous titanium . after freeze - drying the prp , the microscaffold could also stay in the titanium pores because of the expansibility of gelatin when absorbing water . elisa was used to measure the vegf and tgf-1 released over 21 days , with results as shown in figure 2 . it could be determined that there was a triggered release after the first 3 days and the growth factors released would be low and plain at later days , which presented a better sustained release function . all the treated groups showed significantly higher release of vegf and tgf-1 than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . * p < 0.05 versus control . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . after the scaffolds were stored at 25c for 3 months , vegf and tgf-1 were measured for another 21 days , with results as shown in figure 3 . the releasing curve not only seemed irregular compared to fresh scaffolds but also proved that the release function was sustained after 3 months of storage at room temperature . at 3 , 9 , 12 , 15 , 18 , and 21 days time points , treated group exhibited significantly higher than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . * p < 0.05 versus control group . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . after 21 days of culture , the cck-8 cell count of the control and treated groups was significantly higher than that of the blank group ( p = 0.019 and 0.024 , respectively ) . however , there was no significant difference ( p = 0.364 ) between the control group and the treated group after 21 days of culture . cell proliferation assay of the mc3t3-e1 using cell counting kit-8 assay after a 21-day culture . * p < 0.05 versus the blank group . the number of the living cells was determined by the cck-8 assay to detect cytotoxicity [ figure 5 ] . at the 3 days time point of culturing , the value of the cck-8 cell count of the control and treated groups was obviously higher than that of the blank group ( p < 0.05 ) . at 5 day of culture , the value of the cck-8 cell count of the control group remained above the level of the blank group ( p < 0.05 ) , with the treated group almost equal to the blank group . however , at 7 d of culture , cck-8 cell count of the control and treated groups was lower than that of the blank group ( p < 0.05 ) . however , curing bone defects it remains very challenging , not only because of the complex stratified architecture of bone itself but also due to changes in the microenvironment including cytokines and blood supplies . our work is a combination of 3d printing technology that gives the scaffold an accurate structure , and biological treatments with platelets to enhance the osteogenesis effect , thus helping patients with bone defects obtain a better prognosis . the 3d printed scaffold appears gray , bright , and clean . it has been reported that when the pore diameter of a porous structure is 1540 m , fibrous tissue would grow into the interior of the material . when the diameter of pores is 40100 m when the diameter of the pores exceeds 150 m , the bone tissue can infiltrate completely into the pore structure of the scaffold , potentially improving the osteogenesis effect . the 3d geometric structure and interconnected pores of our scaffold offer a quite large internal surface area and a 3d structure , in which preosteoblasts are able to absorb more large molecules , promote cell adhesion , proliferation , and osteogenic capabilities . we tested the vegf and tgf-1 release for 21 days for different times at which the platelets were made immediately and stored for 3 months at 25c [ figures 2 and 3 ] to address whether the control group and treated group had different effects on cytokines control - release . from these results , it was indicated that the scaffolds with platelets adhered on the microscaffold could release growth factors effectively and be restored for a long time via this special preparation method . in the study the cells proliferated further and numerous cells were measured at day 21 via cck-8 assay , the cell count of the control group and treated groups was significantly higher than that of the blank group . however , there was no significant difference between the control group and the treated group . this result demonstrated that the gelatin and platelets in the 3d porous structure of the scaffold could also promote cytokine secretion and the infiltration of nutrients and metabolites in the medium , which enhanced the 3d printed scaffolds biocompatibility . therefore , this kind of scaffold may be used as a bone graft substitute material . a higher cell proliferation rate was associated with the lower toxicity of different types of scaffolds . in this study , detection based on cytotoxicity test standards in vitro showed that preosteoblast proliferation increased rapidly in the early stage ( days 15 ) and then the velocity decreased ; we can see the results in figure 5 . however , the preosteoblasts proliferation seems poor in the treated group compared to the other two groups with scaffolds at day 7 because the cells became confluent on the well surface . therefore , the results indicate that the scaffolds we designed are not toxic to preosteoblast cells and may be safe to implant in animals . in conclusion , the results of this study confirm that our 3d printed scaffolds provided good tissue compatibility and improved the adhesion and proliferation of preosteoblasts . furthermore , the platelets we treated also showed a terrific sustained release effect for vegf and tgf-1 , even after being stored for 3 months . therefore , a 3d printed scaffold with gelatin and platelets is a suitable implant for cell proliferation and may be a good bone graft substitute material for bone defects . in the present study , 3d scaffolds were only observed in vitro for 3 months ; therefore , a longer observation period and in vivo experiments will be performed in future studies .

escherichia coli of serotype o157:h7 was first recognized in 1982 as a human pathogen associated with outbreaks of bloody diarrhea in the united states and is now considered a major cause of foodborne infections ( 1 , 2 ) . the virulence of e. coli o157:h7 depends on the presence of a number of mobile genetic elements ( mge ) , such as shiga toxin ( stx)-converting bacteriophages carrying different genes encoding stx1 and stx2 , the virulence plasmid po157 , the locus of enterocyte effacement ( lee ) , o islands , an arginine translocation system , and various adhesion factors ( 3 ) . in stx - producing e. coli ( stec ) , stx is thought to be responsible for the most severe form of the infection causing the life - threatening hemorrhagic colitis ( hc ) and hemolytic uremic syndrome ( hus ) ( 4 ) . strains causing these clinical symptoms are also known as enterohemorrhagic e. coli ( ehec ) strains ( 5 ) . the genes encoding stxs ( stx1 and stx2 ) are found in lysogenic lambdoid bacteriophages ( 6 ) , which can integrate into the host chromosome via specific insertion sites . integration sites for stx2-converting phages in stec o157:h7 include wrba , argw , sbcb , and yece , whereas stx1-converting phages integrate in the yehv region ( 711 ) . stec o157:h7 strains generally do not ferment sorbitol ( non - sorbitol - fermenting [ nsf ] strains ) , and this feature is widely used to identify these pathogenic strains . nevertheless , sorbitol - fermenting ( sf ) stec o157:nm ( nonmotile ) strains , as an emerging and important pathogen in europe , have been isolated from patients with hus and diarrhea ( 12 ) . both sf and nsf o157:h7/nm strains are thought to have evolved from a common nonpathogenic ancestor of serotype o55:h7 following the modification of the o - antigen gene cluster and the acquisition of a number of virulence - associated mge via horizontal gene transfers ( 13 ) . the most current and accepted evolutionary model proposes that e. coli o157:h7 lost the o55 rfb - gnd gene cluster and acquired the stx2 bacteriophage and the o157 rfb - gnd gene cluster . after the diversification of the two lineages , e. coli o157:h7 acquired stx1 via acquisition of the bacteriophage containing the stx1 gene and lost the ability to ferment sorbitol , while the sorbitol - fermenting stx2-producing e. coli o157 lost its motility and evolved into nonmotile e. coli o157:nm ( 14 , 15 ) . most e. coli o157 isolates produce a large outer membrane protein , intimin ( encoded by the eae gene ) , which is the genetic determinant of the formation of attaching and effacing ( a / e ) lesions , a central mechanism in the pathogenesis of enteropathogenic e. coli ( epec ) ( 16 ) . as described in previous studies , stx - negative e. coli o157:h7/nm isolates were obtained from patients with hus and diarrhea . it was assumed , especially for the hus cases , that these patients were originally infected with an ehec strain and that excision of the stx bacteriophage occurred during the infection . those stx - negative o157:h7/nm isolates appeared to be closely related to stx - positive o157:h7 isolates as determined by conventional molecular typing methods ( 1821 ) . in this study , whole - genome sequencing ( wgs ) was used to obtain a more detailed molecular characterization of stx - negative e. coli o157:h7/nm isolates and to reveal their genetic relationship with stx - positive o157:h7 isolates obtained from patients with gastrointestinal complaints . fecal samples were collected from patients with gastrointestinal complaints in the regions of groningen and rotterdam during the period april 2013 to march 2014 as part of a large multicenter study ( stec - id - net , unpublished data ) . samples were screened for the presence of stx1 , stx2 , and escv ( used as an alternative marker for the lee instead of the eae gene ) and for o - serogroup determination ( o26 , o103 , o104 , o111 , o121 , o145 , and o157 ) by real - time pcr as described previously ( 22 ) . this resulted in the collection of 34 e. coli o157 isolates ( with or without stx ) from 34 different patients . pcr for the detection of the flich7 gene was performed as described before ( 23 ) , and only the isolates positive for flich7 were then subjected to wgs . the publically available genomes of e. coli o157:h7 strains sakai , edl933 , and ss52 , e. coli o157:h45 strain c639_08 , e. coli o127:h6 strain e2348/69 , and e. coli o55:h7 cb9615 were also included in the comparative analysis . moreover , as no complete genomes of sf stec o157:nm and stx - negative o157:h7/nm were available in the ncbi database when the study was performed , we sequenced the genomes of one sf stec o157:nm ( e09/10 ) strain and one stx - negative o157:nm ( e09/224 ) strain and included them as control strains in all the analyses . the information on the isolates used in this study is presented in table s1 in the supplemental material . sorbitol fermentation was determined using ct - smac ( sorbitol macconkey agar with cefixime and tellurite ) plates , and motility was tested using motility test medium with triphenyltetrazolium chloride ( mediaproducts bv , groningen , the netherlands ) . the production of beta - glucuronidase and urease was checked using macconkey ii agar with 4-methylumbelliferryl--d - glucuronide ( mug ) ( bd diagnostics , breda , the netherlands ) and urea - triple sugar iron ( tsi ) agar ( mediaproducts bv , groningen , the netherlands ) , respectively . the o and h serotypes of the isolates were determined by seroagglutination performed at the national institute for public health and the environment ( rivm , bilthoven , the netherlands ) . dna was extracted using the ultraclean microbial dna isolation kit ( mo bio laboratories , carlsbad , ca , usa ) according to the manufacturer 's protocol . a dna library was prepared using the nextera xt kit ( illumina , san diego , ca , usa ) according to the manufacturer 's instructions and then run on a miseq instrument ( illumina ) for generating paired - end 250-bp reads , aiming at a coverage of at least 60-fold . de novo assembly was performed using clc genomics workbench v7.0.3 ( clc bio a / s , aarhus , denmark ) after quality trimming ( qs , 28 ) with optimal word sizes based on the maximum n50 value . annotation was performed by uploading the assembled genome on the rast server version 2.0 ( 24 ) . the sequence type ( st ) was identified by uploading the assembled genomes to the multilocus sequence type ( mlst ) server ( version 1.7 ) ( 25 ) , and the virulence genes and stx subtypes were determined with virulence finder 1.2 ( 26 ) . the serogenotype of the isolates was determined using the cge serotypefinder tool ( 27 ) . to analyze the sequence homology of the lee pathogenicity island in the isolates , the contigs of each sample were subjected to a blast search against the lee region of e. coli o157:h7 strain 71074 ( accession no . gq338312 ) as a reference and plotted by blast ring image generator ( brig ) ( 28 ) . to identify plasmids , po157 ( accession no . nc_009602 ) of the e. coli o157:nm strain 3072/96 were used as references for blast analyses . for identifying proposed marker genes for differentiating nsf stec o157:h7/nm from sf stec o157:nm , including the complete cdt cluster , the complete sequence of efa1 , the tellurite resistance- and adherence - conferring pathogenicity island ( tai ) , and the urease gene cluster ( 29 ) , contigs were either subjected to a blast search using blastn ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) or mapped by clc genomics workbench v7.0.3 using default settings against a reference sequence artificially generated by concatenating sequences of the marker genes . to determine the phylogenetic relationship of the isolates , a gene - by - gene typing approach was performed using seqsphere v1.0 ( ridom gmbh , mnster , germany ) . briefly , an in - house defined mlst+ ( also referred to as core genome [ cg ] mlst ) scheme was developed using the genome of e. coli o157:h7 strain sakai as reference genome to extract open reading frames ( orfs ) from the genome of each isolate by seqsphere . only the orfs without a premature stop codon and ambiguous nucleotides from contigs of assembled genomes were included . the genes shared by the genomes of all isolates analyzed were defined as the core genome for phylogenetic analysis ( 30 , 31 ) . a neighbor - joining ( nj ) tree was constructed based on a distance matrix among the isolates depending on the core genomes of all isolates . the most common and well described stx phage integration sites for stec o157:h7/nm were analyzed in our stx - negative isolates to reveal if any phage was occupying these sites or that they were available for future integration of a phage , including one that contains stx genes . five integration sites were studied : yehv ( for the stx1 phage ) , wrba and argw ( for the stx2a phage ) , sbcb ( for the stx2c phage ) , and yece ( for the stx2a phage of sf stec ) ( 11 ) . these five loci were identified in the contigs , the adjacent regions were extracted , and the presence of phage integrases was detected using the blastn algorithm . the genbank accession numbers of the isolates analyzed in this study are ldoz00000000 , lfua00000000 , lfub00000000 , lgaz00000000 , lfuh00000000 , lgba00000000 , lgbb00000000 , lgbc00000000 , lgbd00000000 , lgbe00000000 , lgbf00000000 , lgbg00000000 , lgbh00000000 , lgbq00000000 , lgbi00000000 , lgbj00000000 , lgbk00000000 , lgbl00000000 , lgbm00000000 , lgbn00000000 , lgbo00000000 , and lgbp00000000 . among 34 e. coli o157 isolates initially obtained in this study , 16 were stec ( all were flich7 positive ) and 18 were categorized as epec ( as they were stx negative but eae positive ) . among the 18 epec isolates , four o157:h7 , for the subsequent comparative study , 20 isolates positive for flich7 ( 16 stec o157:h7 and 4 epec o157:h7/nm ) were used . the phenotypic characteristics of the 20 o157:h7 isolates of this study and the two control strains are shown in table 1 . the 16 stec o157:h7 isolates did not ferment sorbitol ( nsf ) and were beta - glucuronidase negative , whereas among the four epec isolates , one ( epec 287 ) was nsf and beta - glucuronidase negative . the remaining three isolates ( epec 393 , epec 1572 , and epec 1669 ) and the control strain ( e09/224 ) were sorbitol fermenting ( sf ) and beta - glucuronidase positive , as was the sf stec control strain e09/10 ( see table s1 in the supplemental material ) . among the nsf stec isolates , phenotypic and molecular characteristics of the isolates nsf , non - sorbitol fermenting ; sf , sorbitol fermenting ; nm , nonmotile . the type of the eae gene was determined from wgs data using blastn . this isolate was obtained from germany and used as a control strain for sf stec o157:nm isolates . one of these four isolates was obtained from germany and used as a control strain for the stx - negative o157:nm isolates . stx subtyping of stec isolates revealed eight ( 50% ) isolates with stx1a and stx2c subtypes , two ( 12.5% ) with stx1a and stx2a subtypes , and six ( 37.5% ) with the stx2c subtype only . all isolates contained the o - serotyping gene wzxo157 and the flagellar genes flich7 and eae ( type gamma ) and were assigned to st11 ( table 1 ) . all nsf stec isolates had identical virulence profiles , with exception of isolate stec 1109 , which contained the cdt - v gene , encoding cytolethal distending toxin . some of the virulence genes , such as serine protease autotransporter - encoding gene espp , toxin - encoding gene toxb , catalase peroxidase - encoding gene katp , the tellurite resistance- and adherence - conferring island ( tai ) , and the urease gene cluster , were present only in nsf stec and in the only nsf epec isolate but not in any sf isolates . on the other hand , sfpa , the complete efa1 , and the cdt gene cluster were present only in sf isolates . the sf epec isolates carried almost all the virulence genes carried by the german sf stec o157:nm isolate , with the exception of the tccp gene ( encoding tir cytoskeletal coupling protein ) , which was present only in sf epec isolates . distribution of virulence and other genes among stx - positive and stx - negative o157:h7/nm isolates encoding cytolethal distending toxin a , b , and c subunits . tellurite resistance- and adherence - conferring island carrying adhesin gene iha and putative tellurite resistance genes tlra , tlrb , tlrc , and tlrd . ure gene cluster containing urea , ureb , urec , ured , uree , uref , and ureg . all the stec isolates contained an lee region highly similar to the lee of stec o157:h7 strain 71074 , used as a reference . however , some nsf stec isolates lacked two genes encoding the mobile element proteins orfa and orfb located in the insertion sequence is911 . three stec isolates ( stec 2257 , stec 2820 , and stec 2821 ) did not possess the intl gene , which is known to encode an integrase of the putative prophage 933l carried in the lees of other stec o157:h7 isolates . the sequences of the lees of our nsf and sf epec isolates were more similar to those of nsf and sf stec , respectively , than to the lee of epec reference genomes e2348/69 and c639_08 ( fig . 1 ) . comparison of lee pathogenicity islands , showing a blast comparison of stec and epec isolates , depicted by each ring , against the reference lee sequence ( core black circle ) . the colors of different groups as well as the order of the rings for each isolate ( from inner to outer ) with the color gradient for sequence identity are shown at the right . no sequence variation was observed in regions carrying putative virulence genes such as , e.g. , genes involved in the type ii secretion system , hemolysins , toxins , and catalase peroxidase in po157 of nsf stec . however , with the exception of the two stx2a - positive isolates ( stec 2112 and stec 2868 ) , all stec isolates lacked the po157p35 gene , encoding a reverse transcriptase . only three stec isolates ( stec 605 , stec 989 , and stec 1109 ) harbored the plasmid posak1 . among the epec isolates , the nsf one ( epec 287 ) had almost the intact po157 plasmid , lacking only an intact espp gene . the sf epec isolates contained an almost identical copy of plasmid psfo157 of sf stec o157:nm ( fig . 2 ) . comparison of plasmids , showing a blast comparison of stec and epec isolates , depicted by each ring , against the reference plasmid composed of three plasmids shown in the outermost ring by three different colors ( black , blue , and orange represent plasmids po157 , poska1 , and psfo157 , respectively ) . the color of the rings represents sequence identity on a sliding scale ; the more gray the ring is , the lower the percent identity . the colors of different groups as well as the order of the rings for each isolate ( from inner to outer ) with the color gradient for sequence identity are shown at the right . core genome phylogenetic analysis was performed to evaluate the evolutionary relationship between the stx - positive ( stec ) and stx - negative ( epec ) o157:h7/nm isolates . in total , 3,005 orfs were shared by all isolates analyzed in this study , and these were defined as the core genome for phylogenetic analysis . this analysis separated epec c639_08 and epec e2348/69 from the o157:h7/nm isolates in this study ( fig . the latter isolates formed two separated clusters : sf isolates ( cluster 1 ) and nsf isolates ( cluster 2 ) . remarkably , in cluster 1 , four sf epec isolates ( epec 393 , epec 1572 , epec 1669 , and e09/224 ) clustered together with the sf stec isolate . cluster 2 ( nsf o157:h7 isolates ) could be divided into three subclusters : cluster 2a , containing stec o157:nm ( nonmotile ) isolates together with one nsf epec isolate ( epec 287 ) ; cluster 2b , containing six of the motile stec o157:h7 isolates ; and cluster 2c , containing two stx2a - positive isolates ( stec 2112 and stec 2868 ) clustered closely with two previously described stec outbreak isolates , sakai and edl933 ( 1 , 32 ) . the last subcluster also included two other motile isolates ( stec 1109 and stec 989 ) and stec o157:h7 strain ss52 ( stx2a and stx2c positive ) , isolated from super shedder cattle ( 33 ) . taken together , the data indicate that the epec o157:nm isolates clustered with stec isolates but not with epec isolates ( fig . the nj tree was constructed based on a distance matrix among the isolates depending on their core genomes . in the sf epec strains ( epec 393 , epec 1572 , epec 1669 , and epec e09/224 ) , the phage insertion sites analyzed were intact , with the exception of argw , which was occupied in isolates epec 393 and epec 1572 . in epec 287 , although yehv and argw were occupied by phages , the wrba and sbcb loci ( integration sites for stx2a and stx2c phage , respectively ) were unoccupied . a red arrow indicates the presence of a phage integrase adjacent to the integration site . ( a ) sf stec isolate e09/10 ; the yece region is occupied by phage . ( e ) nsf epec isolate epec 287 ; the yehv region occupied by phage . e. coli o157:h7 is one of the major causes of foodborne illness and represents a considerable public health concern worldwide ( 34 ) . this study aimed at determining the phylogenetic relationships and comparing the virulence factors of stx - negative e. coli o157:h7/nm with those of stx - positive e. coli o157:h7 with the highest resolution and greatest possible detail using a wgs approach . the e. coli o157:h7 isolates used in this study were obtained from patients with diarrhea and other gastrointestinal complaints from two different regions in the netherlands ( groningen and rotterdam ) . isolates e09/10 and e09/224 were sequenced and used as control strains for sf stec o157:nm and stx - negative o157:nm isolates , respectively . as the stx - negative isolates were found to be positive for eae but negative for the bfpa gene , they were considered atypical epec . notably , the epec isolates belonged to st11 and contained type gamma intimin , which are not typical features of epec but are frequently associated with stec o157:h7 ( 35 ) . remarkably , all the typical genes ( e.g. , ehxa , asta , lpfa , katp , etpd , espp , the pathogenicity island tai , and the urease gene cluster ) of nsf stec o157:h7 described previously were present in epec 287 . in contrast , sf epec isolates possessed the sfp gene cluster , the cdt gene cluster , and the complete efa1 gene but lacked the genes toxb , katp , espp carried on plasmid po157 , the urease gene cluster , and the pathogenicity island tai , which are typical features of sf stec o157:nm ( 18 , 29 ) . subsequent analyses showed that epec 287 harbored a po157 plasmid almost identical to that of the stec isolates , with sequence variability mostly in mobile genetic elements . the sf epec isolates contained a plasmid similar to the psfo157 present in the sf stec o157:nm isolate strain 3072/96 . additionally , our nsf and sf epec isolates shared an almost identical lee pathogenicity island with the stec isolates containing additional orfs encoding a putative prophage normally not present in the lee of epec ( 36 ) . all these results together suggest that the epec o157:h7/nm isolates investigated shared a virulence profile similar to that of stec isolates rather than to the virulence profile of epec . the genetic relationship of the o157:h7/nm isolates of this study was confirmed by the phylogenetic analysis using a gene - by - gene comparison by core genome mlst . the nsf epec isolate clustered together with the nonmotile nsf stec isolates , and the three sf epec isolates clustered together with the control strain of sf stec o157:nm . it has already been described that the nonmotile characteristic of sf stec o157 is due to a 12-bp deletion in the master regulator of flagellar biosynthesis , the flhc gene ( 37 ) . we also observed this deletion in the same gene in our nonmotile sf epec isolates but not in our nonmotile nsf stec isolates , further strengthening the hypothesis of their derivation from stx - positive sf o157:nm . taken together , the results of our analyses provide further evidence that the nsf and sf epec isolates were mostly related to the stec group , but lacking the stx - converting phages , and might be referred to as ehec strains that have lost the shiga toxin ( ehec - lst ) , as has also been proposed previously ( 19 , 20 ) . studying in more detail the typical stx phage insertion sites ( yece for sf epec and wrba and sbcb for nsf epec ) revealed that they were unoccupied in the stx - negative isolates . such unoccupied regions could be an indication of loss of the stx bacteriophage , e.g. , in the course of infection or during isolation or subculture ( 38 ) . conversely , stx - negative strains could be progenitors of stec prepared to acquire one or more stx - converting phages ( 18 , 39 ) . in our case , the stx genes were not lost in the laboratory during isolation or subculture , as they were not detected in the feces of the patients . therefore , the patients may have been infected with an stx - negative variant , which could explain the mild symptoms they displayed . in routine diagnostic testing , these stx - negative isolates may be missed , as most microbiological laboratories depend on the molecular screening of stec by the detection of the stx genes only . screening for several additional genes ( including eae , saa , and sfpa ) in routine diagnostics to identify these pathogens has already been proposed ( 18 ) . moreover , our findings bring into question whether classifying pathogenic e. coli into stec and epec based on detection of the stx and eae genes , respectively , is reliable enough . due to integrating and interchanging mobile genetic elements , e.g. , the stx - converting bacteriophages , which could integrate into several e. coli pathogroups , it is sometimes complicated to precisely define the classification of pathogenic e. coli ( 40 , 41 ) . obviously , screening a number of accessory virulence genes of stec would be laborious for routine diagnostics . in our opinion , screening of at least the flich7 gene , together with the o157-encoding gene , may help to identify ehec - lst of the most virulent clone of ehec ( o157:h7 ) , as all the flich7-positive isolates were genetically related to stec o157:h7 in this study . our results are consistent with the finding that the proportion of stx - negative variants among sf o157:nm isolates is generally higher than that among nsf o157:h7 isolates ( 20 , 42 ) . nevertheless , the number of isolates studied was too low to draw firm conclusions . however , to our best knowledge , this is the first report where the genetic relationship of stx - negative variants of e. coli o157:h7/nm with stx - positive o157:h7/nm has been confirmed using wgs . in conclusion , stx - negative e. coli o157:h7/nm strains are a cause of gastrointestinal disease in the netherlands , and because of the presence of the complete set of accessory virulence genes , these isolates should be considered of public health concern similar to that for their stx - positive variants . additional diagnostic approaches should be implemented to identify these ehec - lst isolates for surveillance purposes and to allow appropriate treatment measures and for preventing their transmission .

the ability of escherichia coli o157:h7 to induce cellular damage leading to disease in humans is related to numerous virulence factors , most notably the stx gene , encoding shiga toxin ( stx ) and carried by a bacteriophage . loss of the stx - encoding bacteriophage may occur during infection or culturing of the strain . here , we collected stx - positive and stx - negative variants of e. coli o157:h7/nm ( nonmotile ) isolates from patients with gastrointestinal complaints . isolates were characterized by whole - genome sequencing ( wgs ) , and their virulence properties and phylogenetic relationship were determined . because of the presence of the eae gene but lack of the bfpa gene , the stx - negative isolates were considered atypical enteropathogenic e. coli ( aepec ) . however , they had phenotypic characteristics similar to those of the shiga toxin - producing e. coli ( stec ) isolates and belonged to the same sequence type , st11 . furthermore , epec and stec isolates shared similar virulence genes , the locus of enterocyte effacement region , and plasmids . core genome phylogenetic analysis using a gene - by - gene typing approach showed that the sorbitol - fermenting ( sf ) stx - negative isolates clustered together with an sf stec isolate and that one non - sorbitol - fermenting ( nsf ) stx - negative isolate clustered together with nsf stec isolates . therefore , these stx - negative isolates were thought either to have lost the stx phage or to be a progenitor of stec o157:h7/nm . as detection of stec infections is often based solely on the identification of the presence of stx genes , these may be misdiagnosed in routine laboratories . therefore , an improved diagnostic approach is required to manage identification , strategies for treatment , and prevention of transmission of these potentially pathogenic strains .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

the virulence of e. coli o157:h7 depends on the presence of a number of mobile genetic elements ( mge ) , such as shiga toxin ( stx)-converting bacteriophages carrying different genes encoding stx1 and stx2 , the virulence plasmid po157 , the locus of enterocyte effacement ( lee ) , o islands , an arginine translocation system , and various adhesion factors ( 3 ) . in stx - producing e. coli ( stec ) , stx is thought to be responsible for the most severe form of the infection causing the life - threatening hemorrhagic colitis ( hc ) and hemolytic uremic syndrome ( hus ) ( 4 ) . stec o157:h7 strains generally do not ferment sorbitol ( non - sorbitol - fermenting [ nsf ] strains ) , and this feature is widely used to identify these pathogenic strains . nevertheless , sorbitol - fermenting ( sf ) stec o157:nm ( nonmotile ) strains , as an emerging and important pathogen in europe , have been isolated from patients with hus and diarrhea ( 12 ) . the most current and accepted evolutionary model proposes that e. coli o157:h7 lost the o55 rfb - gnd gene cluster and acquired the stx2 bacteriophage and the o157 rfb - gnd gene cluster . after the diversification of the two lineages , e. coli o157:h7 acquired stx1 via acquisition of the bacteriophage containing the stx1 gene and lost the ability to ferment sorbitol , while the sorbitol - fermenting stx2-producing e. coli o157 lost its motility and evolved into nonmotile e. coli o157:nm ( 14 , 15 ) . most e. coli o157 isolates produce a large outer membrane protein , intimin ( encoded by the eae gene ) , which is the genetic determinant of the formation of attaching and effacing ( a / e ) lesions , a central mechanism in the pathogenesis of enteropathogenic e. coli ( epec ) ( 16 ) . as described in previous studies , stx - negative e. coli o157:h7/nm isolates were obtained from patients with hus and diarrhea . it was assumed , especially for the hus cases , that these patients were originally infected with an ehec strain and that excision of the stx bacteriophage occurred during the infection . those stx - negative o157:h7/nm isolates appeared to be closely related to stx - positive o157:h7 isolates as determined by conventional molecular typing methods ( 1821 ) . in this study , whole - genome sequencing ( wgs ) was used to obtain a more detailed molecular characterization of stx - negative e. coli o157:h7/nm isolates and to reveal their genetic relationship with stx - positive o157:h7 isolates obtained from patients with gastrointestinal complaints . fecal samples were collected from patients with gastrointestinal complaints in the regions of groningen and rotterdam during the period april 2013 to march 2014 as part of a large multicenter study ( stec - id - net , unpublished data ) . samples were screened for the presence of stx1 , stx2 , and escv ( used as an alternative marker for the lee instead of the eae gene ) and for o - serogroup determination ( o26 , o103 , o104 , o111 , o121 , o145 , and o157 ) by real - time pcr as described previously ( 22 ) . pcr for the detection of the flich7 gene was performed as described before ( 23 ) , and only the isolates positive for flich7 were then subjected to wgs . the publically available genomes of e. coli o157:h7 strains sakai , edl933 , and ss52 , e. coli o157:h45 strain c639_08 , e. coli o127:h6 strain e2348/69 , and e. coli o55:h7 cb9615 were also included in the comparative analysis . moreover , as no complete genomes of sf stec o157:nm and stx - negative o157:h7/nm were available in the ncbi database when the study was performed , we sequenced the genomes of one sf stec o157:nm ( e09/10 ) strain and one stx - negative o157:nm ( e09/224 ) strain and included them as control strains in all the analyses . the production of beta - glucuronidase and urease was checked using macconkey ii agar with 4-methylumbelliferryl--d - glucuronide ( mug ) ( bd diagnostics , breda , the netherlands ) and urea - triple sugar iron ( tsi ) agar ( mediaproducts bv , groningen , the netherlands ) , respectively . the o and h serotypes of the isolates were determined by seroagglutination performed at the national institute for public health and the environment ( rivm , bilthoven , the netherlands ) . the sequence type ( st ) was identified by uploading the assembled genomes to the multilocus sequence type ( mlst ) server ( version 1.7 ) ( 25 ) , and the virulence genes and stx subtypes were determined with virulence finder 1.2 ( 26 ) . to analyze the sequence homology of the lee pathogenicity island in the isolates , the contigs of each sample were subjected to a blast search against the lee region of e. coli o157:h7 strain 71074 ( accession no . for identifying proposed marker genes for differentiating nsf stec o157:h7/nm from sf stec o157:nm , including the complete cdt cluster , the complete sequence of efa1 , the tellurite resistance- and adherence - conferring pathogenicity island ( tai ) , and the urease gene cluster ( 29 ) , contigs were either subjected to a blast search using blastn ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) or mapped by clc genomics workbench v7.0.3 using default settings against a reference sequence artificially generated by concatenating sequences of the marker genes . to determine the phylogenetic relationship of the isolates , a gene - by - gene typing approach was performed using seqsphere v1.0 ( ridom gmbh , mnster , germany ) . briefly , an in - house defined mlst+ ( also referred to as core genome [ cg ] mlst ) scheme was developed using the genome of e. coli o157:h7 strain sakai as reference genome to extract open reading frames ( orfs ) from the genome of each isolate by seqsphere . the most common and well described stx phage integration sites for stec o157:h7/nm were analyzed in our stx - negative isolates to reveal if any phage was occupying these sites or that they were available for future integration of a phage , including one that contains stx genes . five integration sites were studied : yehv ( for the stx1 phage ) , wrba and argw ( for the stx2a phage ) , sbcb ( for the stx2c phage ) , and yece ( for the stx2a phage of sf stec ) ( 11 ) . these five loci were identified in the contigs , the adjacent regions were extracted , and the presence of phage integrases was detected using the blastn algorithm . the 16 stec o157:h7 isolates did not ferment sorbitol ( nsf ) and were beta - glucuronidase negative , whereas among the four epec isolates , one ( epec 287 ) was nsf and beta - glucuronidase negative . the remaining three isolates ( epec 393 , epec 1572 , and epec 1669 ) and the control strain ( e09/224 ) were sorbitol fermenting ( sf ) and beta - glucuronidase positive , as was the sf stec control strain e09/10 ( see table s1 in the supplemental material ) . among the nsf stec isolates , phenotypic and molecular characteristics of the isolates nsf , non - sorbitol fermenting ; sf , sorbitol fermenting ; nm , nonmotile . the type of the eae gene was determined from wgs data using blastn . one of these four isolates was obtained from germany and used as a control strain for the stx - negative o157:nm isolates . stx subtyping of stec isolates revealed eight ( 50% ) isolates with stx1a and stx2c subtypes , two ( 12.5% ) with stx1a and stx2a subtypes , and six ( 37.5% ) with the stx2c subtype only . all nsf stec isolates had identical virulence profiles , with exception of isolate stec 1109 , which contained the cdt - v gene , encoding cytolethal distending toxin . some of the virulence genes , such as serine protease autotransporter - encoding gene espp , toxin - encoding gene toxb , catalase peroxidase - encoding gene katp , the tellurite resistance- and adherence - conferring island ( tai ) , and the urease gene cluster , were present only in nsf stec and in the only nsf epec isolate but not in any sf isolates . the sf epec isolates carried almost all the virulence genes carried by the german sf stec o157:nm isolate , with the exception of the tccp gene ( encoding tir cytoskeletal coupling protein ) , which was present only in sf epec isolates . distribution of virulence and other genes among stx - positive and stx - negative o157:h7/nm isolates encoding cytolethal distending toxin a , b , and c subunits . all the stec isolates contained an lee region highly similar to the lee of stec o157:h7 strain 71074 , used as a reference . however , some nsf stec isolates lacked two genes encoding the mobile element proteins orfa and orfb located in the insertion sequence is911 . three stec isolates ( stec 2257 , stec 2820 , and stec 2821 ) did not possess the intl gene , which is known to encode an integrase of the putative prophage 933l carried in the lees of other stec o157:h7 isolates . the sequences of the lees of our nsf and sf epec isolates were more similar to those of nsf and sf stec , respectively , than to the lee of epec reference genomes e2348/69 and c639_08 ( fig . however , with the exception of the two stx2a - positive isolates ( stec 2112 and stec 2868 ) , all stec isolates lacked the po157p35 gene , encoding a reverse transcriptase . only three stec isolates ( stec 605 , stec 989 , and stec 1109 ) harbored the plasmid posak1 . core genome phylogenetic analysis was performed to evaluate the evolutionary relationship between the stx - positive ( stec ) and stx - negative ( epec ) o157:h7/nm isolates . in total , 3,005 orfs were shared by all isolates analyzed in this study , and these were defined as the core genome for phylogenetic analysis . remarkably , in cluster 1 , four sf epec isolates ( epec 393 , epec 1572 , epec 1669 , and e09/224 ) clustered together with the sf stec isolate . cluster 2 ( nsf o157:h7 isolates ) could be divided into three subclusters : cluster 2a , containing stec o157:nm ( nonmotile ) isolates together with one nsf epec isolate ( epec 287 ) ; cluster 2b , containing six of the motile stec o157:h7 isolates ; and cluster 2c , containing two stx2a - positive isolates ( stec 2112 and stec 2868 ) clustered closely with two previously described stec outbreak isolates , sakai and edl933 ( 1 , 32 ) . the last subcluster also included two other motile isolates ( stec 1109 and stec 989 ) and stec o157:h7 strain ss52 ( stx2a and stx2c positive ) , isolated from super shedder cattle ( 33 ) . taken together , the data indicate that the epec o157:nm isolates clustered with stec isolates but not with epec isolates ( fig . in the sf epec strains ( epec 393 , epec 1572 , epec 1669 , and epec e09/224 ) , the phage insertion sites analyzed were intact , with the exception of argw , which was occupied in isolates epec 393 and epec 1572 . a red arrow indicates the presence of a phage integrase adjacent to the integration site . e. coli o157:h7 is one of the major causes of foodborne illness and represents a considerable public health concern worldwide ( 34 ) . this study aimed at determining the phylogenetic relationships and comparing the virulence factors of stx - negative e. coli o157:h7/nm with those of stx - positive e. coli o157:h7 with the highest resolution and greatest possible detail using a wgs approach . the e. coli o157:h7 isolates used in this study were obtained from patients with diarrhea and other gastrointestinal complaints from two different regions in the netherlands ( groningen and rotterdam ) . isolates e09/10 and e09/224 were sequenced and used as control strains for sf stec o157:nm and stx - negative o157:nm isolates , respectively . as the stx - negative isolates were found to be positive for eae but negative for the bfpa gene , they were considered atypical epec . , ehxa , asta , lpfa , katp , etpd , espp , the pathogenicity island tai , and the urease gene cluster ) of nsf stec o157:h7 described previously were present in epec 287 . in contrast , sf epec isolates possessed the sfp gene cluster , the cdt gene cluster , and the complete efa1 gene but lacked the genes toxb , katp , espp carried on plasmid po157 , the urease gene cluster , and the pathogenicity island tai , which are typical features of sf stec o157:nm ( 18 , 29 ) . subsequent analyses showed that epec 287 harbored a po157 plasmid almost identical to that of the stec isolates , with sequence variability mostly in mobile genetic elements . the sf epec isolates contained a plasmid similar to the psfo157 present in the sf stec o157:nm isolate strain 3072/96 . all these results together suggest that the epec o157:h7/nm isolates investigated shared a virulence profile similar to that of stec isolates rather than to the virulence profile of epec . the genetic relationship of the o157:h7/nm isolates of this study was confirmed by the phylogenetic analysis using a gene - by - gene comparison by core genome mlst . the nsf epec isolate clustered together with the nonmotile nsf stec isolates , and the three sf epec isolates clustered together with the control strain of sf stec o157:nm . it has already been described that the nonmotile characteristic of sf stec o157 is due to a 12-bp deletion in the master regulator of flagellar biosynthesis , the flhc gene ( 37 ) . we also observed this deletion in the same gene in our nonmotile sf epec isolates but not in our nonmotile nsf stec isolates , further strengthening the hypothesis of their derivation from stx - positive sf o157:nm . taken together , the results of our analyses provide further evidence that the nsf and sf epec isolates were mostly related to the stec group , but lacking the stx - converting phages , and might be referred to as ehec strains that have lost the shiga toxin ( ehec - lst ) , as has also been proposed previously ( 19 , 20 ) . studying in more detail the typical stx phage insertion sites ( yece for sf epec and wrba and sbcb for nsf epec ) revealed that they were unoccupied in the stx - negative isolates . such unoccupied regions could be an indication of loss of the stx bacteriophage , e.g. conversely , stx - negative strains could be progenitors of stec prepared to acquire one or more stx - converting phages ( 18 , 39 ) . in our case , the stx genes were not lost in the laboratory during isolation or subculture , as they were not detected in the feces of the patients . therefore , the patients may have been infected with an stx - negative variant , which could explain the mild symptoms they displayed . in routine diagnostic testing , these stx - negative isolates may be missed , as most microbiological laboratories depend on the molecular screening of stec by the detection of the stx genes only . moreover , our findings bring into question whether classifying pathogenic e. coli into stec and epec based on detection of the stx and eae genes , respectively , is reliable enough . , the stx - converting bacteriophages , which could integrate into several e. coli pathogroups , it is sometimes complicated to precisely define the classification of pathogenic e. coli ( 40 , 41 ) . in our opinion , screening of at least the flich7 gene , together with the o157-encoding gene , may help to identify ehec - lst of the most virulent clone of ehec ( o157:h7 ) , as all the flich7-positive isolates were genetically related to stec o157:h7 in this study . our results are consistent with the finding that the proportion of stx - negative variants among sf o157:nm isolates is generally higher than that among nsf o157:h7 isolates ( 20 , 42 ) . however , to our best knowledge , this is the first report where the genetic relationship of stx - negative variants of e. coli o157:h7/nm with stx - positive o157:h7/nm has been confirmed using wgs . in conclusion , stx - negative e. coli o157:h7/nm strains are a cause of gastrointestinal disease in the netherlands , and because of the presence of the complete set of accessory virulence genes , these isolates should be considered of public health concern similar to that for their stx - positive variants .

despite several decades of research efforts devoted to studying the patterns of chronic kidney disease - end stage renal disease ( ckd - esrd ) progression as well as the impact of acute kidney injury ( aki ) on this continuum of ckd - esrd evolution , a full understanding of the process ( es ) of ckd - esrd progression remains elusive . the common consensus of a predictable , linear , progressive , relentless and time - dependent decline in renal function , with predictably increasing serum creatinine values over time leading inexorably to esrd is widely accepted . aki on ckd is a well - recognized phenomenon , but again this is usually assumed to be transient with usual expected recovery of renal function or at the worst , some loss of residual renal function . we have observed and documented acute yet irreversible renal failure in several ckd patients leading to esrd . subsequently , we prospectively investigated this syndrome of acutely rapid onset yet irreversible esrd further in a 100-patient high - risk ckd cohort , the syndrome of rapid onset esrd ( soro - esrd ) . this accelerated progression to esrd from a - priori stable ckd was precipitated by aki resulting from medical and surgical events that had then quickly and directly led to established irreversible esrd without any renal recovery . of the 15 patients with soro - esrd first described in 2010 , mean age was 68 years , 9 of the 15 ( 60% ) patients were aged 65 years and older and 6 of the 15 ( 40% ) patients were aged 80 years or older . these observations suggested that this syndrome was more common in the older adult ckd patient and that such acute yet irreversible esrd may be related to the changes that occur concurrently in the ageing kidney , otherwise described as renal senescence . since this 100 patient cohort was a high - risk ckd cohort because they were recruited into an angiotensin inhibition withdrawal study after demonstrating worsening renal failure while on concurrent angiotensin inhibition , the question had remained unanswered as to what extent this soro - esrd phenomenon pertains to the general incident united states ( us ) esrd population . from the foregoing therefore , in june 2011 , we investigated the serum creatinine trajectories in consecutive 100 adult esrd patients undergoing out - patient in - center maintenance hemodialysis in a mayo clinic dialysis practice to evaluate the incidence of soro - esrd in a general us esrd population . this is a retrospective analysis of individual patient - level serum creatinine trajectories of the last consecutive 100 adult ( 18 years and older ) esrd patients undergoing out - patient in - center maintenance hemodialysis in four northwestern wisconsin mayo clinic hemodialysis units . we analyzed , in june 2011 , the serum creatinine trajectories of the last 100 consecutive adult esrd patients on rrt for 90 days in four northwestern wisconsin mayo clinic hemodialysis units . the esrd patients were on maintenance out - patient in - center hemodialysis in four mayo clinic hemodialysis units located in the following cities - eau claire , wisconsin , menomonie , wisconsin and barron , wisconsin . they were seen and managed in these four mayo clinic hemodialysis units between january 2010 and february 2011 . we have defined soro - esrd as the unpredictable , unanticipated and accelerated progression from a - priori stable ckd to irreversible esrd , all occurring in one continuum of time without renal recovery in between , requiring permanent rrt , following a new episode of aki , caused by antecedent new medical / surgical events , with the need for rrt usually occurring within 6 - 12 weeks of the aki episode . our working definition of soro - esrd is any patient with an estimated glomerular filtration rate ( egfr ) of 30 ml / min/1.73 m , on or before the 90 day preceding initiation of first rrt for renal failure and who thereafter had remained permanently on rrt for over 90 days and beyond without renal recovery , indefinitely . due to incomplete serum creatinine data , 9 patients were excluded from the analysis . the remaining 91 esrd patients included thirty - one of the 91 ( 34% ) esrd patients satisfied the diagnosis of soro - esrd [ figure 1 ] . these 31 soro - esrd patients included 18 males and 13 females , mean age 72 ( 50 - 92 ) years . this soro - esrd group of 31 patients also included two renal transplant recipients ( rtrs ) [ figure 2 ] . for all 31 soro - esrd patients , the abrupt unanticipated and irreversible soro - esrd followed aki event or events resulting from the following causes - pneumonia , acutely decompensated heart failure , pyelonephritis , post - operative states , general sepsis , contrast - induced nephropathy and others . furthermore , the time interval between the precipitating antecedent aki event and the resulting need for rrt was relatively shorter following surgical aki , when compared with medical causes of aki - usually within days to 1 week , following cardiac surgery [ figure 1 bottom ] . the 31 soro - esrd patients were older compared with the remaining 60 esrd patients who were characterized by the more traditionally accepted pattern of a slow progressive time - dependent loss of egfr over time - 71 12 ( 49 - 91 ) years versus 69 13 , ( 38 - 93 ) p ns . incidentally , we observed that 7 of 31 ( 23% ) soro - esrd patients were concurrently on renin angiotensin aldosterone system ( raas ) blockade at the time of the diagnosis of aki versus 3 of 60 ( 5% ) without soro - esrd , t ( 89 ) = 2.587 , p = 0.0113 . composite figure showing the classic chronic kidney disease - end stage renal disease ( esrd ) progression pattern of serum creatinine trajectory ( top frame ) versus the serum creatinine trajectory pattern in another patient with syndrome of rapid onset - esrd ( bottom frame ) trajectory of epidermal growth factor receptor changes in a renal transplant recipient who developed syndrome of rapid onset - end stage renal disease in january 2011 and who subsequently had a second living - related kidney re - transplantation in january 2012 from the son retrospective analysis demonstrated that in all the patients where infections triggered aki , including pneumonia in 8 , pyelonephritis in 4 , and generalized sepsis in 2 , they all responded to appropriate systemic antimicrobial therapy that was initially broad spectrum and was subsequently adjudicated by culture sensitivity results where applicable . post - aki , in subsequent follow - up , blood pressure control and blood sugar control among the diabetic patients were generally adequate . in patients on concurrent angiotensin inhibition at presentation , this was promptly discontinued , and where applicable , antihypertensive substitution with calcium channel blockers and vasodilators was the practice as we have described previously . we conclude that soro - esrd is not uncommon among the incident us esrd population as characterized in this mayo clinic northwestern wisconsin out - patient in - center chronic hemodialysis population . this retrospective analysis of the individual patient - level serum creatinine trajectories of the last 100 incident adult esrd patients from four northwestern wisconsin mayo clinic hemodialysis units demonstrated an incidence rate of soro - esrd of 34% , about a third of the esrd population . our recent review of the aki literature , 1975 - 2010 , had unearthed 16 individual aki reports that described patients with features consistent with our working diagnosis of soro - esrd . emphatically , an accompanying editorial to one of these 16 studies , published in the quarterly journal of medicine in 1996 , had referred to similar observations of esrd rapidly following aki in patients seen at the general infirmary at leeds , united kingdom as acute irreversible renal failure . we have concluded that this indeed was a very apt description of the syndrome after which we had coined the new name , the syndrome of rapid onset end stage renal disease , or soro - esrd , in 2010 . we suspect that irreversible terminal acute tubular necrosis was the most likely cause of non - recovery in most of our 31 soro - esrd patients . this hypothesis is based on clinical observations as related to paucity of urinalysis findings as well as renal sonographic imaging , where available . renal biopsy was carried out in only one patient , a rtr and the renal pathology demonstrated acute tubular necrosis , changes of chronic glomerulopathy , but without rejection . it is important to acknowledge here that in all 31 patients who developed soro - esrd in this study , the kidney function prior to the aki insult that precipitated the acute yet irreversible esrd was otherwise stable . the examination of individual patient - level serum creatinine trajectories of the 31 soro - esrd patients demonstrated that serum creatinine values were indeed stable and that egfr was not declining , prior to the onset of aki and subsequent development of soro - esrd . such data from four of the 31 patients are shown in the composite figure and it is evident that up until prior to the aki event that precipitated soro - esrd , serum creatinine and therefore egfr was stable and not changing [ figure 3 ] . these findings are consistent with our working diagnosis of soro - esrd as rapid unanticipated irreversible esrd following rapidly on aki in otherwise a - priori stable ckd patients . composite figure showing stable patterns of pre - acute kidney injury serum creatinine trajectories in 4 of the 31 syndrome of rapid onset end stage renal disease ( soro - esrd ) patients , a pattern that is applicable to all 31 soro - esrd patients most importantly , in the more recent nephrology literature , 2011 - 2012 , we have further identified three corroborating new reports that have further substantiated our recent reports of soro - esrd . these three new reports have each further demonstrated that a significant proportion of the incident adult esrd population in both the us and canada , respectively , satisfy the diagnostic criteria for our newly described soro - esrd . in a 2011 report , lee et al . , studied all consecutive patients initiated on maintenance hemodialysis or peritoneal dialysis over several years at two dialysis units . according to the investigators , rapid decline in kidney function to esrd was considered to have occurred if a patient was documented to have estimated gfr > 30 ml / min/1.73 m within 3 months prior to the initiation of chronic dialysis . incidentally , we must observe here that their definition very closely mirrored our standard definition of soro - esrd . lee et al . , revealed that 8 of 105 incident chronic dialysis patients in one dialysis unit ( 7.6% ; 95% confidence interval [ ci ] 3.4%-14.5% ) and 9 of 71 incident patients at another dialysis unit ( 12.7% , 95% ci 6.0%-22.7% ) , suffered rapid decline in kidney function that was the immediate precipitant for the need for permanent renal rrt these observations translate to a soro - esrd incident rate in this northern california hemodialysis unit of 15 of 176 or 9% . it is noteworthy to observe that all these patients with rapid irreversible esrd or soro - esrd were started on hemodialysis for rrt , and that all 15 had relied on hemodialysis catheters for initial vascular access . the authors of this report also observed that the patient - level data documentation submitted to the united states renal data system ( usrds ) did not fully reflect the health status of these patients during their pre - esrd period . as we have continued to argue , the absence of patient - level serum creatinine trajectories in the usrds database would not allow for an analysis of the usrds database to estimate the nation - wide incidence of this soro - esrd in our incident us esrd population . this deficiency in the usrds database had informed the recent paradigm whereby some eminent nephrologists from around the world , including the us , south america , europe and the united kingdom , africa including nigeria and asia and the indian subcontinent , recently met in vancouver , canada , in 2011 , at the world congress of nephrology annual meeting and established the soro - esrd world - wide consortium to further investigate this newly described syndrome on a world - wide platform . analyzed the trajectories of egfr during the 2-year period before dialysis initiation in 5,606 veterans affairs patients who initiated long - term dialysis between 2001 and 2003 . in this report , 9.5% of the esrd patients had accelerated loss of egfr from levels > 60 ml / min/1.73 m ( mean egfr slope , 32.3 13.4 ml / min/1.73 m per year ) and another 3.1% experienced catastrophic loss of egfr from levels > 60 ml / min/1.73 m within 6 months or less to reach irreversible esrd . the authors of this report had concluded that there was substantial heterogeneity in patterns of kidney function loss leading up to the initiation of long - term dialysis , perhaps calling for a more flexible approach toward preparing for esrd . our post - hoc analysis of this seattle report demonstrated a soro - esrd incidence rate in this population of 5,606 veterans affairs esrd patients of 12.6% . in addition , in a 2012 canadian report , siddiqui et al . , had described the increasing use of acute dialysis after cardiac surgery in the period 1995 to 2009 and had demonstrated that the incidence of acute dialysis had increased steadily from 0.2% in 1995 ( 95% ci 0.15 - 0.2 ) to 0.6% in 2009 ( 95% ci 0.6 - 0.7 ) . moreover , this study had validated that among the 1294 patients who received acute dialysis and survived beyond 90 days , 352 patients subsequently required chronic dialysis ( 27.2% , 95% ci 24.8 - 29.7 ) . we have deduced , from a post - hoc analysis of this study that the majority of the cohort of 352 patients requiring chronic dialysis following post - operative aki had developed soro - esrd , giving a soro - esrd incidence rate of 16% . we conclude that soro - esrd is not uncommon among incident adult esrd patients on maintenance hemodialysis in the us and in canada . soro - esrd accounted for 34% of the incident esrd patients in a mayo clinic chronic esrd population , 9% of a northern california incident esrd population , 12.6% of the seattle washington veterans the implications of this phenomenon of the soro - esrd with regards to esrd care planning , av fistula first programs and overall ckd care in general , are huge and warrant further study . for example , in the northern california study , all 15 patients with soro - esrd had started hemodialysis using hemodialysis catheters because there was no time for arteriovenous fistula ( avf ) planning in the first instance . if soro - esrd is shown to be this prevalent among the general incident us ( and world - wide ) esrd population in multi - center studies , major paradigm shifts must be warranted in the way we practice nephrology both here in the us and around the world . maybe , as suggested by lee et al . , the usrds may begin to request for more pre - esrd patient - level data from submitting nephrologists . we must acknowledge here that the impact of aki in ckd - esrd progression and pathogenesis remains fertile grounds for ground - breaking research . furthermore , subgroup analysis of the 31 soro - esrd patients versus the 60 who developed classic esrd revealed that 9 of 31 ( 29% ) soro - esrd patients were 80 years and older . this was not any different from the 17 of 60 ( 28% ) patients who developed classic esrd . the only evident different characteristics observed between these two sub - groups of 80 and over was the higher exposure to angiotensin inhibition in the soro - esrd group ( 4 of 9 [ 44% ] vs. 1 of 17 [ 6% ] , 2 tailed z - test , p = 0.019 ) . overall , 7 of 31 ( 23% ) soro - esrd patients were concurrently on raas blockade at the time of the diagnosis of aki versus 3 of 60 ( 5% ) without soro - esrd , t ( 89 ) = 2.587 , p = 0.0113 . the plausible contribution of older age and the role of exposure to nephrotoxics , potentially including angiotensin converting enzyme ( ace ) inhibitors and angiotensin receptor blockers , to the incidence of soro - esrd remain speculative at this point and require further study . from this analysis , we submit that there is some plausible role for nephrotoxics including raas blockade in the exacerbation of renal outcomes with aki on ckd . we would submit that our findings from this study only further strengthen our previous calls for more research efforts into preventative nephrology practices , especially our recently described the concept of renoprevention . renoprevention is the preemptive withholding of nephrotoxics including raas blockers , the aggressive prevention of perioperative hypotension , the avoidance or minimalization of nephrotoxic exposure from iodinated contrast and relevant antibiotics , during critical illness and in the peri - operative period . such a paradigm , it is hoped , will consequently lead to less aki events and therefore potentially less soro - esrd , better patient outcomes and significant dollar savings as we have demonstrated in a recent analysis in the critical care unit of a northwestern wisconsin mayo clinic health system hospital . such needed paradigm shifts would constitute major rethinking in current nephrology practices , a form of nephrology practice reengineering . besides , the possible interplay of renal senescence and changes in the renal anatomy with ageing such as senile hypofiltration ( glomerulosclerosis , mesangial expansion ) and renal vascular changes ( renal atherosclerosis , vascular dysautonomy , arteriole subendotelial hyalinosis , aglomerular circulation ) and the pathogenesis of this soro - esrd in the older ( > 65 year old ) adult ckd patient is of major research interest to our group . the fact that in our first report of soro - esrd in 2010 , older age appeared to be a significant pathogenetic factor in the development of the syndrome is again acknowledged . the finding in this study that the 31 soro - esrd patients versus the remaining 60 classic esrd patients were older , further strengthened this hypothesis of renal ageing and renal senescence being risk factors for this previously unrecognized syndrome . these observations call for more research in these directions and may imply that the older ckd patient may necessarily be treated differently especially with respect to the use of certain potentially nephrotoxic agents including the raas blocking agents . we have posited the question : does the older ckd adult patient demand to be treated any differently from the younger ckd patient ? there are several plausible explanations that could justify why soro - esrd is commoner among our older or aged patients . on one hand , there are several structural and physiological changes in the aged kidney , such as senile renal blood flow reduction , artery atherosclerosis ( renal arterial stenosis ) and vascular dysautonomia ( impaired vascular autoregulation ) , which can worsen renal parenchymal ischemia when the aged patient is exposed to hypovolemia ( dehydration ) or other renal insults ( sepsis , cardiac failure ) . besides , gfr reduction secondary to senescence ( senile glomerulosclerosis ) , could represent a risk of installing soro - esrd in states of hemodynamic instability , particularly in the oldest old . moreover , aging tubular cells may be more vulnerable to ischemic injury because cellular antioxidant defenses decline with age and oxidant injury may be a critical determinant of ischemic acute renal failure . what 's more , it was also documented that an increased propensity to vasoconstriction may enhance susceptibility of old kidney to toxic substances . this senile tubular frailty predisposes the aged kidney to easily develop acute tubular necrosis , as well as to delay its functional recovery , thus predisposing to the prolonged acute tubular necrosis , or even non - recovery that is usually observed during acute renal failure in elderly patients . furthermore , renal and hepatic senescence , as well as polypharmacy make old people more susceptible to develop severe acute renal failure when they receive potential nephrotoxic substances such as non - steroidal anti - inflammatory or radio - contrast , which for different reasons are more frequently prescribed in this age group . regarding ace inhibitors and angiotensin receptor blockers , even though these drugs are widely used for renoprotection in ckd , it must be taken into account that they can induce acute renal failure in those patients who suffer from disorders , prevalent in the elderly , in which maintenance of gfr is highly dependent on an angiotensin ii - mediated efferent vasoconstriction . this is true for patients with bilateral renal artery stenosis , renal artery stenosis in a solitary kidney , congestive heart failure and severe renal failure , especially when they are volume depleted . finally , several causes of rapidly progressive glomerulopathies , which are frequent among the older patient , such as crescentic glomerulonephritis , systemic vasculitis , atheroembolic disease , could be another causal mechanism of soro - esrd in this older population . lastly , the fact that 2 of the 31 ( 6% ) of the soro - esrd patients in our present study were rtrs raises another significant concern to what extent is renal allograft loss attributable to this soro - esrd ? . the full details of the presentations of these two rtrs who developed soro - esrd , one of whom was re - transplanted in january 2012 , a year after developing soro - esrd , with a living related renal allograft from her 32-year old son at mayo clinic , rochester , the first such report of soro - esrd among rtrs , has just been published in this journal , the indian journal of nephrology , in 2013 . these unanswered questions call for further research and urgent answers . in closing , we must observe that despite decades of painstaking research into the dynamics , processes and mechanisms of ckd - esrd propagation and progression , the medical community remains at a loss in understanding the nuances of these translations . the common ckd staging classification represents an assumed and arguably untested paradigm that in our opinion , had certainly increased awareness of ckd , without truly advancing our knowledge in this field of medicine , nor have we been able to make any major break - through achievements in improving ckd outcomes . tragically , this approach may be counter - productive as it continues to buttress the notion that ckd is a homogenous clinical entity and that all ckd is equal . we suggest a complete reappraisal of current nephrology practices and to begin to develop new models of ckd care that correctly recognize the diversity of ckd as representative of a wide spectrum of disease states . most appropriately , bansal and hsu , in a 2008 analysis of the long - term outcomes of patients with ckd had strongly echoed the observation that the disparate esrd and mortality rates in various ckd populations as reported by various studies in the literature only emphasized the heterogeneity of different ckd populations . nephrologists must not rely on ckd staging alone to direct management of or risk - stratification of patients with ckd , in general , but must always consider the etiology and rate of progression of kidney disease , patient age and a wide array of cardiovascular disease risk factors . the overarching need to always individualize ckd care can not be overemphasized as ckd represents a whole wide spectrum of distinctly different clinical disease entities , with each individual patient often subject to a multitude of aggravating factors , some of which often remain unrecognized . this is in fact how we practice medicine - one patient and only one patient at a time .

despite decades of research , a full understanding of chronic kidney disease ( ckd)-end stage renal disease ( esrd ) progression remains elusive . the common consensus is a predictable , linear , progressive and time - dependent decline of ckd to esrd . acute kidney injury ( aki ) on ckd is usually assumed to be transient , with recovery as the expected outcome . aki - esrd association in current nephrology literature is blamed on the so - called residual confounding . we had previously described a relationship between aki events and rapid onset yet irreversible esrd happening in a continuum in a high - risk ckd cohort . however , the contribution of the syndrome of rapid onset - esrd ( soro - esrd ) to incident united states esrd population remained conjectural . in this retrospective analysis , we analyzed serum creatinine trajectories of the last 100 consecutive esrd patients in 4 mayo clinic chronic hemodialysis units to determine the incidence of soro - esrd . excluding 9 patients , 31 ( 34% ) patients , including two renal transplant recipients , had soro - esrd : 18 males and 13 females age 72 ( range 50 - 92 ) years . precipitating aki followed pneumonia ( 8) , acutely decompensated heart failure ( 7 ) , pyelonephritis ( 4 ) , post - operative ( 5 ) , sepsis ( 3 ) , contrast - induced nephropathy ( 2 ) , and others ( 2 ) . time to dialysis was shortest following surgical procedures . concurrent renin angiotensin aldosterone system blockade was higher with soro - esrd - 23% versus 5% , p = 0.0113 . in conclusion , soro - esrd is not uncommon among the incident general us esrd population . the implications for esrd care planning , av - fistula - first programs , general ckd care and any associations with renal ageing / senescence warrant further study .

Introduction Materials and Methods Results Discussion

despite several decades of research efforts devoted to studying the patterns of chronic kidney disease - end stage renal disease ( ckd - esrd ) progression as well as the impact of acute kidney injury ( aki ) on this continuum of ckd - esrd evolution , a full understanding of the process ( es ) of ckd - esrd progression remains elusive . the common consensus of a predictable , linear , progressive , relentless and time - dependent decline in renal function , with predictably increasing serum creatinine values over time leading inexorably to esrd is widely accepted . aki on ckd is a well - recognized phenomenon , but again this is usually assumed to be transient with usual expected recovery of renal function or at the worst , some loss of residual renal function . we have observed and documented acute yet irreversible renal failure in several ckd patients leading to esrd . subsequently , we prospectively investigated this syndrome of acutely rapid onset yet irreversible esrd further in a 100-patient high - risk ckd cohort , the syndrome of rapid onset esrd ( soro - esrd ) . of the 15 patients with soro - esrd first described in 2010 , mean age was 68 years , 9 of the 15 ( 60% ) patients were aged 65 years and older and 6 of the 15 ( 40% ) patients were aged 80 years or older . these observations suggested that this syndrome was more common in the older adult ckd patient and that such acute yet irreversible esrd may be related to the changes that occur concurrently in the ageing kidney , otherwise described as renal senescence . since this 100 patient cohort was a high - risk ckd cohort because they were recruited into an angiotensin inhibition withdrawal study after demonstrating worsening renal failure while on concurrent angiotensin inhibition , the question had remained unanswered as to what extent this soro - esrd phenomenon pertains to the general incident united states ( us ) esrd population . from the foregoing therefore , in june 2011 , we investigated the serum creatinine trajectories in consecutive 100 adult esrd patients undergoing out - patient in - center maintenance hemodialysis in a mayo clinic dialysis practice to evaluate the incidence of soro - esrd in a general us esrd population . this is a retrospective analysis of individual patient - level serum creatinine trajectories of the last consecutive 100 adult ( 18 years and older ) esrd patients undergoing out - patient in - center maintenance hemodialysis in four northwestern wisconsin mayo clinic hemodialysis units . we analyzed , in june 2011 , the serum creatinine trajectories of the last 100 consecutive adult esrd patients on rrt for 90 days in four northwestern wisconsin mayo clinic hemodialysis units . the esrd patients were on maintenance out - patient in - center hemodialysis in four mayo clinic hemodialysis units located in the following cities - eau claire , wisconsin , menomonie , wisconsin and barron , wisconsin . they were seen and managed in these four mayo clinic hemodialysis units between january 2010 and february 2011 . we have defined soro - esrd as the unpredictable , unanticipated and accelerated progression from a - priori stable ckd to irreversible esrd , all occurring in one continuum of time without renal recovery in between , requiring permanent rrt , following a new episode of aki , caused by antecedent new medical / surgical events , with the need for rrt usually occurring within 6 - 12 weeks of the aki episode . our working definition of soro - esrd is any patient with an estimated glomerular filtration rate ( egfr ) of 30 ml / min/1.73 m , on or before the 90 day preceding initiation of first rrt for renal failure and who thereafter had remained permanently on rrt for over 90 days and beyond without renal recovery , indefinitely . due to incomplete serum creatinine data , 9 patients were excluded from the analysis . the remaining 91 esrd patients included thirty - one of the 91 ( 34% ) esrd patients satisfied the diagnosis of soro - esrd [ figure 1 ] . these 31 soro - esrd patients included 18 males and 13 females , mean age 72 ( 50 - 92 ) years . this soro - esrd group of 31 patients also included two renal transplant recipients ( rtrs ) [ figure 2 ] . for all 31 soro - esrd patients , the abrupt unanticipated and irreversible soro - esrd followed aki event or events resulting from the following causes - pneumonia , acutely decompensated heart failure , pyelonephritis , post - operative states , general sepsis , contrast - induced nephropathy and others . furthermore , the time interval between the precipitating antecedent aki event and the resulting need for rrt was relatively shorter following surgical aki , when compared with medical causes of aki - usually within days to 1 week , following cardiac surgery [ figure 1 bottom ] . the 31 soro - esrd patients were older compared with the remaining 60 esrd patients who were characterized by the more traditionally accepted pattern of a slow progressive time - dependent loss of egfr over time - 71 12 ( 49 - 91 ) years versus 69 13 , ( 38 - 93 ) p ns . incidentally , we observed that 7 of 31 ( 23% ) soro - esrd patients were concurrently on renin angiotensin aldosterone system ( raas ) blockade at the time of the diagnosis of aki versus 3 of 60 ( 5% ) without soro - esrd , t ( 89 ) = 2.587 , p = 0.0113 . composite figure showing the classic chronic kidney disease - end stage renal disease ( esrd ) progression pattern of serum creatinine trajectory ( top frame ) versus the serum creatinine trajectory pattern in another patient with syndrome of rapid onset - esrd ( bottom frame ) trajectory of epidermal growth factor receptor changes in a renal transplant recipient who developed syndrome of rapid onset - end stage renal disease in january 2011 and who subsequently had a second living - related kidney re - transplantation in january 2012 from the son retrospective analysis demonstrated that in all the patients where infections triggered aki , including pneumonia in 8 , pyelonephritis in 4 , and generalized sepsis in 2 , they all responded to appropriate systemic antimicrobial therapy that was initially broad spectrum and was subsequently adjudicated by culture sensitivity results where applicable . we conclude that soro - esrd is not uncommon among the incident us esrd population as characterized in this mayo clinic northwestern wisconsin out - patient in - center chronic hemodialysis population . this retrospective analysis of the individual patient - level serum creatinine trajectories of the last 100 incident adult esrd patients from four northwestern wisconsin mayo clinic hemodialysis units demonstrated an incidence rate of soro - esrd of 34% , about a third of the esrd population . our recent review of the aki literature , 1975 - 2010 , had unearthed 16 individual aki reports that described patients with features consistent with our working diagnosis of soro - esrd . we have concluded that this indeed was a very apt description of the syndrome after which we had coined the new name , the syndrome of rapid onset end stage renal disease , or soro - esrd , in 2010 . we suspect that irreversible terminal acute tubular necrosis was the most likely cause of non - recovery in most of our 31 soro - esrd patients . it is important to acknowledge here that in all 31 patients who developed soro - esrd in this study , the kidney function prior to the aki insult that precipitated the acute yet irreversible esrd was otherwise stable . the examination of individual patient - level serum creatinine trajectories of the 31 soro - esrd patients demonstrated that serum creatinine values were indeed stable and that egfr was not declining , prior to the onset of aki and subsequent development of soro - esrd . such data from four of the 31 patients are shown in the composite figure and it is evident that up until prior to the aki event that precipitated soro - esrd , serum creatinine and therefore egfr was stable and not changing [ figure 3 ] . these findings are consistent with our working diagnosis of soro - esrd as rapid unanticipated irreversible esrd following rapidly on aki in otherwise a - priori stable ckd patients . composite figure showing stable patterns of pre - acute kidney injury serum creatinine trajectories in 4 of the 31 syndrome of rapid onset end stage renal disease ( soro - esrd ) patients , a pattern that is applicable to all 31 soro - esrd patients most importantly , in the more recent nephrology literature , 2011 - 2012 , we have further identified three corroborating new reports that have further substantiated our recent reports of soro - esrd . these three new reports have each further demonstrated that a significant proportion of the incident adult esrd population in both the us and canada , respectively , satisfy the diagnostic criteria for our newly described soro - esrd . according to the investigators , rapid decline in kidney function to esrd was considered to have occurred if a patient was documented to have estimated gfr > 30 ml / min/1.73 m within 3 months prior to the initiation of chronic dialysis . incidentally , we must observe here that their definition very closely mirrored our standard definition of soro - esrd . , revealed that 8 of 105 incident chronic dialysis patients in one dialysis unit ( 7.6% ; 95% confidence interval [ ci ] 3.4%-14.5% ) and 9 of 71 incident patients at another dialysis unit ( 12.7% , 95% ci 6.0%-22.7% ) , suffered rapid decline in kidney function that was the immediate precipitant for the need for permanent renal rrt these observations translate to a soro - esrd incident rate in this northern california hemodialysis unit of 15 of 176 or 9% . it is noteworthy to observe that all these patients with rapid irreversible esrd or soro - esrd were started on hemodialysis for rrt , and that all 15 had relied on hemodialysis catheters for initial vascular access . the authors of this report also observed that the patient - level data documentation submitted to the united states renal data system ( usrds ) did not fully reflect the health status of these patients during their pre - esrd period . as we have continued to argue , the absence of patient - level serum creatinine trajectories in the usrds database would not allow for an analysis of the usrds database to estimate the nation - wide incidence of this soro - esrd in our incident us esrd population . this deficiency in the usrds database had informed the recent paradigm whereby some eminent nephrologists from around the world , including the us , south america , europe and the united kingdom , africa including nigeria and asia and the indian subcontinent , recently met in vancouver , canada , in 2011 , at the world congress of nephrology annual meeting and established the soro - esrd world - wide consortium to further investigate this newly described syndrome on a world - wide platform . in this report , 9.5% of the esrd patients had accelerated loss of egfr from levels > 60 ml / min/1.73 m ( mean egfr slope , 32.3 13.4 ml / min/1.73 m per year ) and another 3.1% experienced catastrophic loss of egfr from levels > 60 ml / min/1.73 m within 6 months or less to reach irreversible esrd . our post - hoc analysis of this seattle report demonstrated a soro - esrd incidence rate in this population of 5,606 veterans affairs esrd patients of 12.6% . , had described the increasing use of acute dialysis after cardiac surgery in the period 1995 to 2009 and had demonstrated that the incidence of acute dialysis had increased steadily from 0.2% in 1995 ( 95% ci 0.15 - 0.2 ) to 0.6% in 2009 ( 95% ci 0.6 - 0.7 ) . we have deduced , from a post - hoc analysis of this study that the majority of the cohort of 352 patients requiring chronic dialysis following post - operative aki had developed soro - esrd , giving a soro - esrd incidence rate of 16% . we conclude that soro - esrd is not uncommon among incident adult esrd patients on maintenance hemodialysis in the us and in canada . soro - esrd accounted for 34% of the incident esrd patients in a mayo clinic chronic esrd population , 9% of a northern california incident esrd population , 12.6% of the seattle washington veterans the implications of this phenomenon of the soro - esrd with regards to esrd care planning , av fistula first programs and overall ckd care in general , are huge and warrant further study . for example , in the northern california study , all 15 patients with soro - esrd had started hemodialysis using hemodialysis catheters because there was no time for arteriovenous fistula ( avf ) planning in the first instance . if soro - esrd is shown to be this prevalent among the general incident us ( and world - wide ) esrd population in multi - center studies , major paradigm shifts must be warranted in the way we practice nephrology both here in the us and around the world . , the usrds may begin to request for more pre - esrd patient - level data from submitting nephrologists . furthermore , subgroup analysis of the 31 soro - esrd patients versus the 60 who developed classic esrd revealed that 9 of 31 ( 29% ) soro - esrd patients were 80 years and older . the only evident different characteristics observed between these two sub - groups of 80 and over was the higher exposure to angiotensin inhibition in the soro - esrd group ( 4 of 9 [ 44% ] vs. 1 of 17 [ 6% ] , 2 tailed z - test , p = 0.019 ) . overall , 7 of 31 ( 23% ) soro - esrd patients were concurrently on raas blockade at the time of the diagnosis of aki versus 3 of 60 ( 5% ) without soro - esrd , t ( 89 ) = 2.587 , p = 0.0113 . the plausible contribution of older age and the role of exposure to nephrotoxics , potentially including angiotensin converting enzyme ( ace ) inhibitors and angiotensin receptor blockers , to the incidence of soro - esrd remain speculative at this point and require further study . from this analysis , we submit that there is some plausible role for nephrotoxics including raas blockade in the exacerbation of renal outcomes with aki on ckd . renoprevention is the preemptive withholding of nephrotoxics including raas blockers , the aggressive prevention of perioperative hypotension , the avoidance or minimalization of nephrotoxic exposure from iodinated contrast and relevant antibiotics , during critical illness and in the peri - operative period . such a paradigm , it is hoped , will consequently lead to less aki events and therefore potentially less soro - esrd , better patient outcomes and significant dollar savings as we have demonstrated in a recent analysis in the critical care unit of a northwestern wisconsin mayo clinic health system hospital . such needed paradigm shifts would constitute major rethinking in current nephrology practices , a form of nephrology practice reengineering . besides , the possible interplay of renal senescence and changes in the renal anatomy with ageing such as senile hypofiltration ( glomerulosclerosis , mesangial expansion ) and renal vascular changes ( renal atherosclerosis , vascular dysautonomy , arteriole subendotelial hyalinosis , aglomerular circulation ) and the pathogenesis of this soro - esrd in the older ( > 65 year old ) adult ckd patient is of major research interest to our group . the fact that in our first report of soro - esrd in 2010 , older age appeared to be a significant pathogenetic factor in the development of the syndrome is again acknowledged . the finding in this study that the 31 soro - esrd patients versus the remaining 60 classic esrd patients were older , further strengthened this hypothesis of renal ageing and renal senescence being risk factors for this previously unrecognized syndrome . there are several plausible explanations that could justify why soro - esrd is commoner among our older or aged patients . besides , gfr reduction secondary to senescence ( senile glomerulosclerosis ) , could represent a risk of installing soro - esrd in states of hemodynamic instability , particularly in the oldest old . this is true for patients with bilateral renal artery stenosis , renal artery stenosis in a solitary kidney , congestive heart failure and severe renal failure , especially when they are volume depleted . finally , several causes of rapidly progressive glomerulopathies , which are frequent among the older patient , such as crescentic glomerulonephritis , systemic vasculitis , atheroembolic disease , could be another causal mechanism of soro - esrd in this older population . lastly , the fact that 2 of the 31 ( 6% ) of the soro - esrd patients in our present study were rtrs raises another significant concern to what extent is renal allograft loss attributable to this soro - esrd ? the full details of the presentations of these two rtrs who developed soro - esrd , one of whom was re - transplanted in january 2012 , a year after developing soro - esrd , with a living related renal allograft from her 32-year old son at mayo clinic , rochester , the first such report of soro - esrd among rtrs , has just been published in this journal , the indian journal of nephrology , in 2013 . in closing , we must observe that despite decades of painstaking research into the dynamics , processes and mechanisms of ckd - esrd propagation and progression , the medical community remains at a loss in understanding the nuances of these translations . the common ckd staging classification represents an assumed and arguably untested paradigm that in our opinion , had certainly increased awareness of ckd , without truly advancing our knowledge in this field of medicine , nor have we been able to make any major break - through achievements in improving ckd outcomes . tragically , this approach may be counter - productive as it continues to buttress the notion that ckd is a homogenous clinical entity and that all ckd is equal . we suggest a complete reappraisal of current nephrology practices and to begin to develop new models of ckd care that correctly recognize the diversity of ckd as representative of a wide spectrum of disease states . most appropriately , bansal and hsu , in a 2008 analysis of the long - term outcomes of patients with ckd had strongly echoed the observation that the disparate esrd and mortality rates in various ckd populations as reported by various studies in the literature only emphasized the heterogeneity of different ckd populations . nephrologists must not rely on ckd staging alone to direct management of or risk - stratification of patients with ckd , in general , but must always consider the etiology and rate of progression of kidney disease , patient age and a wide array of cardiovascular disease risk factors . the overarching need to always individualize ckd care can not be overemphasized as ckd represents a whole wide spectrum of distinctly different clinical disease entities , with each individual patient often subject to a multitude of aggravating factors , some of which often remain unrecognized .

some instances of new gene evolution from previously non - coding sequence have now been discovered [ 1 , 2 ] . also , new genes can be formed by shuffling of pre - existing nucleotide sequences . the relatively recent discovery of large numbers of taxonomically restricted genes also demands a closer investigation of their mode(s ) of origin . nevertheless , a major mechanism for the generation of new genes is via duplication . such duplicates are called paralogues , to reflect their homologous relationship being due to a duplication event rather than a speciation event ( see figure 1 ) . since the first animal whole genome sequence of the nematode caenorhabditis elegans , the number of animal whole genome sequences has been increasing at an impressive rate . it should , however , be kept in mind that there is a high level of variability in the quality of these genome sequences ; quality here referring to the depth of sequence coverage of the genome , levels of effort to fill gaps in the sequence , and amount of independent mapping data to inform and confirm the assembly . as a result , many of the animal whole genome sequences that are available must be handled with caution when estimating the extent and nature of duplication events . furthermore , most animal genome sequences can only be assembled to a subchromosomal scale , with genomic scaffolds covering only fragments of chromosomes . this becomes important when trying to assess duplication and translocation mechanisms and distinguishing intra- and interchromosomal events . inevitably , the organisms with the largest research communities and the most intensively studied genomes tend to have the highest quality genome assemblies and annotations . most studies of gene and genome duplications , and hypotheses about mechanisms , stem from analyses of such organisms as vertebrates ( including humans , other mammals , and fish ) and insect and nematode model systems , as will become clear below . here we review the current terminology used for duplicated genes and then discuss the role of whole genome duplication , particularly within the context of vertebrate evolution , and review the current understanding of modes of subchromosomal duplications and recent data on mechanisms for distribution of these duplicated sequences around the genome . the terminology used to define the evolutionary relationships between duplicated genes has become increasingly detailed . the precise inference of the evolutionary relationships between duplicated genes is fundamental for most comparative genomic studies , but it can be complicated because duplication is often combined with speciation and subsequent gene loss . the most widely used terms for describing evolutionary relationships between genes are homologous , orthologous , and paralogous . a subset of homologous genes are orthologous , these being the genes separated only by speciation and not by a duplication event ( figure 1(a ) ) . another subset of homologous genes are paralogous , which are those resulting from a duplication event ( figure 1(b ) ) . pro - orthology denotes the relationship of a gene to one of the descendants of its orthologue after duplication of that orthologue ( figure 1(c ) ) . conversely , semi - orthology is the relationship of one of a set of duplicated genes to a gene that is orthologous to the ancestor of the whole set ( figure 1(d ) ) . sharman also proposed the term trans - homology to describe members of the same gene family descendant from an ancestral gene via two independent gene duplication events . a further important term connected with paralogy is the one proposed by wolfe , who coined the term ohnologue for those paralogues stemming from a whole genome duplication ( figure 1(f ) ) . two years later , sonnhammer and koonin highlighted that the definition of a paralogous relationship can be related to a speciation event . inparalogues are paralogues in a given lineage that all evolved by gene duplications that happened after a speciation event that separated the given lineage from the other lineage under consideration ( figure 1(e ) ) . outparalogues are paralogues in a given lineage that evolved by gene duplications that happened before a speciation event ( figure 1(e ) ) . careful consideration must be taken when using the terms such as inparalogues , outparalogues , and ohnologues . the specification of the relation of the duplication event to the speciation event must be included when these terms are used , otherwise evolutionary interpretations and use of terminology can easily be confused . finally , a new umbrella term , duplogs , has been thrown into the duplication terminology pool to define intraspecies paralogues . this term amalgamates all the types of paralogues within a species , including inparalogues , outparalogues , and ohnologues . sonnhammer and koonin also defined co - orthologues , which are synonymous with sharman 's definition of trans - homologues , and are inparalogues of one lineage which are homologous to another set of inparalogues in a second lineage . artifacts stemming from phylogenetic inference , such as lineage - specific gene loss , can mislead the deduction of the evolutionary relationship of genes . for this purpose , koonin devised the term pseudo - orthologue to accommodate those genes that are essentially paralogues but appear to be orthologues due to differential , lineage - specific gene loss ( figure 1(g ) ) . xenologues are homologues acquired through horizontal gene transfer by one or both species that are being compared , but appearing to be orthologues when pairwise comparison of the genomes is performed ( figure 1(h ) ) . pseudo - paralogues are homologues that through the analysis in a single genome are interpreted as paralogues ; however , these homologues originated by a combination of vertical inheritance and horizontal gene transfer ( figure 1(h ) ) . recently a new term , toporthology , has been specified , which aims to include another aspect of the concept of orthology , that of positional orthology . toporthology describes the evolutionary relationship of orthologues that retain their ancestral genomic positions . in the context of gene duplications , a duplication event is said to be symmetric if deletion of either of the copies of the duplicated sequences would return the gene order to the original , ancestral state . a duplication event is asymmetric if deleting only one of the copies could return the gene order to its original , ancestral state . two genes are positionally homologous , topohomologous , if they are homologous and neither gene comes from an asymmetric duplication since the time of their common ancestor . the term toporthology and its associated derivations need to be used with extreme caution . the value , and aim , of distinguishing toporthologues / topoparalogues is to distinguish those genes ( which are not necessarily one - to - one orthologues ) that are most comparable in terms of their evolutionary history . however , being able to distinguish toporthology obviously requires reliable , accurate genome assemblies and hinges on distinguishing parent / source locations from daughter / target locations of duplicated regions . also , the distinction of toporthology can obviously be complicated by genomic rearrangements that occur after the duplication event and which can obscure whether a duplication was symmetric or asymmetric . currently , the complications introduced by such postduplication genomic rearrangements lead to some counterintuitive uses of the terminology . one might assume that toporthology / topoparalogy simply refers to orthologues / paralogues that are both in the ancestral locations , and conversely that atoporthology / atopoparalogy simply describes the situation in which at least one of the genes is no longer in the ancestral location . the use of the terminology is not so straight - forward , however , as can be seen by a close inspection of figure 2 in , in which ya1 and ya2 are topoparalogues rather than atopoparalogues despite ya2 no longer being in the ancestral location . the classification of ya1 and ya2 as topoparalogues arises because they were not produced by an asymmetric duplication , but then the subsequent change of position of ya2 has obscured this . consequently the precision of the data ( taxonomic sampling and quality of genome assembly ) severely compromises the utility of this terminology . despite the apparent use of the terms to reflect relationships relative to ancestral locations within the genome , in fact the movement of genes to new , nonancestral locations subsequent to the duplication event is not accommodated . firstly , there is the complexity of the evolutionary processes involved in production of duplicates and the care that must thus be exercised when comparing genes between species . secondly , there is currently an over - abundance of terminology , some of which is redundant and some of which is counterintuitive . it is to be hoped that with time the terminology will settle on a consensus of selected terms and those that are impractical or potentially misleading will be abandoned . we now turn from the terminology of gene duplication to the biological processes and evolutionary events . one of the most striking features of the human genome , which is shared with the other members of our subphylum , the vertebrata , is the extensive occurrence of paralogons : homologous regions of chromosomes that are related via duplication events rather than speciation events . this observation is usually attributed to the occurrence of two rounds of whole genome duplication at the origin of the vertebrates ( the so - called 2r hypothesis ) , because of the preponderance of four paralogons for each region of the human genome being considered . thus , one copy of the diploid genome duplicated to give two copies , and this tetraploid state then duplicated a second time to effectively give an octoploid state , which with time has been diploidized again but with the remnants of the octoploid state being detectable from analyses of the paralogons . the 2r events were inevitably followed by extensive gene loss , as would be expected given the inevitable high levels of genetic redundancy that would ensue from such large - scale duplications , such that less than 30% of the 2r paralogous genes are estimated to remain . this means that 2r paralogue families now consist of between two to four members , thus providing a significant pool of extra genes that have made a significant contribution to the evolution and diversification of the vertebrates . this 2r hypothesis has its roots in the ideas of susumu ohno , and it then began to gain increasing support from molecular genetic work , principally from the invertebrate chordate amphioxus . for example , amphioxus has a single hox gene cluster whilst humans have four [ 17 , 18 ] . the 2r hypothesis was not universally accepted at first , largely on the grounds of differing interpretations of molecular phylogenetic trees and the assessment of branching topologies within different gene families and amongst paralogues . the topology argument that formed the basis for challenging the 2r hypothesis requires the trees to be interpreted in a very restricted fashion , with the four paralogues adopting a symmetrical topology of ( ( a , b)(c , d ) ) . this was supposed to represent the first wgd producing two paralogues , which were the precursors to ab and cd , followed by the second wgd producing the a and b as well as c and d genes . however , it is far from clear that duplicated genes always behave in the expected post - duplication way , with daughters evolving at equal rates post - duplication . in fact there is increasing evidence for asymmetric evolution of duplicated genes , often with disruptions to tree topology that tend to arise from long branch attraction . also , as analyses progressed to genome - scale data the controversy has largely subsided with the ever - increasing evidence in favour of 2r . this is typified by the sequencing of the whole genome of the american amphioxus , branchiostoma floridae , and analyses not just of paralogue phylogenies but also patterns of gene synteny across chordates . the trend for a single locus in amphioxus matching four loci in humans ( and other vertebrates , with some notable exceptions mentioned below ) , which was originally developed from work on the hox gene cluster(s ) was found to extend to large - scale , genome - wide quadruple conserved synteny . there have still been one or two dissenting voices , such as arguing instead for segmental duplications occurring at different times rather than whole genome duplications ( and hence simultaneous origins of paralogons ) . however , we note that the interpretation of the molecular phylogenies in contains a number of errors , including deductions based on support values at inappropriate nodes as well as nodes that do not have significant support values . questionable rooting strategies are employed in several of the trees in and incomplete datasets are used for some genes , such as the sp transcription factors . the analyses of abbasi in fact do not challenge the 2r hypothesis , but in fact often support it as soon as one accepts that some gene loss occurred after 2r . that gene loss is a common phenomenon is now without doubt [ 15 , 2630 ] . also , since both wgd events occurred close together in time , and via autotetraploidy in both cases , then it is to be expected that the phylogenies of the paralogues do not in fact adopt the ( ( a , b)(c , d ) ) topology , as explained by furlong and holland . tree topologies should thus not still be being used as a test of 2r with the view that divergence from the ( ( a , b)(c , d ) ) topology is in conflict with 2r . furthermore , the 2r hypothesis no longer relies solely upon the topology of individual gene trees , but instead gains its most convincing support from conserved synteny arrangements that cover over 90% of the human genome and extends to the genomes of birds and fish ( including chicken , stickleback , and puffer fish ) . therefore , we hold the view that the 2r hypothesis ( with subsequent gene loss ) is definitely the most parsimonious explanation for the origin and evolution of vertebrate genomes . the plausibility of the 2r hypothesis is further strengthened by the discoveries of whole genome duplications elsewhere in the animal kingdom , thus demonstrating that the process can certainly occur , and do so with reasonable frequency ( see table 1 ) [ 31 , 32 ] . for example , the origin of the teleost fish coincides with another wgd , the 3r event . again , this hypothesis is strongly supported by the patterns of synteny relative to other vertebrates and the existence of extensive paralogons matching the topology expected for a 3r event . whole genome duplications and polyploidization events are constantly coming to light within the animal kingdom , and are clearly a significant mode of duplication that has shaped animal evolution . duplications that encompass sections of dna smaller than whole chromosomes are given the generic name of segmental duplications ( sds ) . these can vary enormously in size , from a few base pairs up to many megabases , and may or may not contain intact , functional genes . they can also be found in several different arrangements , which are important for considerations as to how these sds might form . sds can be adjacent ( tandem duplications ) , separated , or interspersed along a particular chromosome ( intrachromosomal ) or on distinct chromosomes ( interchromosomal ) . the detection of sds in these different categories obviously depends upon the quality of a genome sequence assembly , but the prevalence of sds in the human genome , for example , tend to be estimated at about 5 - 6% ( for sds 1 kb , with 90% sequence identity , and filtered for transposable elements and other high - copy repeats ) . estimates of sd prevalence in other mammals tends to produce slightly lower levels than in humans , although in the case of mouse that has recently been revised upwards to almost 5% and hence is now thought to be comparable to the levels in humans [ 62 , 63 ] . a striking aspect of the comparisons between rates and distributions of sds in various mammalian genome sequences is that tandem duplications are by far the most prevalent category of sd , comprising 7590% of sds in the cow for example . this preponderance of tandem duplicates in mammals as diverse as cows , rodents , and dogs does not , however , reflect the situation in humans , in which sds are much more frequently interspersed [ 6467 ] . the interspersed distribution of human sds is possibly the result of an expansion of alu transposable elements within primates [ 62 , 68 ] . moving outside of the mammals , the fruit fly drosophila melanogaster has the majority of its sds in the intrachromosomal category ( 86% ) , and of these most are situated close together in the genome ( 50% and < 14 kb apart ) . the different categories of sds ( tandem , interspersed intrachromosomal , and interchromosomal ) may well reflect different mechanisms of dna - based duplication . non - homologous end - joining ( nhej ) is more likely to account for adjacent duplications [ 7072 ] with the repair of dna breaks being more likely to occur between ends in close proximity . the alternative of nonallelic homologous recombination ( nahr ) is likely mediated via repetitive sequences dispersed around the genome and hence is a route to interspersed duplications . this process has been given the name duplication - dependent strand annealing ( ddsa ) by fiston - lavier et al . , who also noted that in d. melanogaster the mean size of intrachromosomal events is larger than the average size of interchromosomal events ( 3.1 kb versus 2.1 kb , respectively ) . this contrasts with the average size of sds in humans being approximately 18.6 kb and 14.8 kb for the intrachromosomal and interchromosomal categories respectively . in addition to this observation that intrachromosomal sds tend to be longer than interchromosomal sds possibly reflecting different mechanisms being the cause of their origin , it is striking that the size of sds varies in different species . a further data point is provided by the nematode caenorhabditis elegans , in which the average size of sds is only 1.4 kb . this implies that the size of duplication is not necessarily determined by physical properties of the dna or possibly the duplication mechanism ( unless mechanisms differ between the taxa thus far examined ) , but instead is likely to relate to the structure and organization of the genome . density and distribution of repetitive sequences will be one factor , and these vary across different species . in addition , strong selective pressures are likely to come into operation when genes are duplicated within sds , often disrupting genetic networks and pathways if a gene is duplicated and then expressed ( e.g. , via dosage imbalance ) . thus there will tend to be selective pressure against duplications that encompass genes ( and their regulatory elements ) , thus reducing the average size of segmental duplicates in taxa with smaller , more compact genes . alongside consideration of the duplication mechanisms within the context of determining the organisation of duplicated genes , it follows that one must also consider processes by which segments of dna or genes can be translocated around the genome . although these mechanisms are not necessarily leading to generation of duplications ( and in fact often are not ) they are still crucial in understanding the subsequent distribution of genes , which in the present context happen to be duplicates . retrotransposition is one of the duplication mechanisms that does not necessarily lead to generation of functional duplicated genes , but is crucial in distributing duplicated single genes , especially in an inter - chromosomal fashion [ 7679 ] . inversions are very common and help to scatter duplicated genes along a particular chromosome arm [ 71 , 80 ] . also large - scale events such as inversions between arms involving the centromere or chromosome fusions and fissions are also known to play a prominent role in karyotype evolution , and reciprocal translocations between chromosome arms are very common . surprisingly high rates of reciprocal translocations occur in humans , with estimates of around one in 500 newborns carrying such large - scale rearrangements [ 8184 ] . this is not necessarily unusual to humans , as cattle reciprocal translocations have been estimated to occur at a rate of 1.4 per 1000 animals . these high rates of translocations are thought to be mediated via nahr using duplicated or repetitive segments located in different chromosomes , that is interchromosomal low - copy repeats ( lcrs ) . . characterized several hundred interchromosomal lcrs in the human genome , ranging in size from 5 kb to over 50 kb , all of which they suggest can act as the substrates for reciprocal translocations . in addition , hermetz et al . described a translocation occurring via homologous recombination between herv elements on different chromosomes . in combination all of these routes to rearrangement of genome organisation often make it difficult to accurately determine between likely mechanisms of duplicate origin . this is because it is difficult to determine whether the locations of any two duplicated sequences reflect their organisation at their point of origin , or instead is the end point of originating by a process such as tandem duplication and then subsequently being dispersed . attempts to address this problem have involved estimating the age of duplicates by calculating the rates of synonymous substitutions ( ks ) . this has led to observations that younger genes tend to be closer together in the genome , particularly being more highly represented in the intrachromosomal category of duplicates relative to the interchromosomal category [ 74 , 88 ] . however , such estimates of gene age can be confounded by the process of gene conversion , which can homogenise gene sequence after the origin of the duplicates [ 89 , 90 ] . since gene conversion is more likely to occur between genes that are in close proximity then there will be a degree of misjudging the age of duplicates as inappropriately young , and this effect will be most pronounced in the categories of closely linked genes such as tandem duplicates . furthermore , the positive correlation between age and dispersal in the genome has recently been questioned with the proposal of a process named drift duplication . ezawa and colleagues ' comparisons of duplicate age and genomic location in human , mouse , zebrafish , c. elegans , d. melanogaster , and drosophila pseudoobscura suggest that interspersed intrachromosomal duplications can be generated at once , rather than originating as tandem duplicates which are subsequently relocated away from each other , and this can happen at comparable rates to tandem duplication . the precise mechanism leading to drift duplication is not specified by ezawa et al . , and is likely to involve a combination of processes . one of these could well be the recently discovered process of duplication via circular dna - based translocation . witrik cows a translocation of 492 kb occurred which was then followed by a repatriation of a 575 kb segment , including the kit gene that is involved in the pigmentation patterning of the cows and their distinctive the intriguing aspect to these translocations is the order of sequences within the translocated segment , which is consistent with translocation via a circular dna intermediate which is opened up for re - insertion at a different point in the circle from the boundaries of the original excision ( figure 2 ) . also , since the repatriated segment was larger than the originally translocated segment then some sequence duplication results ( figure 2 ) . further examples of duplications via circular dna intermediates are being found , such as the vasa genes of tilapia . the difference between the cow and tilapia examples however is that the cow circular dna intermediate is repatriated into an ancestral locus , presumably due to homologous recombination , whereas the tilapia vasa duplicates that arose via circular intermediates have gone to new locations . the tilapia vasa example is thus more reminiscent of drift duplication , but it remains to be seen how prevalent such circular dna translocation events are and how the reintegration sites are selected . given the range of genomic rearrangement mechanisms and their apparent frequencies , it is perhaps surprising that syntenic arrangements can be conserved for vast evolutionary timespans , for example , from humans to the origin of chordates and beyond , to even some basal lineages of animals such as the cnidarian nematostella vectensis and the placozoan trichoplax adhaerens [ 92 , 93 ] . what is also striking is that this phenomenon of long - term general synteny conservation is not detected uniformly across the animal kingdom . some lineages and groups of animals seem to have particularly derived genome organisations relative to other animals ( e.g. , oikopleura and urochordates in general ; drosophila and other diptera ; nematodes like c.elegans [ 8 , 94 , 95 ] ) . one could speculate that this might reflect different abundances of repetitive elements , for example , which can have a role in facilitating genomic rearrangements . another possibility is that gene sizes , and perhaps more importantly gene densities within the chromosomes , vary significantly across the animal kingdom . this variation might not just be the number of nucleotides spanned by the coding sequence , but also by the regulatory elements , which will influence how frequently rearrangement mutations can occur that are still compatible with organismal viability . regardless of this , some animal genomes seem to be more tolerant of , or prone to , rearrangements than others . with the burgeoning amounts of human genome sequence data , particularly in relation to disease and cancer genomics , a new phenomenon involving a catastrophic rearrangement of the genome has recently been described : chromothripsis [ 96 , 97 ] . perhaps the process of chromothripsis has a relevance beyond the realms of cancer and disease biology and may be comparable to processes whereby some animal genomes become extensively rearranged relative to other lineages . the variety of mechanisms by which such duplications occur , as well as the various means by which the duplicated segments are subsequently rearranged ( and sometimes partially lost ) , requires careful analysis and consistent use of biologically informed terminology . obviously a major goal for the future will be to expand the taxonomic coverage of high - quality genome assemblies to enable the deduction of more accurate and more widely applicable , general conclusions about such phenomena as gene and genome duplications . this should be complemented by the continued development of in silico tools and models to estimate duplication and rearrangement rates . such tools then need to be applied across an increased range of genomes in order to distinguish general mechanisms and principles from lineage - specific oddities , such as lack of synteny between urochordates and vertebrates or the paucity of tandem duplications in humans relative to other mammals .

duplication of genetic material is clearly a major route to genetic change , with consequences for both evolution and disease . a variety of forms and mechanisms of duplication are recognised , operating across the scales of a few base pairs upto entire genomes . with the ever - increasing amounts of gene and genome sequence data that are becoming available , our understanding of the extent of duplication is greatly improving , both in terms of the scales of duplication events as well as their rates of occurrence . an accurate understanding of these processes is vital if we are to properly understand important events in evolution as well as mechanisms operating at the level of genome organisation . here we will focus on duplication in animal genomes and how the duplicated sequences are distributed , with the aim of maintaining a focus on principles of evolution and organisation that are most directly applicable to the shaping of our own genome .

1. Introduction 2. Terminology: Beware Overlap, Synonyms, and Ambiguity (and Use with Care) 3. Whole Genome Duplications (WGDs): Origin of Vertebrates and 2R 4. Subchromosomal Duplications: Variable Sizes, Rates, and Mechanisms 5. Conclusion

nevertheless , a major mechanism for the generation of new genes is via duplication . such duplicates are called paralogues , to reflect their homologous relationship being due to a duplication event rather than a speciation event ( see figure 1 ) . since the first animal whole genome sequence of the nematode caenorhabditis elegans , the number of animal whole genome sequences has been increasing at an impressive rate . it should , however , be kept in mind that there is a high level of variability in the quality of these genome sequences ; quality here referring to the depth of sequence coverage of the genome , levels of effort to fill gaps in the sequence , and amount of independent mapping data to inform and confirm the assembly . as a result , many of the animal whole genome sequences that are available must be handled with caution when estimating the extent and nature of duplication events . furthermore , most animal genome sequences can only be assembled to a subchromosomal scale , with genomic scaffolds covering only fragments of chromosomes . this becomes important when trying to assess duplication and translocation mechanisms and distinguishing intra- and interchromosomal events . inevitably , the organisms with the largest research communities and the most intensively studied genomes tend to have the highest quality genome assemblies and annotations . most studies of gene and genome duplications , and hypotheses about mechanisms , stem from analyses of such organisms as vertebrates ( including humans , other mammals , and fish ) and insect and nematode model systems , as will become clear below . here we review the current terminology used for duplicated genes and then discuss the role of whole genome duplication , particularly within the context of vertebrate evolution , and review the current understanding of modes of subchromosomal duplications and recent data on mechanisms for distribution of these duplicated sequences around the genome . the precise inference of the evolutionary relationships between duplicated genes is fundamental for most comparative genomic studies , but it can be complicated because duplication is often combined with speciation and subsequent gene loss . pro - orthology denotes the relationship of a gene to one of the descendants of its orthologue after duplication of that orthologue ( figure 1(c ) ) . conversely , semi - orthology is the relationship of one of a set of duplicated genes to a gene that is orthologous to the ancestor of the whole set ( figure 1(d ) ) . sharman also proposed the term trans - homology to describe members of the same gene family descendant from an ancestral gene via two independent gene duplication events . two years later , sonnhammer and koonin highlighted that the definition of a paralogous relationship can be related to a speciation event . outparalogues are paralogues in a given lineage that evolved by gene duplications that happened before a speciation event ( figure 1(e ) ) . the specification of the relation of the duplication event to the speciation event must be included when these terms are used , otherwise evolutionary interpretations and use of terminology can easily be confused . sonnhammer and koonin also defined co - orthologues , which are synonymous with sharman 's definition of trans - homologues , and are inparalogues of one lineage which are homologous to another set of inparalogues in a second lineage . artifacts stemming from phylogenetic inference , such as lineage - specific gene loss , can mislead the deduction of the evolutionary relationship of genes . for this purpose , koonin devised the term pseudo - orthologue to accommodate those genes that are essentially paralogues but appear to be orthologues due to differential , lineage - specific gene loss ( figure 1(g ) ) . xenologues are homologues acquired through horizontal gene transfer by one or both species that are being compared , but appearing to be orthologues when pairwise comparison of the genomes is performed ( figure 1(h ) ) . recently a new term , toporthology , has been specified , which aims to include another aspect of the concept of orthology , that of positional orthology . in the context of gene duplications , a duplication event is said to be symmetric if deletion of either of the copies of the duplicated sequences would return the gene order to the original , ancestral state . a duplication event is asymmetric if deleting only one of the copies could return the gene order to its original , ancestral state . two genes are positionally homologous , topohomologous , if they are homologous and neither gene comes from an asymmetric duplication since the time of their common ancestor . the value , and aim , of distinguishing toporthologues / topoparalogues is to distinguish those genes ( which are not necessarily one - to - one orthologues ) that are most comparable in terms of their evolutionary history . currently , the complications introduced by such postduplication genomic rearrangements lead to some counterintuitive uses of the terminology . one might assume that toporthology / topoparalogy simply refers to orthologues / paralogues that are both in the ancestral locations , and conversely that atoporthology / atopoparalogy simply describes the situation in which at least one of the genes is no longer in the ancestral location . the use of the terminology is not so straight - forward , however , as can be seen by a close inspection of figure 2 in , in which ya1 and ya2 are topoparalogues rather than atopoparalogues despite ya2 no longer being in the ancestral location . the classification of ya1 and ya2 as topoparalogues arises because they were not produced by an asymmetric duplication , but then the subsequent change of position of ya2 has obscured this . consequently the precision of the data ( taxonomic sampling and quality of genome assembly ) severely compromises the utility of this terminology . despite the apparent use of the terms to reflect relationships relative to ancestral locations within the genome , in fact the movement of genes to new , nonancestral locations subsequent to the duplication event is not accommodated . firstly , there is the complexity of the evolutionary processes involved in production of duplicates and the care that must thus be exercised when comparing genes between species . it is to be hoped that with time the terminology will settle on a consensus of selected terms and those that are impractical or potentially misleading will be abandoned . we now turn from the terminology of gene duplication to the biological processes and evolutionary events . one of the most striking features of the human genome , which is shared with the other members of our subphylum , the vertebrata , is the extensive occurrence of paralogons : homologous regions of chromosomes that are related via duplication events rather than speciation events . this observation is usually attributed to the occurrence of two rounds of whole genome duplication at the origin of the vertebrates ( the so - called 2r hypothesis ) , because of the preponderance of four paralogons for each region of the human genome being considered . thus , one copy of the diploid genome duplicated to give two copies , and this tetraploid state then duplicated a second time to effectively give an octoploid state , which with time has been diploidized again but with the remnants of the octoploid state being detectable from analyses of the paralogons . the 2r events were inevitably followed by extensive gene loss , as would be expected given the inevitable high levels of genetic redundancy that would ensue from such large - scale duplications , such that less than 30% of the 2r paralogous genes are estimated to remain . this means that 2r paralogue families now consist of between two to four members , thus providing a significant pool of extra genes that have made a significant contribution to the evolution and diversification of the vertebrates . this 2r hypothesis has its roots in the ideas of susumu ohno , and it then began to gain increasing support from molecular genetic work , principally from the invertebrate chordate amphioxus . the 2r hypothesis was not universally accepted at first , largely on the grounds of differing interpretations of molecular phylogenetic trees and the assessment of branching topologies within different gene families and amongst paralogues . the topology argument that formed the basis for challenging the 2r hypothesis requires the trees to be interpreted in a very restricted fashion , with the four paralogues adopting a symmetrical topology of ( ( a , b)(c , d ) ) . this was supposed to represent the first wgd producing two paralogues , which were the precursors to ab and cd , followed by the second wgd producing the a and b as well as c and d genes . however , it is far from clear that duplicated genes always behave in the expected post - duplication way , with daughters evolving at equal rates post - duplication . also , as analyses progressed to genome - scale data the controversy has largely subsided with the ever - increasing evidence in favour of 2r . this is typified by the sequencing of the whole genome of the american amphioxus , branchiostoma floridae , and analyses not just of paralogue phylogenies but also patterns of gene synteny across chordates . the trend for a single locus in amphioxus matching four loci in humans ( and other vertebrates , with some notable exceptions mentioned below ) , which was originally developed from work on the hox gene cluster(s ) was found to extend to large - scale , genome - wide quadruple conserved synteny . however , we note that the interpretation of the molecular phylogenies in contains a number of errors , including deductions based on support values at inappropriate nodes as well as nodes that do not have significant support values . questionable rooting strategies are employed in several of the trees in and incomplete datasets are used for some genes , such as the sp transcription factors . that gene loss is a common phenomenon is now without doubt [ 15 , 2630 ] . also , since both wgd events occurred close together in time , and via autotetraploidy in both cases , then it is to be expected that the phylogenies of the paralogues do not in fact adopt the ( ( a , b)(c , d ) ) topology , as explained by furlong and holland . tree topologies should thus not still be being used as a test of 2r with the view that divergence from the ( ( a , b)(c , d ) ) topology is in conflict with 2r . furthermore , the 2r hypothesis no longer relies solely upon the topology of individual gene trees , but instead gains its most convincing support from conserved synteny arrangements that cover over 90% of the human genome and extends to the genomes of birds and fish ( including chicken , stickleback , and puffer fish ) . the plausibility of the 2r hypothesis is further strengthened by the discoveries of whole genome duplications elsewhere in the animal kingdom , thus demonstrating that the process can certainly occur , and do so with reasonable frequency ( see table 1 ) [ 31 , 32 ] . for example , the origin of the teleost fish coincides with another wgd , the 3r event . again , this hypothesis is strongly supported by the patterns of synteny relative to other vertebrates and the existence of extensive paralogons matching the topology expected for a 3r event . whole genome duplications and polyploidization events are constantly coming to light within the animal kingdom , and are clearly a significant mode of duplication that has shaped animal evolution . duplications that encompass sections of dna smaller than whole chromosomes are given the generic name of segmental duplications ( sds ) . these can vary enormously in size , from a few base pairs up to many megabases , and may or may not contain intact , functional genes . sds can be adjacent ( tandem duplications ) , separated , or interspersed along a particular chromosome ( intrachromosomal ) or on distinct chromosomes ( interchromosomal ) . the detection of sds in these different categories obviously depends upon the quality of a genome sequence assembly , but the prevalence of sds in the human genome , for example , tend to be estimated at about 5 - 6% ( for sds 1 kb , with 90% sequence identity , and filtered for transposable elements and other high - copy repeats ) . estimates of sd prevalence in other mammals tends to produce slightly lower levels than in humans , although in the case of mouse that has recently been revised upwards to almost 5% and hence is now thought to be comparable to the levels in humans [ 62 , 63 ] . a striking aspect of the comparisons between rates and distributions of sds in various mammalian genome sequences is that tandem duplications are by far the most prevalent category of sd , comprising 7590% of sds in the cow for example . moving outside of the mammals , the fruit fly drosophila melanogaster has the majority of its sds in the intrachromosomal category ( 86% ) , and of these most are situated close together in the genome ( 50% and < 14 kb apart ) . the different categories of sds ( tandem , interspersed intrachromosomal , and interchromosomal ) may well reflect different mechanisms of dna - based duplication . non - homologous end - joining ( nhej ) is more likely to account for adjacent duplications [ 7072 ] with the repair of dna breaks being more likely to occur between ends in close proximity . the alternative of nonallelic homologous recombination ( nahr ) is likely mediated via repetitive sequences dispersed around the genome and hence is a route to interspersed duplications . this process has been given the name duplication - dependent strand annealing ( ddsa ) by fiston - lavier et al . this contrasts with the average size of sds in humans being approximately 18.6 kb and 14.8 kb for the intrachromosomal and interchromosomal categories respectively . this implies that the size of duplication is not necessarily determined by physical properties of the dna or possibly the duplication mechanism ( unless mechanisms differ between the taxa thus far examined ) , but instead is likely to relate to the structure and organization of the genome . , via dosage imbalance ) . alongside consideration of the duplication mechanisms within the context of determining the organisation of duplicated genes , it follows that one must also consider processes by which segments of dna or genes can be translocated around the genome . although these mechanisms are not necessarily leading to generation of duplications ( and in fact often are not ) they are still crucial in understanding the subsequent distribution of genes , which in the present context happen to be duplicates . retrotransposition is one of the duplication mechanisms that does not necessarily lead to generation of functional duplicated genes , but is crucial in distributing duplicated single genes , especially in an inter - chromosomal fashion [ 7679 ] . surprisingly high rates of reciprocal translocations occur in humans , with estimates of around one in 500 newborns carrying such large - scale rearrangements [ 8184 ] . these high rates of translocations are thought to be mediated via nahr using duplicated or repetitive segments located in different chromosomes , that is interchromosomal low - copy repeats ( lcrs ) . . characterized several hundred interchromosomal lcrs in the human genome , ranging in size from 5 kb to over 50 kb , all of which they suggest can act as the substrates for reciprocal translocations . in addition , hermetz et al . in combination all of these routes to rearrangement of genome organisation often make it difficult to accurately determine between likely mechanisms of duplicate origin . this is because it is difficult to determine whether the locations of any two duplicated sequences reflect their organisation at their point of origin , or instead is the end point of originating by a process such as tandem duplication and then subsequently being dispersed . attempts to address this problem have involved estimating the age of duplicates by calculating the rates of synonymous substitutions ( ks ) . this has led to observations that younger genes tend to be closer together in the genome , particularly being more highly represented in the intrachromosomal category of duplicates relative to the interchromosomal category [ 74 , 88 ] . however , such estimates of gene age can be confounded by the process of gene conversion , which can homogenise gene sequence after the origin of the duplicates [ 89 , 90 ] . since gene conversion is more likely to occur between genes that are in close proximity then there will be a degree of misjudging the age of duplicates as inappropriately young , and this effect will be most pronounced in the categories of closely linked genes such as tandem duplicates . furthermore , the positive correlation between age and dispersal in the genome has recently been questioned with the proposal of a process named drift duplication . the precise mechanism leading to drift duplication is not specified by ezawa et al . , and is likely to involve a combination of processes . one of these could well be the recently discovered process of duplication via circular dna - based translocation . witrik cows a translocation of 492 kb occurred which was then followed by a repatriation of a 575 kb segment , including the kit gene that is involved in the pigmentation patterning of the cows and their distinctive the intriguing aspect to these translocations is the order of sequences within the translocated segment , which is consistent with translocation via a circular dna intermediate which is opened up for re - insertion at a different point in the circle from the boundaries of the original excision ( figure 2 ) . the tilapia vasa example is thus more reminiscent of drift duplication , but it remains to be seen how prevalent such circular dna translocation events are and how the reintegration sites are selected . given the range of genomic rearrangement mechanisms and their apparent frequencies , it is perhaps surprising that syntenic arrangements can be conserved for vast evolutionary timespans , for example , from humans to the origin of chordates and beyond , to even some basal lineages of animals such as the cnidarian nematostella vectensis and the placozoan trichoplax adhaerens [ 92 , 93 ] . what is also striking is that this phenomenon of long - term general synteny conservation is not detected uniformly across the animal kingdom . , oikopleura and urochordates in general ; drosophila and other diptera ; nematodes like c.elegans [ 8 , 94 , 95 ] ) . another possibility is that gene sizes , and perhaps more importantly gene densities within the chromosomes , vary significantly across the animal kingdom . this variation might not just be the number of nucleotides spanned by the coding sequence , but also by the regulatory elements , which will influence how frequently rearrangement mutations can occur that are still compatible with organismal viability . regardless of this , some animal genomes seem to be more tolerant of , or prone to , rearrangements than others . with the burgeoning amounts of human genome sequence data , particularly in relation to disease and cancer genomics , a new phenomenon involving a catastrophic rearrangement of the genome has recently been described : chromothripsis [ 96 , 97 ] . perhaps the process of chromothripsis has a relevance beyond the realms of cancer and disease biology and may be comparable to processes whereby some animal genomes become extensively rearranged relative to other lineages . the variety of mechanisms by which such duplications occur , as well as the various means by which the duplicated segments are subsequently rearranged ( and sometimes partially lost ) , requires careful analysis and consistent use of biologically informed terminology . obviously a major goal for the future will be to expand the taxonomic coverage of high - quality genome assemblies to enable the deduction of more accurate and more widely applicable , general conclusions about such phenomena as gene and genome duplications .

the first transplantation of mechanical heart valve performed in august 1960 has saved the lives of thousands of heart patients . since that time , each year , the success in valve replacement depends on the myocardial function , health condition , technical skills of the surgical team , and the quality of postoperative care . kortke reports that patients recovery process after heart replacement is slower than in other heart surgeries . in addition , nassar , in his descriptive study , revealed that the mother mortality rate in mechanical valve transplantation was 3 - 4% . during the long years of waiting for the valve replacement , these patients had catastrophic experiences . postoperative complications ( e.g. long - term hospitalization , social isolation , and the outcomes of valve replacement ) are of upmost significance . hence , protecting the replaced valve and taking care of it are of greatest importance and require the mutual support of family and the patient . after the valve replacement , these patients need long - life care and are at risk for thromboembolism , which obligate them to use anticoagulants . the annual thromboembolic risk among warfarin users and non - users is 1% and 4% , respectively . international normalized ratio ( inr ) for the patients with mechanical aortic and mitral prosthesis is 2 - 3 and 2.5 - 3.5 , respectively . according to menendez , the late - onset complications of valve replacement are : anticoagulant - induced bleeding , prosthesis malfunction , infectious endocarditis , recurrent thrombosis , and valvular insufficiency , which represent the outcomes of failure in the care system . more than the half of the patients did not use the right dose of anticoagulants and had no information on the drug side effects and on the relevant lifestyle . in the present condition , christensen points out that the rate of complications and problems related to treatment with anticoagulants due to self - care and conventional management method are 2.5% and 7.3% , respectively . warfarin use in pregnancy results in some complications ( e.g. abortion and physical anomalies ) . there is no agreement on the right dose of warfarin during pregnancy for supporting both mother and fetus . the doses of warfarin prescribed for valve - replaced patients depend on different factors , which by themselves have different effects on inr . the administration of herbal drugs along with warfarin has different effects on inr . in deccache and aujoulat 's report on hospitalized patients , 20% of the cases had received adequate information on their disease / health condition through advising , 20% of them were not satisfied with their care condition , and the remaining 60% were required to receive more education . replaced patients , due to their susceptibility and the risky outcomes of the procedures , have different levels of worry and anxiety , and continue to live with anxiety , doubt , and worry . the preoperative physical problems and also the postoperative physical complications in valve - replaced patients are associated with a feeling of worry . hence , identification of the needs of such patients from their own experiences is necessary for providing nursing care and lessening the fear and anxiety in them . it seems that the worries , experiences , and attitudes of the patients are ignored by the nursing staff as they are classified as their least important priorities and usually are not considered in routine care planning . assessing the potential sources for fear , worry , and emotional status ( doubt , denial , aggressiveness , and isolation , often as a general reaction to illness or self - awareness ) is of importance . moreover , to our knowledge , till the present time , no qualitative study has been reported on the experiences of these patients while facing the problems following heart valve replacement . given the special cultural , social , and religious contexts in which the heart valve replaced patients are cared for , the present study was done with a qualitative approach to describe the stressful experiences of heart valve in this qualitative study , qualitative purposeful sampling was performed to obtain the perceived experiences of the heart valve subjects consisted of cases with experiences of valve replacement and the tendency to cooperate and report their experiences . data collection was done by conducting a deep , semi - structured interview using an mp4 voice recorder . each interview was started with an open question , what were your experiences in facing and living with the reality of valve replacement ? in addition , to receive further information and for a deep perception of words , some explorative questions like can you give more explanations ? and data collection was done until saturation was reached . using the graneheim and lundman content analysis for qualitative data , the synchronous description and analysis of the interviews were done as follows : ( 1 ) typing the entire interview script immediately after each session ; ( 2 ) reading through the whole text for a general perception of its content ; ( 3 ) determining the meaning units and the initial codes ; ( 4 ) classifying similar initial codes into more comprehensive categories ; and ( 5 ) determining the main themes of categories . the explanation and analysis were done in the following sequence : immediately after each interview session , its content was re - written in the word software , imported to maxqda - v7 , and then the scripts were read several times to find general perceptions of the patients based on the aim of the research . then the meaning units / initial codes were derived , merged , and classified based on similarities . all efforts were taken to keep maximal homogeneity within the categories and also maximal heterogeneity between categories , and avoid the inclusion of the same data in two categories . ranking was done for new category based on aims and the main concepts were taken for the validity and reliability of results . based on polit and beck 's approval , for the accuracy of research findings in addition , for the acceptance of the data , all academic members with an experience of qualitative research were requested to evaluate the codes derived . through reviewing the handwritten scripts by the participants and peer - reviewing it by the research team colleagues , the validity of data was checked . for transferability , the participants were requested to declare some items for completing and correcting the codes . the written informed consent and the signed agreement of having the right to withdraw from the study or continue with the study whenever they wanted to were obtained from all participants . their demographic data are shown in table 1 . from the rich and informative description of participants , then , based on similarities , they were classified and summarized into 14 subcategories . based on the conceptual and abstract exploration of the themes and using analysis and comparison , five subsidiary themes and a main theme of a life associated with fear and worry were extracted . most therapeutic centers in iran lack the facilities for valve replacement surgery , and this is also the case regarding experiences of the care team . these patients were often referred to the main centers . for removing my womb , i was hospitalized for 2 months . i was referred to one of them ( doctors ) for surgery most of the doctors rejected me due to the lack of therapeutic faccilities . ( participant # 3 , 32 years female , mitral valve replacement ) . in case of need for treatment or surgery the second source of worry for participants is the carelessness and failure of the therapeutic team . so , the patients do not refer to the doctors unless they are assured about them the reason for their worry is that if these patients face a problem , what do they do ? they desperately refer to other doctors and therapeutic centers which gives them a sort of worry . i fear everyone and everything and i told myself about the failure in surgery and its bad outcomes , i.e. dying for nothing . ( participant # 2 , 47-year - old female , mitral valve replacement done twice ) . for a cheeper pt , i often refer to hospitals , i always have the anxiety of a wrong pt . these worries are : worry of unexpected events , worry of the outcome of warfarin use , worry of the functioning of prosthetic valve , and worry of coagulatory test results . all the time the patients are worried about an unexpected event happening for different reasons , e.g. shortage of drugs and the discontinuty of valve function . sometimes i no longer bear it and have anxiety . for instance , when i have no warfarin , i think if the valves were non - functional , what can i do for it , how long really it works for me . these worries are intensified with a high risk of endocarditis , angiography , tooth extraction , or less education . i have a hard job , i must lift heavy loads of construction materials as a constitutional worker . ( participant # 12 , 34-year - old male , both mitral and aortic valve replacement ) . ( participant # 2 , 47-year - old male , mitral valve replaced twice ) . sometimes these worries result in the patient not following the treatment . when they performed angiography for me , i feared of dying for no reasons . despite the instructions of the doctor , i left the hospital for home , but desperately i came here again . ( participant # 8 , 50-year - old male , both mitral and aortic valve replacement ) . these patients should continue using warfarin until the end of life and all team members emphasize on this . so , patients are worried about forgetting the drug or its overdose , especially in some conditions like pregnancy and/or surgery . now , i fear everything , even going to a dentist and a bad luck . when patients face a new situation ( a change in weather , travel and food regimen , using new drugs , and change in body metabolism , all of which have an effect on inr ) , they have to repeat the test and report its results to their doctors . the facilities are not available all the time and in case of inadequte education , it results in recurrent referrals and longer hospitalizations . disturbances in daily living and transmission of the disease to offsprings are one of the worries . for a long time during their life , these patients are involved in rheumatic heart disease , valvular involvement and replacement , and its outcomes to the extent that the condition afffects their routine life which results in a busy mind and constant worry . there was a great fear and threat in my mind on living with this sort of valve . another source of worry among the valve - replaced patients , especially affecting the offspring , is the repetitive nature of the exacerbation of the disease and the results of lab reports . however , in the present time , majority of the society members and their families , for different reasons , do not provide the required support . one thing that made me have more fear on operation and reject its proposal was my children 's fear of losing me and dying , and hence i bore the pain and rejected the operation . , ( participant # 8 , 50-year - old male , both aortic and mitral valve replacement ) . for instance , during pregnancy , a change in the anticoagulant regimen ( e.g. warfarin , heparin ) should be made . another stressor that worsened my conditions was that everyday i heard words on the condition of my newborn baby . will it be healthy or have some problems in its heart ? ( participant # 4 , 40-year - old female , mitral valve replacement ) . when these pateints use the teratogenic drug warfarin , especially in the initial stages of the pregnancy , they have a troublesome life . because all the time , they hear about the teratogenicity of these agents and are worried about the defects it causes in their babies . fear of hospital is one of the themes that consisted of the following : fear of re - operation , fear of hospital milieu and the facilities of the hospital . the patients are not aware of most of the therapeutic / diagnostic methods used for valve - replaced pateints during their life period to the extent that they think that most of their problems could be solved with some ordinary lab tests . sometimes the unfavorable outcomes of these tests made them reject the diagnostic procedures or re - operation . my fear 's origin is in having no information , forgetting the instructions and not obeying the orders . sometimes the patients fear originated from their previous experiences or , in most cases , the information they received in relation to dying or related to the matter that they thought that they died because of their inadequate search for a better hospital and hurry in operation . so , they are much worried about the facilities and the capability of the surgeons . i was so feared of signing the informed consent and took the responsibility of discharging from hospital . ( participant # 8 , 50-year - old male , both aortic and mitral valve replacement ) . these pateints are asking and searching much for their obligatory operation valve surgery and also the alternatives , especially the non - surgical ones . one of their concerns in this regard is the carelessness of the surgeons , their insufficient experience , or the lack of facilities of the care team . the common source of fear for these patients is the unfamiliar environment and pepole . among the multiple sources of fear , one important thing is the pateints worry on the conflict and disagreement between the therapeutic team , mostly on diagnostic and treatment issues . the patients minds are occupied with problems on the follow - up and post - treatment risks and outcomes , which mostly begin after the valve replacement . in such big cities , like tehran , you can find the tactful doctors , only a few of them are here and they are busy all the time . ( participant # 11 , 45-year - old male , mitral valve replacement ) . because of their worry , pateints are trying to communicate with the nurses and doctors and ask them about the disease , treatment , and lifestyle . but because of inadequate education and the lack of opportunities to communicate with the therapeutic team , the pateints receive information from other sources which results in having doubts about the treatment or the fundamental errors that , by itself , aggravates the their anxiety . the theme of fear for unknowns consists of fear of the unexpected and unfavorable events and fear of the establishment of new condition the cause of fear is often similar previous experience of the patients , i.e. the risk of infection , disturbances in inr , bleeding , and clotting , that the probability of their occurrence increases in exposure to the new situation . in addition , often in therapeutic settings , the newly arrived non - professional personnel lack the required capability to care for the pateints . these situations are fearful for patients ; especially on hospitalization and during the diagnostic / therapeutic procedures , the pateints are worried about the occurrence of unknown events . i am afraid of being hospitalized , i am afraid of the doctors act , they do in a way that my valve be occluded and instruct me to the operation . in this case , it would be my third operation . when the pateints are worried , they try not to get hospitalized as much as possible ; otherwise , they try to get discharged earlier , all of which signify the lack of confidence in the therapeutic / health system that definitely has some negative effects on the patients treatment . knowing nothing about the problems , their reasons , and problem - solving strategies spontaneously , i felt a sort of post - operative anxiety in myself , i thought maybe my condition got worse , i am feared of the noise in my valves , it causes me not to have a comfortable sleep overnight , all the time it occupies my mind . ( participant # 7 , 67-year - old female , mitral valve replacement ) . sometimes fear following the valve replacement is productive in nature and acts as a motive for following the lab results and the efficacy of treatment and education . for example , it results in a feeling of lack of confidence on the therapeutic team , which causes worry , injury , and anxiety . most therapeutic centers in iran lack the facilities for valve replacement surgery , and this is also the case regarding experiences of the care team . these patients were often referred to the main centers . for removing my womb , i was hospitalized for 2 months . i was referred to one of them ( doctors ) for surgery most of the doctors rejected me due to the lack of therapeutic faccilities . ( participant # 3 , 32 years female , mitral valve replacement ) . in case of need for treatment or surgery the second source of worry for participants is the carelessness and failure of the therapeutic team . so , the patients do not refer to the doctors unless they are assured about them . the reason for their worry is that if these patients face a problem , what do they do ? they desperately refer to other doctors and therapeutic centers which gives them a sort of worry . i fear everyone and everything and i told myself about the failure in surgery and its bad outcomes , i.e. dying for nothing . ( participant # 2 , 47-year - old female , mitral valve replacement done twice ) . ( participant # 7 , 67-year - old female , mitral valve replacement ) . for a cheeper pt , i often refer to hospitals , i always have the anxiety of a wrong pt . these worries are : worry of unexpected events , worry of the outcome of warfarin use , worry of the functioning of prosthetic valve , and worry of coagulatory test results . all the time the patients are worried about an unexpected event happening for different reasons , e.g. shortage of drugs and the discontinuty of valve function . sometimes i no longer bear it and have anxiety . for instance , when i have no warfarin , i think if the valves were non - functional , what can i do for it , how long really it works for me . these worries are intensified with a high risk of endocarditis , angiography , tooth extraction , or less education . i have a hard job , i must lift heavy loads of construction materials as a constitutional worker . ( participant # 12 , 34-year - old male , both mitral and aortic valve replacement ) . ( participant # 2 , 47-year - old male , mitral valve replaced twice ) . sometimes these worries result in the patient not following the treatment . when they performed angiography for me , i feared of dying for no reasons . despite the instructions of the doctor , i left the hospital for home , but desperately i came here again . ( participant # 8 , 50-year - old male , both mitral and aortic valve replacement ) . these patients should continue using warfarin until the end of life and all team members emphasize on this . so , patients are worried about forgetting the drug or its overdose , especially in some conditions like pregnancy and/or surgery . now , i fear everything , even going to a dentist and a bad luck . when patients face a new situation ( a change in weather , travel and food regimen , using new drugs , and change in body metabolism , all of which have an effect on inr ) , they have to repeat the test and report its results to their doctors . the facilities are not available all the time and in case of inadequte education , it results in recurrent referrals and longer hospitalizations . disturbances in daily living and transmission of the disease to offsprings are one of the worries . for a long time during their life , these patients are involved in rheumatic heart disease , valvular involvement and replacement , and its outcomes to the extent that the condition afffects their routine life which results in a busy mind and constant worry . there was a great fear and threat in my mind on living with this sort of valve . another source of worry among the valve - replaced patients , especially affecting the offspring , is the repetitive nature of the exacerbation of the disease and the results of lab reports . however , in the present time , majority of the society members and their families , for different reasons , do not provide the required support . one thing that made me have more fear on operation and reject its proposal was my children 's fear of losing me and dying , and hence i bore the pain and rejected the operation . , ( participant # 8 , 50-year - old male , both aortic and mitral valve replacement ) . the other source of worry is the transmission of disease to the children . for instance , during pregnancy , a change in the anticoagulant regimen ( e.g. warfarin , heparin ) should be made . another stressor that worsened my conditions was that everyday i heard words on the condition of my newborn baby . will it be healthy or have some problems in its heart ? ( participant # 4 , 40-year - old female , mitral valve replacement ) . when these pateints use the teratogenic drug warfarin , especially in the initial stages of the pregnancy , they have a troublesome life . because all the time , they hear about the teratogenicity of these agents and are worried about the defects it causes in their babies . fear of hospital is one of the themes that consisted of the following : fear of re - operation , fear of hospital milieu and the facilities of the hospital . the patients are not aware of most of the therapeutic / diagnostic methods used for valve - replaced pateints during their life period to the extent that they think that most of their problems could be solved with some ordinary lab tests . sometimes the unfavorable outcomes of these tests made them reject the diagnostic procedures or re - operation . my fear 's origin is in having no information , forgetting the instructions and not obeying the orders . sometimes the patients fear originated from their previous experiences or , in most cases , the information they received in relation to dying or related to the matter that they thought that they died because of their inadequate search for a better hospital and hurry in operation . so , they are much worried about the facilities and the capability of the surgeons . it took 6 months that i decided for the operation . i was so feared of signing the informed consent and took the responsibility of discharging from hospital . ( participant # 8 , 50-year - old male , both aortic and mitral valve replacement ) . these pateints are asking and searching much for their obligatory operation valve surgery and also the alternatives , especially the non - surgical ones . one of their concerns in this regard is the carelessness of the surgeons , their insufficient experience , or the lack of facilities of the care team . the common source of fear for these patients is the unfamiliar environment and pepole . among the multiple sources of fear , one important thing is the pateints worry on the conflict and disagreement between the therapeutic team , mostly on diagnostic and treatment issues . the patients minds are occupied with problems on the follow - up and post - treatment risks and outcomes , which mostly begin after the valve replacement . in such big cities , like tehran , you can find the tactful doctors , only a few of them are here and they are busy all the time . ( participant # 11 , 45-year - old male , mitral valve replacement ) . because of their worry , pateints are trying to communicate with the nurses and doctors and ask them about the disease , treatment , and lifestyle . but because of inadequate education and the lack of opportunities to communicate with the therapeutic team , the pateints receive information from other sources which results in having doubts about the treatment or the fundamental errors that , by itself , aggravates the their anxiety . the theme of fear for unknowns consists of fear of the unexpected and unfavorable events and fear of the establishment of new condition , the cause of fear is often similar previous experience of the patients , i.e. the risk of infection , disturbances in inr , bleeding , and clotting , that the probability of their occurrence increases in exposure to the new situation . in addition , often in therapeutic settings , the newly arrived non - professional personnel lack the required capability to care for the pateints . these situations are fearful for patients ; especially on hospitalization and during the diagnostic / therapeutic procedures , the pateints are worried about the occurrence of unknown events . i am afraid of being hospitalized , i am afraid of the doctors act , they do in a way that my valve be occluded and instruct me to the operation . in this case when the pateints are worried , they try not to get hospitalized as much as possible ; otherwise , they try to get discharged earlier , all of which signify the lack of confidence in the therapeutic / health system that definitely has some negative effects on the patients treatment . knowing nothing about the problems , their reasons , and problem - solving strategies spontaneously , i felt a sort of post - operative anxiety in myself , i thought maybe my condition got worse , i am feared of the noise in my valves , it causes me not to have a comfortable sleep overnight , all the time it occupies my mind . ( participant # 7 , 67-year - old female , mitral valve replacement ) . sometimes fear following the valve replacement is productive in nature and acts as a motive for following the lab results and the efficacy of treatment and education . for example , it results in a feeling of lack of confidence on the therapeutic team , which causes worry , injury , and anxiety . findings from the main themes of fear and worry signified the actual experience of the patients . analysis of the participants experiences showed that the patients got their fear and worry from the following sources : worry on care condition , worry of the specified conditions of the patients , worry on the instability in life , fear of hospital , and fear of unknowns . of these fear sources , three sources are related to human sources and the remaining two sources to non - human ( environmental and facilities ) sources . these novel findings signify the role of therapeutic team , especially nurses , its presence , quality , and communication with patients for eliminating their fear and worry . the findings of our study reveal that when the participants are unable to communicate with the therapeutic team , they do have a feeling of fear and worry . on facing the stressful conditions , patients require nursing care followed by their understanding of the condition . in situations where the nurses had no intimate and emotional relations with their clients , the patients feared and were worried . these findings are in line with the studies of hawley who revealed that a positive conversation between the emergency ward nurses and patients resulted in confidence , sympathy , insurance , and relieving the worry on the unknowns of the disease . baldursdottir and jonsdottir pointed out that the patients reported capability as the most important care behavior of the emergency ward nurses . in our study , we found from the participants experiences that worry on the therapeutic team and worry on the carelessness of the team are the sources of unexpected events . revealed that the most important worries among chinese patients were about delay in administering the drug , no on - time treatment , and the non - constant sitting on patient 's bedside . also , jooybari et al . showed that being in the same room , patients could have an intimate relationship among them and in the absence of the nurse , this relationship develops a sort of robust motivation for cooperation and social interactions . badir stated that family members have a significant role in patients health and recovery process . tensions in valve - replaced patients are the psychosocial effects of the problem ( e.g. waiting for the result of clinical test , fear of losing the valve , etc . ) . these sources of tension in everyday life require adaptation and , in some cases , are among the routine problems , like using warfarin or not using it and also its effects on the body . these usual events , by themselves , are not catastrophic and ailing , but due to their cumulative effects on the individual 's life , they could result in some somatic problems for the patient . other stressors with regard to patient 's condition are the ones that have an effect on his / her life , e.g. life events that , in some cases , cause social / psychological crises like physical disability , permanent disability , or problematic life with a prosthetic valve . revealed that some factors ( e.g. physical inactivity , low salary due to inactivity , lack of entertainment , and lower satisfaction in emotional support ) are among the factors causing post - heart transplantation disappointment in patients . the constant noise of prosthetic valve , the financial problems , and the physical disability result in more anxiety . losing the role function or perceived aim in life can cause tension and discomfort . each of these specified variables or the extra needs of the patients results in ineffective adaptation which can be resolved with post - replacement recovery and rehabilitation . in line with these findings , , in their study on heart recipients , revealed that the group of recipients who followed a regular training program and received emotional support postoperatively had a significant improvement in psychological function . gross and coworkers reported that anxiety is an inseparable problem of chronic disease and these patients have no favorable quality of life . hence , their anxiety must be assessed and managed because of its effect on treatment and in following the prescriptions . in their study , corruble et al . emphasized on anxiety as a leading issue which , especially in post - transplantation period , could be due to the drug misuse and other factors and results in an increased mortality rate . overall , the actual assessment of the stressful situation and its sources can improve the care condition of such patients . the worry and fear after heart valve replacement originate from two main sources , namely human sources or the personnel involved in care and the non - human sources which relate to the environment and logistic facilities . being aware of these fears and worry sources and stressors helps the caregivers in providing care , essential education , and promoting the quality of life and survival . identifying the actual problems of the heart valve replaced patients and detection of the unknowns of these patients signify their needs for support , advice , and psychological rehabilitation .

background : few attempts were made for alleviating the physical / psychological problems among the cardiac valve replaced patients and no comprehensive study was done based on the experiences of such patients . this study was undertaken to describe the stressful experiences of the heart valve - replaced patients.materials and methods : in this qualitative study performed during 2012 - 2013 with a content analysis approach , 13 patients from tehran and kashan therapeutic centers participated . the study sampling was accomplished with purposeful sampling using a semi - structured interview that continued until data saturat ion . all interviews were recorded , and were immediately handwritten word by word and finally typewritten . description and analysis of the data were done by graneheim and lundman content analysis.results:one hundred and seventy - five primary codes were derived among the 680 codes taken from the participants interviewed . using abstract and deep perception of the categories , 14 subcategories and 5 themes were derived . the themes are as follows : worry of care conditions , worry of life with the ongoing condition of having prosthetic cardiac valve , worry regarding the instability in life , fear of hospital , and fear of unknown factors . each theme consisted of special subsidiary themes with specific functions.conclusions:the main themes of fear and worry about on losing the valve were identified and introduced in the cardiac valve - replaced patients . as the nature and function of these themes are different in different societies , recognition and discrete definition of them are necessary for care planning and promotion .

I M R Worry on care condition Worry on the effect of disease on life stability D C

since that time , each year , the success in valve replacement depends on the myocardial function , health condition , technical skills of the surgical team , and the quality of postoperative care . during the long years of waiting for the valve replacement , these patients had catastrophic experiences . long - term hospitalization , social isolation , and the outcomes of valve replacement ) are of upmost significance . after the valve replacement , these patients need long - life care and are at risk for thromboembolism , which obligate them to use anticoagulants . according to menendez , the late - onset complications of valve replacement are : anticoagulant - induced bleeding , prosthesis malfunction , infectious endocarditis , recurrent thrombosis , and valvular insufficiency , which represent the outcomes of failure in the care system . more than the half of the patients did not use the right dose of anticoagulants and had no information on the drug side effects and on the relevant lifestyle . in the present condition , christensen points out that the rate of complications and problems related to treatment with anticoagulants due to self - care and conventional management method are 2.5% and 7.3% , respectively . there is no agreement on the right dose of warfarin during pregnancy for supporting both mother and fetus . the doses of warfarin prescribed for valve - replaced patients depend on different factors , which by themselves have different effects on inr . in deccache and aujoulat 's report on hospitalized patients , 20% of the cases had received adequate information on their disease / health condition through advising , 20% of them were not satisfied with their care condition , and the remaining 60% were required to receive more education . replaced patients , due to their susceptibility and the risky outcomes of the procedures , have different levels of worry and anxiety , and continue to live with anxiety , doubt , and worry . the preoperative physical problems and also the postoperative physical complications in valve - replaced patients are associated with a feeling of worry . hence , identification of the needs of such patients from their own experiences is necessary for providing nursing care and lessening the fear and anxiety in them . it seems that the worries , experiences , and attitudes of the patients are ignored by the nursing staff as they are classified as their least important priorities and usually are not considered in routine care planning . assessing the potential sources for fear , worry , and emotional status ( doubt , denial , aggressiveness , and isolation , often as a general reaction to illness or self - awareness ) is of importance . moreover , to our knowledge , till the present time , no qualitative study has been reported on the experiences of these patients while facing the problems following heart valve replacement . given the special cultural , social , and religious contexts in which the heart valve replaced patients are cared for , the present study was done with a qualitative approach to describe the stressful experiences of heart valve in this qualitative study , qualitative purposeful sampling was performed to obtain the perceived experiences of the heart valve subjects consisted of cases with experiences of valve replacement and the tendency to cooperate and report their experiences . data collection was done by conducting a deep , semi - structured interview using an mp4 voice recorder . in addition , to receive further information and for a deep perception of words , some explorative questions like can you give more explanations ? and data collection was done until saturation was reached . using the graneheim and lundman content analysis for qualitative data , the synchronous description and analysis of the interviews were done as follows : ( 1 ) typing the entire interview script immediately after each session ; ( 2 ) reading through the whole text for a general perception of its content ; ( 3 ) determining the meaning units and the initial codes ; ( 4 ) classifying similar initial codes into more comprehensive categories ; and ( 5 ) determining the main themes of categories . the explanation and analysis were done in the following sequence : immediately after each interview session , its content was re - written in the word software , imported to maxqda - v7 , and then the scripts were read several times to find general perceptions of the patients based on the aim of the research . then the meaning units / initial codes were derived , merged , and classified based on similarities . all efforts were taken to keep maximal homogeneity within the categories and also maximal heterogeneity between categories , and avoid the inclusion of the same data in two categories . ranking was done for new category based on aims and the main concepts were taken for the validity and reliability of results . based on polit and beck 's approval , for the accuracy of research findings in addition , for the acceptance of the data , all academic members with an experience of qualitative research were requested to evaluate the codes derived . through reviewing the handwritten scripts by the participants and peer - reviewing it by the research team colleagues , the validity of data was checked . for transferability , the participants were requested to declare some items for completing and correcting the codes . the written informed consent and the signed agreement of having the right to withdraw from the study or continue with the study whenever they wanted to were obtained from all participants . from the rich and informative description of participants , then , based on similarities , they were classified and summarized into 14 subcategories . based on the conceptual and abstract exploration of the themes and using analysis and comparison , five subsidiary themes and a main theme of a life associated with fear and worry were extracted . most therapeutic centers in iran lack the facilities for valve replacement surgery , and this is also the case regarding experiences of the care team . i was referred to one of them ( doctors ) for surgery most of the doctors rejected me due to the lack of therapeutic faccilities . in case of need for treatment or surgery the second source of worry for participants is the carelessness and failure of the therapeutic team . they desperately refer to other doctors and therapeutic centers which gives them a sort of worry . these worries are : worry of unexpected events , worry of the outcome of warfarin use , worry of the functioning of prosthetic valve , and worry of coagulatory test results . ( participant # 2 , 47-year - old male , mitral valve replaced twice ) . sometimes these worries result in the patient not following the treatment . despite the instructions of the doctor , i left the hospital for home , but desperately i came here again . these patients should continue using warfarin until the end of life and all team members emphasize on this . when patients face a new situation ( a change in weather , travel and food regimen , using new drugs , and change in body metabolism , all of which have an effect on inr ) , they have to repeat the test and report its results to their doctors . disturbances in daily living and transmission of the disease to offsprings are one of the worries . for a long time during their life , these patients are involved in rheumatic heart disease , valvular involvement and replacement , and its outcomes to the extent that the condition afffects their routine life which results in a busy mind and constant worry . there was a great fear and threat in my mind on living with this sort of valve . another source of worry among the valve - replaced patients , especially affecting the offspring , is the repetitive nature of the exacerbation of the disease and the results of lab reports . however , in the present time , majority of the society members and their families , for different reasons , do not provide the required support . one thing that made me have more fear on operation and reject its proposal was my children 's fear of losing me and dying , and hence i bore the pain and rejected the operation . another stressor that worsened my conditions was that everyday i heard words on the condition of my newborn baby . when these pateints use the teratogenic drug warfarin , especially in the initial stages of the pregnancy , they have a troublesome life . fear of hospital is one of the themes that consisted of the following : fear of re - operation , fear of hospital milieu and the facilities of the hospital . the patients are not aware of most of the therapeutic / diagnostic methods used for valve - replaced pateints during their life period to the extent that they think that most of their problems could be solved with some ordinary lab tests . sometimes the unfavorable outcomes of these tests made them reject the diagnostic procedures or re - operation . one of their concerns in this regard is the carelessness of the surgeons , their insufficient experience , or the lack of facilities of the care team . the common source of fear for these patients is the unfamiliar environment and pepole . among the multiple sources of fear , one important thing is the pateints worry on the conflict and disagreement between the therapeutic team , mostly on diagnostic and treatment issues . the patients minds are occupied with problems on the follow - up and post - treatment risks and outcomes , which mostly begin after the valve replacement . in such big cities , like tehran , you can find the tactful doctors , only a few of them are here and they are busy all the time . because of their worry , pateints are trying to communicate with the nurses and doctors and ask them about the disease , treatment , and lifestyle . but because of inadequate education and the lack of opportunities to communicate with the therapeutic team , the pateints receive information from other sources which results in having doubts about the treatment or the fundamental errors that , by itself , aggravates the their anxiety . the theme of fear for unknowns consists of fear of the unexpected and unfavorable events and fear of the establishment of new condition the cause of fear is often similar previous experience of the patients , i.e. these situations are fearful for patients ; especially on hospitalization and during the diagnostic / therapeutic procedures , the pateints are worried about the occurrence of unknown events . i am afraid of being hospitalized , i am afraid of the doctors act , they do in a way that my valve be occluded and instruct me to the operation . in this case , it would be my third operation . when the pateints are worried , they try not to get hospitalized as much as possible ; otherwise , they try to get discharged earlier , all of which signify the lack of confidence in the therapeutic / health system that definitely has some negative effects on the patients treatment . knowing nothing about the problems , their reasons , and problem - solving strategies spontaneously , i felt a sort of post - operative anxiety in myself , i thought maybe my condition got worse , i am feared of the noise in my valves , it causes me not to have a comfortable sleep overnight , all the time it occupies my mind . sometimes fear following the valve replacement is productive in nature and acts as a motive for following the lab results and the efficacy of treatment and education . for example , it results in a feeling of lack of confidence on the therapeutic team , which causes worry , injury , and anxiety . most therapeutic centers in iran lack the facilities for valve replacement surgery , and this is also the case regarding experiences of the care team . i was referred to one of them ( doctors ) for surgery most of the doctors rejected me due to the lack of therapeutic faccilities . in case of need for treatment or surgery the second source of worry for participants is the carelessness and failure of the therapeutic team . they desperately refer to other doctors and therapeutic centers which gives them a sort of worry . these worries are : worry of unexpected events , worry of the outcome of warfarin use , worry of the functioning of prosthetic valve , and worry of coagulatory test results . these worries are intensified with a high risk of endocarditis , angiography , tooth extraction , or less education . ( participant # 2 , 47-year - old male , mitral valve replaced twice ) . sometimes these worries result in the patient not following the treatment . despite the instructions of the doctor , i left the hospital for home , but desperately i came here again . when patients face a new situation ( a change in weather , travel and food regimen , using new drugs , and change in body metabolism , all of which have an effect on inr ) , they have to repeat the test and report its results to their doctors . disturbances in daily living and transmission of the disease to offsprings are one of the worries . for a long time during their life , these patients are involved in rheumatic heart disease , valvular involvement and replacement , and its outcomes to the extent that the condition afffects their routine life which results in a busy mind and constant worry . there was a great fear and threat in my mind on living with this sort of valve . another source of worry among the valve - replaced patients , especially affecting the offspring , is the repetitive nature of the exacerbation of the disease and the results of lab reports . however , in the present time , majority of the society members and their families , for different reasons , do not provide the required support . one thing that made me have more fear on operation and reject its proposal was my children 's fear of losing me and dying , and hence i bore the pain and rejected the operation . for instance , during pregnancy , a change in the anticoagulant regimen ( e.g. another stressor that worsened my conditions was that everyday i heard words on the condition of my newborn baby . when these pateints use the teratogenic drug warfarin , especially in the initial stages of the pregnancy , they have a troublesome life . because all the time , they hear about the teratogenicity of these agents and are worried about the defects it causes in their babies . fear of hospital is one of the themes that consisted of the following : fear of re - operation , fear of hospital milieu and the facilities of the hospital . the patients are not aware of most of the therapeutic / diagnostic methods used for valve - replaced pateints during their life period to the extent that they think that most of their problems could be solved with some ordinary lab tests . sometimes the unfavorable outcomes of these tests made them reject the diagnostic procedures or re - operation . one of their concerns in this regard is the carelessness of the surgeons , their insufficient experience , or the lack of facilities of the care team . the common source of fear for these patients is the unfamiliar environment and pepole . among the multiple sources of fear , one important thing is the pateints worry on the conflict and disagreement between the therapeutic team , mostly on diagnostic and treatment issues . the patients minds are occupied with problems on the follow - up and post - treatment risks and outcomes , which mostly begin after the valve replacement . in such big cities , like tehran , you can find the tactful doctors , only a few of them are here and they are busy all the time . because of their worry , pateints are trying to communicate with the nurses and doctors and ask them about the disease , treatment , and lifestyle . but because of inadequate education and the lack of opportunities to communicate with the therapeutic team , the pateints receive information from other sources which results in having doubts about the treatment or the fundamental errors that , by itself , aggravates the their anxiety . the theme of fear for unknowns consists of fear of the unexpected and unfavorable events and fear of the establishment of new condition , the cause of fear is often similar previous experience of the patients , i.e. i am afraid of being hospitalized , i am afraid of the doctors act , they do in a way that my valve be occluded and instruct me to the operation . in this case when the pateints are worried , they try not to get hospitalized as much as possible ; otherwise , they try to get discharged earlier , all of which signify the lack of confidence in the therapeutic / health system that definitely has some negative effects on the patients treatment . knowing nothing about the problems , their reasons , and problem - solving strategies spontaneously , i felt a sort of post - operative anxiety in myself , i thought maybe my condition got worse , i am feared of the noise in my valves , it causes me not to have a comfortable sleep overnight , all the time it occupies my mind . sometimes fear following the valve replacement is productive in nature and acts as a motive for following the lab results and the efficacy of treatment and education . for example , it results in a feeling of lack of confidence on the therapeutic team , which causes worry , injury , and anxiety . findings from the main themes of fear and worry signified the actual experience of the patients . analysis of the participants experiences showed that the patients got their fear and worry from the following sources : worry on care condition , worry of the specified conditions of the patients , worry on the instability in life , fear of hospital , and fear of unknowns . these novel findings signify the role of therapeutic team , especially nurses , its presence , quality , and communication with patients for eliminating their fear and worry . the findings of our study reveal that when the participants are unable to communicate with the therapeutic team , they do have a feeling of fear and worry . on facing the stressful conditions , patients require nursing care followed by their understanding of the condition . in situations where the nurses had no intimate and emotional relations with their clients , the patients feared and were worried . these findings are in line with the studies of hawley who revealed that a positive conversation between the emergency ward nurses and patients resulted in confidence , sympathy , insurance , and relieving the worry on the unknowns of the disease . baldursdottir and jonsdottir pointed out that the patients reported capability as the most important care behavior of the emergency ward nurses . in our study , we found from the participants experiences that worry on the therapeutic team and worry on the carelessness of the team are the sources of unexpected events . revealed that the most important worries among chinese patients were about delay in administering the drug , no on - time treatment , and the non - constant sitting on patient 's bedside . showed that being in the same room , patients could have an intimate relationship among them and in the absence of the nurse , this relationship develops a sort of robust motivation for cooperation and social interactions . tensions in valve - replaced patients are the psychosocial effects of the problem ( e.g. waiting for the result of clinical test , fear of losing the valve , etc . ) these sources of tension in everyday life require adaptation and , in some cases , are among the routine problems , like using warfarin or not using it and also its effects on the body . these usual events , by themselves , are not catastrophic and ailing , but due to their cumulative effects on the individual 's life , they could result in some somatic problems for the patient . life events that , in some cases , cause social / psychological crises like physical disability , permanent disability , or problematic life with a prosthetic valve . physical inactivity , low salary due to inactivity , lack of entertainment , and lower satisfaction in emotional support ) are among the factors causing post - heart transplantation disappointment in patients . the constant noise of prosthetic valve , the financial problems , and the physical disability result in more anxiety . losing the role function or perceived aim in life can cause tension and discomfort . each of these specified variables or the extra needs of the patients results in ineffective adaptation which can be resolved with post - replacement recovery and rehabilitation . gross and coworkers reported that anxiety is an inseparable problem of chronic disease and these patients have no favorable quality of life . overall , the actual assessment of the stressful situation and its sources can improve the care condition of such patients . the worry and fear after heart valve replacement originate from two main sources , namely human sources or the personnel involved in care and the non - human sources which relate to the environment and logistic facilities . being aware of these fears and worry sources and stressors helps the caregivers in providing care , essential education , and promoting the quality of life and survival . identifying the actual problems of the heart valve replaced patients and detection of the unknowns of these patients signify their needs for support , advice , and psychological rehabilitation .

cerebral ischemic stroke ( cis ) is a major cause of morbidity and mortality , and is expected to remain so until at least 2030 . high plasma levels of low - density lipoprotein cholesterol ( ldl - c ) have consistently been shown to be a risk factor for the development of atherosclerosis . plasma concentrations of ldl - c are determined primarily by the activity of the ldl receptor ( ldlr ) in the liver . proprotein convertase subtilisin - like kexin type 9 ( pcsk9 ) was recently discovered to be a major factor in cholesterol homeostasis through enhanced degradation of ldlr [ 36 ] and possibly in neural development . however , both rare mutations and common variants in the coding regions of pcsk9 can affect ldl cholesterol levels and stroke risk . recent studies identified several pcsk9 variants influencing circulating ldl - c levels . since the first identification mutation of pcsk9 was implicated in autosomal dominant hypercholesterolemia by abifadel , more than 53 missense variants have been identified . a common snp , e670 g ( rs505151 ) in exon 12 of pcsk9 , results in the substitution of glutamate for a glycine residue at position 670 in the protein carriers of 670 gln in the general population presented increased plasma tc , ldl - c , and apob levels . another study suggested a key role played by the e670 g polymorphism in determining plasma ldl - c levels and the severity of coronary atherosclerosis in the united states . more recently , the presence of the 670 g allele was significantly associated with an increased risk of large - vessel atherosclerosis ( lva ) stroke and intimal media thickness ( imt ) . however , these studies were inconsistent with previous studies [ 1416 ] , which were conducted in caucasian and african populations and failed to find this association . furthermore , the carriers of 670 g showed significantly increased ldl in men but not in women in a european population . in addition , the rs72555377 insertion polymorphism in exon 1 of pcsk9 is associated with lower ldl - c in caucasian populations , while the l11 allele , with insertion of 2 leucines , is associated with higher ldl - c , and rs562556 ( ile474val ) in exon9 of the pcsk9 gene is associated with approximately 7% lower ldl cholesterol levels in carriers in a japanese population . in our study , we used a single - base terminal extension ( snapshot ) method to detect the genotypes of the 20 single - nucleotide polymorphisms ( snp ) in the pcsk9 gene to assess the association between the human pcsk9 gene polymorphism and cis in members of the han and uygur populations of china . all participants explicitly provided permission for dna analyses as well as collection of relevant clinical data . this study was approved by the ethics committee of the first affiliated hospital of xinjiang medical university , urumqi , china ( no . 20120510 ) . subjects were from a han population and a uygur population who lived in the xinjiang uygur autonomous region of china . we recruited the cis group from the first affiliated hospital of xinjiang medical university neurology department between since october 2011 and may 2012 , and the control group came from the same hospital in the same period . in the cis group , there were 408 cis patients ( 158 uygur , 250 han ) , mean age 61.9711.80 years . inclusion criteria were : ( 1 ) diagnosed in accordance with the standards set at 10 international classifications of diseases ( icd10 ) ; ( 2 ) confirmed by mri . exclusion criteria were : ( 1 ) patients with chd ; ( 2 ) hemorrhagic cerebrovascular disease confirmed by ct or mri ; ( 3 ) refused to participate in trials . in the control group there were 348 of healthy controls ( 149 uygur , 199 han ) , mean age 61.8411.65 years . inclusion criteria were : ( 1 ) aged > 40 ; ( 2 ) no known family history of cerebrovascular disease ; ( 3 ) the cardiopulmonary physical examination and nervous system examination did not find abnormalities ; ( 4 ) mri negative except for cerebrovascular disease . all patients completed the standard test registration form , and disclosed the following data : ( 1 ) general information : age , sex , race . ( 2 ) personal history : smoking history ( daily average smoking , smoking an average of 1 day or more , time > 1 year , defined as smoking ) , ( drinking alcohol an average of 3 times per week , more than 50 g each time > 1 year , defined as drinking ) , hypertension , diabetes , hyperlipidemia , transient ischemic attack ( tia ) , atrial fibrillation ( af ) , heart valve disease , heart valve replacement , peripheral vascular disease . hypertension : the seventh world health organization /international society of hypertension league conference defined the new standard for the diagnosis of hypertension ; in our study , the diagnosis of hypertension was established if patients were treated with antihypertensive medication or if the mean of 3 measurements of systolic blood pressure ( sbp ) > 140 mm hg or diastolic blood pressure ( dbp ) > 90 mm hg , respectively . mmol / l or after fasting glucose 7.0mmol / l or glucose in line 2 h 11.1mmol / l or with a history of diabetes or treatment with insulin were considered diabetic . ( 3 ) medical history prior to admission : treatment with antihypertensive drugs , antiplatelet drugs and anticoagulants , diabetes , lipid drug , anti - seizure medication , birth control pills , hormones . ( 4 ) family history : whether grandparents , parents , siblings , and children had hypertension , diabetes , cerebral hemorrhage , cerebral infarction , myocardial infarction , coronary heart disease , or arrhythmia incidence . ( 5 ) physical examination : height , weight , blood pressure , pulse , temperature . ( 6 ) special tests : electrocardiogram , chest x - ray , heart neck ultrasound , blood routine , blood glucose , blood lipids . serum concentrations of total cholesterol ( tc ) , triglyceride ( tg ) , glucose ( glu ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) , apolipo protein a1 ( apoa1 ) , apolipo protein b ( apob ) , and apolipo lipoprotein a ( apolpa ) were measured using standard methods in the central laboratory of first affiliated hospital of xinjiang medical university . fasting blood samples ( 5 ml ) drawn by venipuncture were taken from all participants early in the morning . the blood samples were drawn into a 5-ml ethylene diamine tetraacetic acid ( edta ) tube and centrifuged at 4000g for 5 min to separate the plasma content . the dna samples were stored at 80c until use , then diluted to 50 ng/l concentration . we selected the genotypes of the 20 snps in the pcsk9 gene using the haploview 4.2 software and the hapmap phrase ii database by using minor allele frequency ( maf ) 0.1 and linkage disequilibrium patterns with r2 0.5 as a cut - off . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end ( table 1 ) . snapshot reactions were performed as described by the manufacturer ( applied biosystems , warrington , uk ) . briefly , 4.0-l of pcr product was incubated at 37c for 60 min with 2-u shrimp alkaline phosphatase ( sap ) and 2-u exonuclease i ( exoi ) . following a 15-min incubation to inactivate the enzymes , 1 ul of digested pcr product was mixed with 5 ul of ready reaction premix , 1 ul of 1.0- um primer ( table 1 ) , and 3 ul of dh2o . this mixture was placed in the thermal cycler and underwent 25 cycles of 96c for 10 s , 50c for 5 s , and 60c for 30 s. when completed , 0.5-u sap was added and the reaction mixture was incubated for 60 min . prior to loading onto the prism 310 , 10 ul of formamide was added to 1 ul of reaction mixture and samples were heated to 95c for 5 min . all continuous variables ( e.g. , age , bmi , pulse , and cholesterol levels ) are presented as means standard deviation ( s.d . ) . the difference between the cis and control groups was analyzed using an independent - sample t - test . the differences in the frequencies of sex , hypertension , diabetes mellitus , smoking , drinking , and genotypes were analyzed using chi - square test or fisher s exact test , as appropriate . logistic regression analyses with effect ratios ( odds ratio [ or ] and 95% ci ) were used to assess the contribution of the major risk factors . all statistical analyses were performed using spss 17.0 for windows ( spss institute , chicago , usa ) . all participants explicitly provided permission for dna analyses as well as collection of relevant clinical data . this study was approved by the ethics committee of the first affiliated hospital of xinjiang medical university , urumqi , china ( no . 20120510 ) . subjects were from a han population and a uygur population who lived in the xinjiang uygur autonomous region of china . we recruited the cis group from the first affiliated hospital of xinjiang medical university neurology department between since october 2011 and may 2012 , and the control group came from the same hospital in the same period . in the cis group , there were 408 cis patients ( 158 uygur , 250 han ) , mean age 61.9711.80 years . inclusion criteria were : ( 1 ) diagnosed in accordance with the standards set at 10 international classifications of diseases ( icd10 ) ; ( 2 ) confirmed by mri . exclusion criteria were : ( 1 ) patients with chd ; ( 2 ) hemorrhagic cerebrovascular disease confirmed by ct or mri ; ( 3 ) refused to participate in trials . in the control group there were 348 of healthy controls ( 149 uygur , 199 han ) , mean age 61.8411.65 years . inclusion criteria were : ( 1 ) aged > 40 ; ( 2 ) no known family history of cerebrovascular disease ; ( 3 ) the cardiopulmonary physical examination and nervous system examination did not find abnormalities ; ( 4 ) mri negative except for cerebrovascular disease . all patients completed the standard test registration form , and disclosed the following data : ( 1 ) general information : age , sex , race . ( 2 ) personal history : smoking history ( daily average smoking , smoking an average of 1 day or more , time > 1 year , defined as smoking ) , ( drinking alcohol an average of 3 times per week , more than 50 g each time > 1 year , defined as drinking ) , hypertension , diabetes , hyperlipidemia , transient ischemic attack ( tia ) , atrial fibrillation ( af ) , heart valve disease , heart valve replacement , peripheral vascular disease . hypertension : the seventh world health organization /international society of hypertension league conference defined the new standard for the diagnosis of hypertension ; in our study , the diagnosis of hypertension was established if patients were treated with antihypertensive medication or if the mean of 3 measurements of systolic blood pressure ( sbp ) > 140 mm hg or diastolic blood pressure ( dbp ) > 90 mm hg , respectively . mmol / l or after fasting glucose 7.0mmol / l or glucose in line 2 h 11.1mmol / l or with a history of diabetes or treatment with insulin were considered diabetic . ( 3 ) medical history prior to admission : treatment with antihypertensive drugs , antiplatelet drugs and anticoagulants , diabetes , lipid drug , anti - seizure medication , birth control pills , hormones . ( 4 ) family history : whether grandparents , parents , siblings , and children had hypertension , diabetes , cerebral hemorrhage , cerebral infarction , myocardial infarction , coronary heart disease , or arrhythmia incidence . ( 5 ) physical examination : height , weight , blood pressure , pulse , temperature . ( 6 ) special tests : electrocardiogram , chest x - ray , heart neck ultrasound , blood routine , blood glucose , blood lipids . serum concentrations of total cholesterol ( tc ) , triglyceride ( tg ) , glucose ( glu ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) , apolipo protein a1 ( apoa1 ) , apolipo protein b ( apob ) , and apolipo lipoprotein a ( apolpa ) were measured using standard methods in the central laboratory of first affiliated hospital of xinjiang medical university . fasting blood samples ( 5 ml ) drawn by venipuncture were taken from all participants early in the morning . the blood samples were drawn into a 5-ml ethylene diamine tetraacetic acid ( edta ) tube and centrifuged at 4000g for 5 min to separate the plasma content . the dna samples were stored at 80c until use , then diluted to 50 ng/l concentration . we selected the genotypes of the 20 snps in the pcsk9 gene using the haploview 4.2 software and the hapmap phrase ii database by using minor allele frequency ( maf ) 0.1 and linkage disequilibrium patterns with r2 0.5 as a cut - off . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end ( table 1 ) . snapshot reactions were performed as described by the manufacturer ( applied biosystems , warrington , uk ) . briefly , 4.0-l of pcr product was incubated at 37c for 60 min with 2-u shrimp alkaline phosphatase ( sap ) and 2-u exonuclease i ( exoi ) . following a 15-min incubation to inactivate the enzymes , 1 ul of digested pcr product was mixed with 5 ul of ready reaction premix , 1 ul of 1.0- um primer ( table 1 ) , and 3 ul of dh2o . this mixture was placed in the thermal cycler and underwent 25 cycles of 96c for 10 s , 50c for 5 s , and 60c for 30 s. when completed , 0.5-u sap was added and the reaction mixture was incubated for 60 min . prior to loading onto the prism 310 , 10 ul of formamide was added to 1 ul of reaction mixture and samples were heated to 95c for 5 min . all continuous variables ( e.g. , age , bmi , pulse , and cholesterol levels ) are presented as means standard deviation ( s.d . ) . the difference between the cis and control groups the differences in the frequencies of sex , hypertension , diabetes mellitus , smoking , drinking , and genotypes were analyzed using chi - square test or fisher s exact test , as appropriate . logistic regression analyses with effect ratios ( odds ratio [ or ] and 95% ci ) were used to assess the contribution of the major risk factors . all statistical analyses were performed using spss 17.0 for windows ( spss institute , chicago , usa ) . table 2 showed the clinical characteristics of the cis patients ( n=408 ) and control participants ( n=348 ) . for all han and uygur subjects , there were no significant differences in age and sex between cis patients and control subjects , indicating the study was an age- and sex - matched case - control study . as expected , several common risk factors for cis were significantly different between the 2 subgroups : glu , low hdl - c , high ldl - c , eh , and dm . other cis risk factors , such as high tc , tg levels , and cigarette smoking and drinking , were not significantly different . table 1 shows the basic information and the distribution of genotypes and alleles of the 20 snps for the pcsk9 gene . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end . we observed that the distribution of genotypes and alleles of 3 snps ( snp1 , snp2 , and snp8 ) were significantly different between cis group and control participants . the 3 snps among the 3 groups ( total , han , and uyghur ) were examined by hardy - weinberg equilibrium test and no significant differences were found in these 3 groups ( data not shown ) . in the study , we confirmed the distribution of genotypes and alleles of the 3 snps ( snp1 , snp2 , and snp8 ) for the pcsk9 gene . for snp1 ( rs17111503 ) , the distribution of alleles showed a significant difference between cis and control participants ( p=0.028 ) in the han group , but not in the total group and uygur group . for snp2 ( rs2479408 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in the uygur group . c allele of rs2479408 was significantly higher in cis patients than in control participants ( total : 96.94% vs. 94.97% ; han : 99.80% vs. 98.24% ) . for snp3 ( rs529787 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in the total and han groups , but not in the uygur group . c allele of rs529787 was significantly higher in cis patients than in control participants ( total : 96.81% vs. 94.68% ; han : 99.80% vs. 97.99% ) ( data no shown ) . table 3 and figure 1 show patterns of linkage disequilibrium in the pcsk9 gene , with their |d| and r values . |d| values from 0.25 to 0.7 indicate moderate ld and |d| values of 00.25 indicate low ld . in the study , 3 strong ld patterns were observed between snp1 and snp2 ( |d|=0.999 ) , snp2 and snp8 ( |d|=0.983 ) , and snp1 and snp8 ( |d|=0.999 ) . the r value of snp2snp8 > 0.5 means the snp2 and snp8 can replace each other and they can not construct haplotypes simultaneously . therefore , given that the position of snp1 and snp2 are both in 2 kb upstream of pcsk9 gene and the position of snp8 is in intron3 , we constructed the haplotypes using snp1 and snp2 . the overall distribution of the haplotypes were significantly different between the cis patients and the control subjects ( all p<0.05 ) . the most frequent haplotype in this study was a - c haplotype . for han , the frequency of a - c was significantly higher in the cis patients than in the control subjects ( p=0.0011 ) . in addition , the frequency of the g - c haplotype was lower in the cis patients than in the control subjects ( p=0.0011 ) . table 5 showed that multiple logistic regression analyses were performed with age , sex , bmi , hdl - c , ldl - c , tc , tg , apoa1 apob , apolpa , eh , dm , and smoking and drinking , because these variables were the major confounding factors for cis . the significant difference of the dominant model ( cc vs. cg + gg ) of rs2479408 was retained after adjustment for covariates in the han , but not in the uygur group ( or : 75.262 , 95% confidence interval [ ci ] : 7.232783.278 , p<0.001 ) . pcsk9 , also known as neural apoptosis - regulated convertase 1 ( narc1 ) , is the ninth member of the proprotein convertase ( pc ) family . the human pcsk9 gene is located on chromosome 1p32.3 ; it encompasses 12 exons and encodes a 692 amino acid glycoprotein . pcsk9 is synthesized as an inactive zymogen , pro - pcsk9 ( 73 kda ) and contains a signal peptide , a prodomain ( residues 31152 ) and a catalytic domain ( residues 153451 ) followed by a c - terminal domain ( residues 452692 ) . pcsk9 acts as a serine protease and molecular chaperone that reduces both hepatic and extrahepatic low - density lipoprotein receptor levels through an endosomal / lysosomal pathway and increases plasma ldl cholesterol . recent advances revealed a large number of genetic variants of pcsk9 that may modulate plasma cholesterol levels either positively or negatively . gain of function missense mutations in pcsk9 were associated with autosomal - dominant hypercholesterolemia ( adh ) , a rare form of familial hypercholesterolemia ( fh ) in which neither the ldlr nor the ligand binding domain of apolipoprotein ( apo ) b100 are mutated . loss of function nonsense mutations in pcsk9 were associated with low plasma ldl - c levels and a reduced incidence of cardiovascular disease . later , many in vitro and in vivo overexpression and knockout / knockdown studies confirmed that pcsk9 targets the ldlr for degradation [ 3234 ] . studies have confirmed that both rare mutations and common variants in the coding regions of pcsk9 affect ldl cholesterol levels and stroke risk . in this study , we selected 20 snps of pcsk9 and used case - control analyses to assess the association between the human pcsk9 gene polymorphism and cis in the han and uygur populations . our findings showed the distribution of snp8 ( rs529787 ) genotypes were significantly different between cis and control participants ( p=0.049 ) . however , when analyzing han and uygur groups separately , we found that only in the han population was the distribution of snp1 ( rs1711503 ) , snp2 ( rs2479408 ) , and snp8 ( rs529787 ) alleles significantly different between cis and control participants ( p=0.028 , p=0.013 , p=0.006 , respectively ) . for snp1 ( rs17111503 ) , the frequency of a allele was higher in cis than in control participants ( p=0.028 , 39.80% vs. 32.66% ) in the han group , indicating that the risk of cis was increased with the a allele of rs17111503 . for snp2 ( rs2479408 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in the uygur group . c allele of rs2479408 was significantly higher in cis patients than in control participants ( total : 96.94% vs. 94.97% ; han : 99.80% vs. 98.24% ) . moreover , the significant difference of the dominant model ( cc vs. cg + gg ) of rs2479408 was retained after adjustment for covariates : age , sex , bmi , hdl - c , ldl - c , tc , tg , apoa1 apob , apolpa , eh , dm , and smoking and drinking in the han group ( or : 75.262 , 95% confidence interval [ ci ] : 7.232783.278 , p<0.001 ) , indicating that the risk of cis was increased with the c allele of rs2479408 . for snp3 ( rs529787 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in uygurs . c allele of rs529787 was significantly higher in cis patients than in control participants ( total : 96.81% vs. 94.68% ; han : 99.80% vs. 97.99% ) . when we constructed the haplotypes using snp1 and snp2 , we found that the most frequent haplotype in this study was a - c haplotype . for han , the frequency of a - c was significantly higher in the cis patients than in the control subjects ( p=0.0011 ) , but the frequency of the g - c haplotype was lower in the cis patients than in the control subjects ( p=0.0011 ) . this fully showed that a allele of rs17111503 and c allele of rs2479408 may be the risk factor of cis , and g allele of rs17111503 and g allele of rs2479408 may be the protective factor of cis . snp20 ( rs505151 ) was observed in the exon12 of the pcsk9 gene and the polymorphisms caused the substitution of glutamate for a glycine residue at position 670 in the protein . the studies about the association between rs505151 of pcsk9 gene polymorphisms ( e670 g ) and the cardiovascular risk have provided inconsistent results , as the introduction of description . our study was consistent with previous studies [ 1416 ] showing no significant association between the polymorphism of pcsk9 ( rs505151 ) and cis . by comparison , we found the age of our control subjects was higher than the other studies and the study by afef slimani . in our study , there were no significant difference in age between cis patients ( age : 63.5611.37 ) and control subjects ( age : 62.3511.79 ) ( p=0.269 ) , but in the study by afef slimani , there were significant difference in age between cis patients ( age : 66/54.576.50 ) and control subjects ( age : 49/4555 ) ( p<0.0001 ) . age is a risk factor for stroke , and this may be why our conclusions were not consistent with their conclusions . in addition , there may be differences in populations and geographical factors that explain some differences . we found that both rs1711503 and 2479408 of pcsk9 were associated with cis in the han population of china . a - c haplotype may be a risk genetic marker of cis in han in china . a allele of rs17111503 and c allele of rs2479408 may be the risk marker of cis . studies with statistically significant numbers of clinical samples are needed for further research in china .

backgroundcerebral ischemic stroke ( cis ) is a major cause of morbidity and mortality . its main pathological basis is atherosclerosis ( as ) ; in turn , the main risk factor in as is dyslipidemia . human proprotein convertase subtilisin / kexin9 ( pcsk9 ) plays a key role in regulating plasma low - density lipoprotein ( ldl ) cholesterol levels . we sought to assess the association between pcsk9 and cis in chinese han and uygur populations.material/methodswe selected 408 cis patients and 348 control subjects and used a single - base terminal extension ( snapshot ) method to detect the genotypes of the 20 single - nucleotide polymorphisms ( snps ) in pcsk9.resultsdistribution of snp8 ( rs529787 ) genotypes showed a significant difference between cis and control participants ( p=0.049 ) . however , when analyzing han and uygur populations separately , we found that only han subjects showed distribution of snp1 ( rs1711503 ) , snp2 ( rs2479408 ) , and snp8 ( rs529787 ) alleles that was significantly different between cis and control participants ( p=0.028 , p=0.013 , p=0.006 , respectively ) , and distribution of snp2 ( rs2479408 ) in the dominant model ( cc vs. cg + gg ) was significantly different between cis and control participants ( p=0.013 ) , even after adjustment for covariates ( or : 75.262 , 95% confidence interval [ ci ] : 7.232783.278 , p<0.001 ) . distribution of the 2 haplotypes ( a - c and g - c ) ( rs1711503 and rs2479408 ) was significantly different between cis and control participants ( both , p=0.011).conclusionsboth rs1711503 and rs2479408 of pcsk9 genes were associated with cis in the han population of china . a - c haplotype may be a genetic marker of cis risk in this population .

Background Material and Methods Ethics approval of the study protocol Subjects Clinical characteristics of the study participants Biochemical analysis Blood collection and DNA extraction SNaPshot Reactions Statistical analysis Results Discussion Conclusions

cerebral ischemic stroke ( cis ) is a major cause of morbidity and mortality , and is expected to remain so until at least 2030 . high plasma levels of low - density lipoprotein cholesterol ( ldl - c ) have consistently been shown to be a risk factor for the development of atherosclerosis . plasma concentrations of ldl - c are determined primarily by the activity of the ldl receptor ( ldlr ) in the liver . proprotein convertase subtilisin - like kexin type 9 ( pcsk9 ) was recently discovered to be a major factor in cholesterol homeostasis through enhanced degradation of ldlr [ 36 ] and possibly in neural development . however , both rare mutations and common variants in the coding regions of pcsk9 can affect ldl cholesterol levels and stroke risk . since the first identification mutation of pcsk9 was implicated in autosomal dominant hypercholesterolemia by abifadel , more than 53 missense variants have been identified . a common snp , e670 g ( rs505151 ) in exon 12 of pcsk9 , results in the substitution of glutamate for a glycine residue at position 670 in the protein carriers of 670 gln in the general population presented increased plasma tc , ldl - c , and apob levels . another study suggested a key role played by the e670 g polymorphism in determining plasma ldl - c levels and the severity of coronary atherosclerosis in the united states . more recently , the presence of the 670 g allele was significantly associated with an increased risk of large - vessel atherosclerosis ( lva ) stroke and intimal media thickness ( imt ) . in addition , the rs72555377 insertion polymorphism in exon 1 of pcsk9 is associated with lower ldl - c in caucasian populations , while the l11 allele , with insertion of 2 leucines , is associated with higher ldl - c , and rs562556 ( ile474val ) in exon9 of the pcsk9 gene is associated with approximately 7% lower ldl cholesterol levels in carriers in a japanese population . in our study , we used a single - base terminal extension ( snapshot ) method to detect the genotypes of the 20 single - nucleotide polymorphisms ( snp ) in the pcsk9 gene to assess the association between the human pcsk9 gene polymorphism and cis in members of the han and uygur populations of china . this study was approved by the ethics committee of the first affiliated hospital of xinjiang medical university , urumqi , china ( no . subjects were from a han population and a uygur population who lived in the xinjiang uygur autonomous region of china . we recruited the cis group from the first affiliated hospital of xinjiang medical university neurology department between since october 2011 and may 2012 , and the control group came from the same hospital in the same period . in the cis group , there were 408 cis patients ( 158 uygur , 250 han ) , mean age 61.9711.80 years . in the control group there were 348 of healthy controls ( 149 uygur , 199 han ) , mean age 61.8411.65 years . hypertension : the seventh world health organization /international society of hypertension league conference defined the new standard for the diagnosis of hypertension ; in our study , the diagnosis of hypertension was established if patients were treated with antihypertensive medication or if the mean of 3 measurements of systolic blood pressure ( sbp ) > 140 mm hg or diastolic blood pressure ( dbp ) > 90 mm hg , respectively . serum concentrations of total cholesterol ( tc ) , triglyceride ( tg ) , glucose ( glu ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) , apolipo protein a1 ( apoa1 ) , apolipo protein b ( apob ) , and apolipo lipoprotein a ( apolpa ) were measured using standard methods in the central laboratory of first affiliated hospital of xinjiang medical university . we selected the genotypes of the 20 snps in the pcsk9 gene using the haploview 4.2 software and the hapmap phrase ii database by using minor allele frequency ( maf ) 0.1 and linkage disequilibrium patterns with r2 0.5 as a cut - off . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end ( table 1 ) . following a 15-min incubation to inactivate the enzymes , 1 ul of digested pcr product was mixed with 5 ul of ready reaction premix , 1 ul of 1.0- um primer ( table 1 ) , and 3 ul of dh2o . this mixture was placed in the thermal cycler and underwent 25 cycles of 96c for 10 s , 50c for 5 s , and 60c for 30 s. when completed , 0.5-u sap was added and the reaction mixture was incubated for 60 min . , age , bmi , pulse , and cholesterol levels ) are presented as means standard deviation ( s.d . ) the difference between the cis and control groups was analyzed using an independent - sample t - test . the differences in the frequencies of sex , hypertension , diabetes mellitus , smoking , drinking , and genotypes were analyzed using chi - square test or fisher s exact test , as appropriate . logistic regression analyses with effect ratios ( odds ratio [ or ] and 95% ci ) were used to assess the contribution of the major risk factors . subjects were from a han population and a uygur population who lived in the xinjiang uygur autonomous region of china . we recruited the cis group from the first affiliated hospital of xinjiang medical university neurology department between since october 2011 and may 2012 , and the control group came from the same hospital in the same period . in the cis group , there were 408 cis patients ( 158 uygur , 250 han ) , mean age 61.9711.80 years . in the control group there were 348 of healthy controls ( 149 uygur , 199 han ) , mean age 61.8411.65 years . hypertension : the seventh world health organization /international society of hypertension league conference defined the new standard for the diagnosis of hypertension ; in our study , the diagnosis of hypertension was established if patients were treated with antihypertensive medication or if the mean of 3 measurements of systolic blood pressure ( sbp ) > 140 mm hg or diastolic blood pressure ( dbp ) > 90 mm hg , respectively . ( 4 ) family history : whether grandparents , parents , siblings , and children had hypertension , diabetes , cerebral hemorrhage , cerebral infarction , myocardial infarction , coronary heart disease , or arrhythmia incidence . serum concentrations of total cholesterol ( tc ) , triglyceride ( tg ) , glucose ( glu ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) , apolipo protein a1 ( apoa1 ) , apolipo protein b ( apob ) , and apolipo lipoprotein a ( apolpa ) were measured using standard methods in the central laboratory of first affiliated hospital of xinjiang medical university . we selected the genotypes of the 20 snps in the pcsk9 gene using the haploview 4.2 software and the hapmap phrase ii database by using minor allele frequency ( maf ) 0.1 and linkage disequilibrium patterns with r2 0.5 as a cut - off . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end ( table 1 ) . following a 15-min incubation to inactivate the enzymes , 1 ul of digested pcr product was mixed with 5 ul of ready reaction premix , 1 ul of 1.0- um primer ( table 1 ) , and 3 ul of dh2o . this mixture was placed in the thermal cycler and underwent 25 cycles of 96c for 10 s , 50c for 5 s , and 60c for 30 s. when completed , 0.5-u sap was added and the reaction mixture was incubated for 60 min . , age , bmi , pulse , and cholesterol levels ) are presented as means standard deviation ( s.d . ) the difference between the cis and control groups the differences in the frequencies of sex , hypertension , diabetes mellitus , smoking , drinking , and genotypes were analyzed using chi - square test or fisher s exact test , as appropriate . logistic regression analyses with effect ratios ( odds ratio [ or ] and 95% ci ) were used to assess the contribution of the major risk factors . table 2 showed the clinical characteristics of the cis patients ( n=408 ) and control participants ( n=348 ) . for all han and uygur subjects , there were no significant differences in age and sex between cis patients and control subjects , indicating the study was an age- and sex - matched case - control study . as expected , several common risk factors for cis were significantly different between the 2 subgroups : glu , low hdl - c , high ldl - c , eh , and dm . other cis risk factors , such as high tc , tg levels , and cigarette smoking and drinking , were not significantly different . table 1 shows the basic information and the distribution of genotypes and alleles of the 20 snps for the pcsk9 gene . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end . we observed that the distribution of genotypes and alleles of 3 snps ( snp1 , snp2 , and snp8 ) were significantly different between cis group and control participants . in the study , we confirmed the distribution of genotypes and alleles of the 3 snps ( snp1 , snp2 , and snp8 ) for the pcsk9 gene . for snp1 ( rs17111503 ) , the distribution of alleles showed a significant difference between cis and control participants ( p=0.028 ) in the han group , but not in the total group and uygur group . for snp2 ( rs2479408 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in the uygur group . c allele of rs2479408 was significantly higher in cis patients than in control participants ( total : 96.94% vs. 94.97% ; han : 99.80% vs. 98.24% ) . for snp3 ( rs529787 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in the total and han groups , but not in the uygur group . c allele of rs529787 was significantly higher in cis patients than in control participants ( total : 96.81% vs. 94.68% ; han : 99.80% vs. 97.99% ) ( data no shown ) . in the study , 3 strong ld patterns were observed between snp1 and snp2 ( |d|=0.999 ) , snp2 and snp8 ( |d|=0.983 ) , and snp1 and snp8 ( |d|=0.999 ) . therefore , given that the position of snp1 and snp2 are both in 2 kb upstream of pcsk9 gene and the position of snp8 is in intron3 , we constructed the haplotypes using snp1 and snp2 . the overall distribution of the haplotypes were significantly different between the cis patients and the control subjects ( all p<0.05 ) . the most frequent haplotype in this study was a - c haplotype . for han , the frequency of a - c was significantly higher in the cis patients than in the control subjects ( p=0.0011 ) . in addition , the frequency of the g - c haplotype was lower in the cis patients than in the control subjects ( p=0.0011 ) . table 5 showed that multiple logistic regression analyses were performed with age , sex , bmi , hdl - c , ldl - c , tc , tg , apoa1 apob , apolpa , eh , dm , and smoking and drinking , because these variables were the major confounding factors for cis . the significant difference of the dominant model ( cc vs. cg + gg ) of rs2479408 was retained after adjustment for covariates in the han , but not in the uygur group ( or : 75.262 , 95% confidence interval [ ci ] : 7.232783.278 , p<0.001 ) . pcsk9 , also known as neural apoptosis - regulated convertase 1 ( narc1 ) , is the ninth member of the proprotein convertase ( pc ) family . pcsk9 acts as a serine protease and molecular chaperone that reduces both hepatic and extrahepatic low - density lipoprotein receptor levels through an endosomal / lysosomal pathway and increases plasma ldl cholesterol . recent advances revealed a large number of genetic variants of pcsk9 that may modulate plasma cholesterol levels either positively or negatively . gain of function missense mutations in pcsk9 were associated with autosomal - dominant hypercholesterolemia ( adh ) , a rare form of familial hypercholesterolemia ( fh ) in which neither the ldlr nor the ligand binding domain of apolipoprotein ( apo ) b100 are mutated . loss of function nonsense mutations in pcsk9 were associated with low plasma ldl - c levels and a reduced incidence of cardiovascular disease . studies have confirmed that both rare mutations and common variants in the coding regions of pcsk9 affect ldl cholesterol levels and stroke risk . in this study , we selected 20 snps of pcsk9 and used case - control analyses to assess the association between the human pcsk9 gene polymorphism and cis in the han and uygur populations . our findings showed the distribution of snp8 ( rs529787 ) genotypes were significantly different between cis and control participants ( p=0.049 ) . however , when analyzing han and uygur groups separately , we found that only in the han population was the distribution of snp1 ( rs1711503 ) , snp2 ( rs2479408 ) , and snp8 ( rs529787 ) alleles significantly different between cis and control participants ( p=0.028 , p=0.013 , p=0.006 , respectively ) . for snp1 ( rs17111503 ) , the frequency of a allele was higher in cis than in control participants ( p=0.028 , 39.80% vs. 32.66% ) in the han group , indicating that the risk of cis was increased with the a allele of rs17111503 . for snp2 ( rs2479408 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in the uygur group . c allele of rs2479408 was significantly higher in cis patients than in control participants ( total : 96.94% vs. 94.97% ; han : 99.80% vs. 98.24% ) . moreover , the significant difference of the dominant model ( cc vs. cg + gg ) of rs2479408 was retained after adjustment for covariates : age , sex , bmi , hdl - c , ldl - c , tc , tg , apoa1 apob , apolpa , eh , dm , and smoking and drinking in the han group ( or : 75.262 , 95% confidence interval [ ci ] : 7.232783.278 , p<0.001 ) , indicating that the risk of cis was increased with the c allele of rs2479408 . for snp3 ( rs529787 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in uygurs . c allele of rs529787 was significantly higher in cis patients than in control participants ( total : 96.81% vs. 94.68% ; han : 99.80% vs. 97.99% ) . when we constructed the haplotypes using snp1 and snp2 , we found that the most frequent haplotype in this study was a - c haplotype . for han , the frequency of a - c was significantly higher in the cis patients than in the control subjects ( p=0.0011 ) , but the frequency of the g - c haplotype was lower in the cis patients than in the control subjects ( p=0.0011 ) . this fully showed that a allele of rs17111503 and c allele of rs2479408 may be the risk factor of cis , and g allele of rs17111503 and g allele of rs2479408 may be the protective factor of cis . snp20 ( rs505151 ) was observed in the exon12 of the pcsk9 gene and the polymorphisms caused the substitution of glutamate for a glycine residue at position 670 in the protein . the studies about the association between rs505151 of pcsk9 gene polymorphisms ( e670 g ) and the cardiovascular risk have provided inconsistent results , as the introduction of description . our study was consistent with previous studies [ 1416 ] showing no significant association between the polymorphism of pcsk9 ( rs505151 ) and cis . by comparison , we found the age of our control subjects was higher than the other studies and the study by afef slimani . in our study , there were no significant difference in age between cis patients ( age : 63.5611.37 ) and control subjects ( age : 62.3511.79 ) ( p=0.269 ) , but in the study by afef slimani , there were significant difference in age between cis patients ( age : 66/54.576.50 ) and control subjects ( age : 49/4555 ) ( p<0.0001 ) . age is a risk factor for stroke , and this may be why our conclusions were not consistent with their conclusions . we found that both rs1711503 and 2479408 of pcsk9 were associated with cis in the han population of china . a - c haplotype may be a risk genetic marker of cis in han in china . a allele of rs17111503 and c allele of rs2479408 may be the risk marker of cis .

however , the development of antivascular endothelial growth factor ( anti - vegf ) has revolutionised the treatment of a plethora of ocular angiogenic disease processes . it has become the favoured therapy for conditions such as choroidal neovascularisation , diabetic macula oedema , vein occlusions , myopic choroidal neovascularisation , and retinopathy of prematurity to name a few . in 2013 , avastin ( bevacizumab ) and lucentis ( ranibizumab ) were ranked 9th and 19th , respectively , in terms of top global sales of pharmaceutical products emphasising their impact in medicine as a whole . it could be argued that the evolution of anti - vegf therapy can be traced back to 1948 , where michaelson hypothesised that a diffusible , hypoxia - induced , angiogenic factor x was responsible for iris and retinal neovascularisation associated with ischaemic retinopathies . further research extended our understanding of this endothelial cell - specific glycoprotein ; however , it was only in 1989 ; leung et al . isolated an endothelial mitogen from pituitary follicular cells leading to it being branded , vascular endothelial growth factor ( vegf ) . at the same time , keck et al . discovered a tumour - derived factor named vascular permeability factor ( vpf ) , which was responsible for inducing vascular permeability . since then , researchers strongly suggest that this diffusible , hypoxia - induced , endothelial cell - specific factor vegf conceivably represents michelson 's retinal tissue factor x . subsequent sequencing of these two genes led to the realisation that the factors were in fact identical . in a further study , where the retinas of primates were rendered ischaemic by laser photocoagulation of the veins , neovascularisation of the iris developed suggesting the presence of a diffusible molecule . that diffusible molecule was found to be vegf mrna . furthermore , elevated levels of vegf in ocular fluids from patients with active neovascular ocular disease were found compared with ocular fluids with no vascularisation . all the evidence of angiogenesis points to the role of vegf in ocular neovascularisation . in 1997 , genetech initiated phase 1 trials for the development of an anti - vegf molecule named avastin ( bevacizumab ) . subsequent successful results from phases 2 and 3 trials led to fda approval in february 2004 for the treatment of colon cancer in combination with chemotherapy . furthermore , with the knowledge that vegf played a significant role in neovascular amd , fda approved pegaptanib ( macugen ) was created , making it the first antiangiogenic therapy for ocular neovascularisation . after approval of bevacizumab for cancer therapy and vegf 's role in wet amd , systemic iv bevacizumab was used as an off - label medication . ophthalmologists soon began to inject bevacizumab into the vitreous cavity leading to positive results virtually eliminating the systemic side effects . believing that bevacizumab would not efficiently diffuse through the retina to reach the choroid , genetech decided to generate a truncated alternative molecule . ranibizumab ( lucentis ) was determined effective by two pivotal trials : the marina ( minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular amd ) and anchor ( anti - vegf antibody for the treatment of predominantly classic choroidal neovascularisation in amd ) trials . both of these trials were the first phase 3 trials to show improvement in visual outcomes for all forms of choroidal neovascularisation and were given fda approval in 2006 [ 10 , 11 ] . a recent anti - vegf strategy , developed by regeneron , consisted of a chimeric fusion protein that acted as a decoy receptor to sequester vegf and thereby block its biological effects . aflibercept was developed to improve the pharmacokinetics of vegf binding with reduced frequency of dosing . based on the view study , aflibercept was approved by the fda in november 2011 . in this paper , we will explore the current indications of anti - vegf in a variety of ocular angiogenic conditions that has changed the visual outcomes of patients . diabetic macular oedema ( dmo or dme ) is the leading cause of visual impairment in patients aged 20 to 74 and represents a significant burden of disease with the increasing incidence and prevalence of diabetes worldwide [ 13 , 14 ] . the development of dmo occurs as a result of vascular endothelial damage with breakdown of the blood - retinal barrier . hypoxia caused by microvascular disease stimulates the release of vascular endothelial growth factor - a ( vegf - a ) which is a major contributor to this vascular permeability and angiogenesis . ranibizumab ( ivr ) ( lucentis ; genentech inc . ; marketed by novartis in europe ) belongs to a class of drugs that block the action of vegf - a , thus reducing oedema and stabilising or improving vision . it has a european marketing authorisation for the treatment of visual impairment due to macular oedema in adults and has been recommended by the national institute for health and care excellence ( nice ) in april 2013 as a treatment option if the eye has a central retinal thickness of 400 micrometres ( m ) or more at the start of treatment . this is also conditional on the manufacturer providing ivr with the discount agreed upon in the patient access scheme . the four randomised controlled trials ( rcts ) submitted to nice as evidence for clinical effectiveness included restore ( ranibizumab monotherapy or combined with laser versus laser monotherapy for diabetic macular edema ) , diabetic retinopathy clinical research network protocol i ( drcr.net ) , resolve ( safety and efficacy of ranibizumab in diabetic macular edema ) , and read-2 ( ranibizumab for edema of the macula in diabetes ) [ 1721 ] . the restore and drcr.net ( diabetic retinopathy clinical research network ) rcts received detailed attention whilst the others were judged to be of less direct relevance . the restore trial concluded ivr monotherapy and combined with laser provided superior visual acuity ( measured with early treatment diabetic retinopathy study- ( etdrs- ) like charts ) gain over patients treated with laser alone . at month 12 , the visual acuity of eyes randomised to ivr monotherapy rose by a mean average of 6.1 letters and eyes randomised to ivr plus laser photocoagulation gained a mean average of 5.9 letters . eyes randomised to laser photocoagulation alone gained fewer letters ( 0.8 ) than eyes randomised to either ivr - containing arm ( p < 0.001 ) . mean central thickness was significantly reduced from baseline with ivr ( 118.7 m ) and ivr plus laser ( 128.3 m ) versus laser ( 61.3 m ) ; both p < 0.001 . ivr monotherapy combined with laser had a safety profile in dmo similar to that in age - related macular degeneration with no endophthalmitis cases reported and one reported patient with increased intraocular pressure in each ivr arm . the drcr.net trial was funded by the us national institute of health and was a multicentre randomised ( by eye rather than participant ) to sham injection and prompt laser ( n = 293 ) , ivr and prompt laser ( n = 187 ) , ivr and deferred ( 24 weeks ) laser ( n = 188 ) , or triamcinolone and prompt laser ( n = 186 ) . only the results of 12-month follow - up were available for the nice technology appraisal , although two - year follow - up is now published . compared with the sham injection plus prompt laser group , the mean change in the visual acuity ( etdrs ) letter score from baseline was 3.7 letters greater in the ivr plus prompt laser group , 5.8 letters greater in the ivr plus deferred laser group , and 1.5 letters worse in the triamcinolone plus prompt laser group . although none of the rcts of ivr in dmo were designed primarily to assess safety outcomes , no significant differences were observed between arms in the frequency of ocular and nonocular adverse events . the approval of ivr by the u.s . food and drug administration ( fda ) for dmo was based on genentech 's phase iii trials , ride , and rise . these trials were identically designed , parallel , double - blind , multicentre , three - year trials . subjects received 0.3 mg ivr ( n = 250 ) , 0.5 mg ivr ( n = 252 ) , or sham injection ( n = 247 ) . all patients were evaluated monthly for the need for macular laser according to protocol - specified criteria including central foveal thickness 250 m . in rise , 18% of sham patients gained 15 letters versus 45% of 0.3 mg ( p < 0.0001 ; difference versus sham adjusted for randomisation stratification factors , 24% ) and 39% of 0.5 mg ivr patients ( p < 0.001 ; adjusted difference , 21% ) . in ride , 12% of sham patients gained 15 letters versus 34% of 0.3 mg patients ( p < 0.0001 ; adjusted difference , 21% ) and 46% of 0.5 mg ivr patients ( p < 0.0001 ; adjusted difference , 33% ) . ivr patients underwent significantly fewer macular laser procedures and ocular safety was consistent with other ivr studies ( endophthalmitis in 4 ivr patients ) . guidance by nice states that up to 0.5 mg of ivr should be given monthly and continued until maximum visual acuity is reached , defined as stable visual acuity for three consecutive months . the fda has approved the lower dose of 0.3 mg , once monthly injections of ivr for dmo . it appears that the lower dose of 0.3 mg has a similar success profile to the higher 0.5 mg dose ; however the nhs is able to obtain the drug at a discounted rate through a patient access scheme and therefore prescribe up to 0.5 mg for the same cost . bevacizumab ( ivb , avastin ; genentech inc . ) is a full - length humanised antibody that binds all forms of vegf - a . it is not licensed for intraocular use but is a much less costly alternative with a good evidence base . the decision support unit ( dsu ) is commissioned by nice to provide research to support the institute 's technology appraisal programme . as such , they have evaluated the efficacy of ivb for the treatment of dmo . based on seven rcts , they conclude that efficacy measures for visual acuity ( bcva etdrs 15 letters ) favoured ivb compared with laser therapy , although the effect size is diminished as follow - up time is increased . bcva logmar scores indicate that only longer - term treatment is advantageous over laser therapy , whilst changes in central retinal thickness did not indicate that ivb confers a sustained advantage over laser therapy . of note , some trials were not included within the report such as the 24-month data from the bolt study . this was a prospective , masked , single centre , two - arm trial with subjects randomised to either ivb or macular laser therapy . at two years , the ivb group gained a mean of 8.6 etdrs letters , whereas the laser group lost a mean of 0.5 etdrs letters ( p = 0.005 ) . mean reduction in central retinal thickness was 146 m in the ivb arm versus 118 m in the laser arm . it is likely that the use of ivb is limited in nhs patients mainly as a result of nice guidance in favour of an alternative therapy . aflibercept ( iva , eyelea ; regeneron / bayer healthcare ) is a recent addition to the anti - vegf class . it is a fully human , soluble vegf receptor fusion protein that targets all forms of vegf - a and placental growth factor . the da vinci study group published one - year outcomes comparing different doses and dosing regimens of iva with macular laser in patients with dmo . the study was an industry sponsored double - masked , multicentre phase 2 clinical trial which randomised patients into one of five groups . mean improvements in bcva in the iva groups at week 52 were 11.0 , 13.1 , 9.7 , and 12.0 letters for different dosing regimens ( 0.5 mg every 4 weeks , 2 mg every 4 weeks , 2 mg every 8 weeks after 3 initial monthly doses , and 2 mg as needed after 3 initial monthly doses , resp . ) versus 1.3 letters for the laser group ( p 0.0001 versus laser ) . mean reductions in central retinal thickness in the iva groups at week 52 were 165.4 m , 227.4 m , 187.8 m , and 180.3 m versus 58.4 m for laser ( p < 0.0001 versus laser ) . two similarly designed , double - masked , randomised phase 3 trials ( vista and vivid ) compared iva ( 2 mg every 4 weeks ( 2q4 ) and 2 mg every 8 weeks ( 2q8 ) groups ) with laser treatment for dmo . mean bcva gains from baseline to week 52 in the iva 2q4 and 2q8 groups versus the laser group were 12.5 and 10.7 versus 0.2 letters ( p < 0.0001 ) in vista and 10.5 and 10.7 versus 1.2 letters ( p < 0.0001 ) in vivid . similarly , mean reductions in central retinal thickness were 185.9 and 183.1 versus 73.3 m ( p < 0.0001 ) in vista and 195.0 and 192.4 versus 66.2 m ( p < 0.0001 ) in vivid . further studies comparing iva , ivr , and ivb are being performed by the drcr.net group with expected reporting date january 2016 . iva has very recently been approved by the european commission for the treatment of visual impairment as a result of dmo . treatment is initiated with one injection per month for five consecutive doses , followed by one injection every two months without any requirement for monitoring between injections . after the first 12 months of treatment , the treatment interval may be extended based on visual and anatomic outcomes . a full discussion with regard to the role of intravitreal steroids or laser photocoagulation is beyond the scope of this report . it is worth mentioning that a nice technology appraisal recommends fluocinolone acetonide intravitreal implant ( iluvien , alimera sciences inc . ) for pseudophakic patients with dmo unresponsive to other treatment options . ozurdex ( dexamethasone intravitreal implant ) has recently received a european marketing license for the use in adult patients with visual impairment due to dmo who are pseudophakic or who are considered insufficiently responsive to or unsuitable for noncorticosteroid therapy . steroid implants may reduce the frequency of intravitreal injections but have recognised complications of increased intraocular pressure and cataract formation . overall , there is high quality evidence that anti - vegf drugs provide benefit compared with other therapeutic options for dmo . this is supported by systematic reviews published in the cochrane library and a similar review produced by the american academy of ophthalmology . this concluded that anti - vegf pharmacotherapy , delivered by intravitreal injection , is reasonably safe and effective in the treatment of dme . a statement by the royal college of ophthalmologists in their published diabetic retinopathy guidelines confirms that anti - vegf injections are considered the new gold standard of therapy for eyes with centre - involving macular oedema and reduced vision [ 30 , 31 ] . future research should now compare these drugs and treatment regimens to help refine clinical care pathways . recently , the targeted therapy of vegf has revolutionised the treatment of neovascular amd . in 2004 , the u.s . food and drug administration approved the first treatment targeting vegf , pegaptanib sodium injection ( macugen ; eyetech , new york , ny ) . the vision ( vegf inhibition study in ocular neovascularisation ) study was a prospective randomised double - masked trial to assess the benefit of treating early subfoveal cnv secondary to amd with pegaptanib sodium . it randomised 1186 subjects to sham versus 3 doses of the drug , with each receiving an injection every 6 weeks . 70% of those treated with the lowest ( 0.3 mg ) dose avoided 3 or more lines of visual loss at 1 year , compared to 55% of controls . the antibody fragment , ranibizumab , was approved for use in neovascular amd by the fda . the landmark anchor and marina studies aimed to access the efficacy of ranibizumab in both classic and minimally classic / occult neovascular amd , respectively . anchor was a 2-year , randomised , double - blind trial comparing ranibizumab with pdt in predominantly classic subfoveal cnv [ 11 , 33 ] . patients had to be over 50 , bcva 20/40 to 20/320 , and with a lesion size of less than 5400 microns . 423 subjects were randomised into 3 groups ( 3 monthly pdt and monthly sham intravitreal injections ; monthly 0.3 mg ranibizumab with 3 monthly sham pdt ; monthly 0.5 mg ranibizumab with 3 monthly sham pdt ) . after 1 year , 94.3% of 0.3 mg ranibizumab and 96.4% of the 0.5 mg ranibizumab groups lost less than 15 letters compared to 64.3% of those in the pdt group ( p < 0.001 ) . va improved by 15 letters or more in 35.7% of the 0.3 mg group and 40.3% of the 0.5% group , versus 5.6% of the pdt group ( p < 0.001 ) . these improvements were maintained at 2 years . on average , va improved over 8.1 to 10.7 letters from baseline , compared to an average loss of 9.8 letters in the pdt group . it concluded that both doses of ranibizumab were superior to pdt for classic neovascular amd . its sister study , the marina trial , was a similar 2-year multicentre , randomised , double - blind , placebo - controlled study comparing ranibizumab versus sham in minimally classic or occult cnv . patients had to be over 50 , bcva 20/40 to 20/320 , and with a lesion of less than 12 disc diameters . 716 subjects were randomised to monthly sham , 0.3 mg ranibizumab , and 0.5 mg ranibizumab injections . at 1 year , 94.5% of the 0.3 mg group and 94.6% of the 0.5 mg group lost less than 15 letters , compared to 62.2% of the placebo group . va improved by 15 letters in 24.5% of the 0.3 mg group and 33.8% of the 0.5 mg group , versus 5% of the sham group . the average improvement in va was 6.5 letters in the 0.3 mg group and 7.2 letters in the 0.5 mg group , compared to a loss of 10.4 letters in the sham group . both studies demonstrated that ranibizumab was effective at treating both classic and occult neovascular amd . following the encouraging results of anchor and marina , focus then shifted to investigating potential dosing regimens for ranibizumab in an attempt to reduce the treatment burden of monthly injections . the pier study was a 2-year rct involving 184 patients given quarterly injections after an initial 24-month period with a subsequent phase of monthly injections in the latter part of year 2 . it found that although average va improved in the treatment groups for the first 3 months , there was a gradual decline in va ( approximately 2 letters ) from months 4 to 24 when on quarterly dosing . the excite trial then directly compared monthly versus quarterly ranibizumab injections over 1 year . it again found that va improved in both regimes over the first 3 months and declined in the quarterly groups over the next 9 months . average letters gained at 1 year were 8.3 letters in the monthly group , 4.9 letters in the 0.3 mg group , and 3.8 letters in the 0.5 mg group . this further supported the notion that although quarterly injections improved va in neovascular amd , it was not as effective as monthly injections . the pronto ( prospective optical coherence tomography imaging of patients with neovascular amd treated with intraocular ranibizumab ) trial was a small , nonrandomised , uncontrolled , open - label study which used oct to vary ranibizumab dosing following a 3-month loading phase . thirty - seven patients were retreated if there was persistence or increase of intraretinal fluid and decrease in va of 5 or more letters or a new haemorrhage / area of cnv . over 2 years , it found an average improvement of 11.1 letters , with a mean number of injections of 9.9 . these results were comparable to anchor and marina but were limited by the study design and small sample size . the sustain ( safety and efficacy of a flexible dosing regimen of ranibizumab in neovascular age - related macular degeneration ) study was a larger , 1 year single arm study which again involved prn ranibizumab dosing following a 3-month loading phase . five hundred and thirteen patients were recruited , with parameters for retreatment being loss of more than 5 letters or increase of 100 microns in crt . mean bcva was + 5.8 letters at month 3 , decreasing to + 3.6 letters at 12 months . this suggested that although va improvement does decline slightly on prn dosing , it may not worsen as much as the pier study had reported . 1098 patients were randomised to receive 0.5 mg or 2 mg ranibizumab on a monthly or prn basis . patients had to be over 50 with cnv < 12 disk diameters and bcva 20/40 to 20/320 . at 12 months , it found that the average gain in va letters was + 10.1 ( 0.5 mg monthly ) , + 9.2 ( 2 mg monthly ) , + 8.2 mg ( 0.5 mg prn ) , and + 8.6 mg ( 2 mg prn ) . the mean change from baseline in crt in the 4 groups was 172.0 m , 161.2 m , 163.3 m , and 172.4 m , respectively . this study demonstrated that monthly doses of 0.5 mg ranibizumab produce the optimum visual results for patients with neovascular amd , although prn dosing does lead to clinically meaningful visual improvement not significantly different than monthly dosing . treat and extend regime , which involves treating monthly until the macula is dry and then incrementally increasing time between injections whilst the macular remains dry [ 39 , 40 ] . this seems to lead to stabilised va with a reduction in injections but has not been assessed with a prospective , randomised controlled trial . in 2004 , intravenous bevacizumab was approved for the treatment of metastatic colon cancer , and this leads to a number of ophthalmologists using the drug off - label as an intravitreal injection . this offered a cheaper alternative to ranibizumab , although there were concerns over its safety and lack of trial data . in response to this , the catt ( comparison of amd treatments trials ) and ivan ( lucentis and avastin effective in treating wet amd ) studies aimed to compare the safety and efficacy of using ranibizumab versus bevacizumab [ 41 , 42 ] . the catt trial was a multicentre , randomised trial involving 1208 subjects comparing the efficacy and side effects of monthly or prn regimes of ranibizumab 0.5 mg and bevacizumab 1.25 mg . at 1 year , catt could not demonstrate that prn bevacizumab was not inferior to monthly ranibizumab ( rbz monthly : + 8.5 letters ; bvz monthly : + 8 letters ; rbz prn : + 6.8 letters ; bvz prn : + 5.9 letters ) , although anatomically rbz led to a greater decrease in crt . whilst it found that there was no difference in rates of death , stroke , or myocardial infarction , it did find that there was increased rate of hospitalization in the bvz group ( 24.1% versus 19% ) . at 2 years , mean gain was again similar between the 2 groups , although monthly dosing performed better than prn dosing . rates of death and thrombotic events were the same , although numbers of patients with one or more serious systemic adverse events was higher in the bvz group ( 39.9% versus 31.7% ) . ivan was a smaller , similarly designed nhs - funded trial , recruiting 628 patients . at 2 years , it found that bcva was similar between the rbz and bvz groups and between the monthly and prn regimes , although the primary outcome of bvz being noninferior to rbz was not met . pooled safety estimates of both trials found that there was no difference in death or thrombotic events between the 2 drugs but risk of systemic adverse events was higher in the bvz group . unexpectedly , it also found that the risk of death was higher in the prn regime versus monthly regime . the authors suggested that this may be due to an immunological reaction and that further investigation was needed . in 2011 , aflibercept ( vegf trap - eye ) was approved for use by the fda . this is a fusion protein that binds to all isoforms of vegf , with greater affinity than rbz and bvz . there was a hope that this increased vegf - binding activity would decrease the frequency of injections . 2419 patients were randomised to aflibercept 0.5 mg monthly ; aflibercept 2 mg monthly ; aflibercept 2 mg two monthly ( after loading phase ) ; and rbz 0.5 mg monthly . the study found that at year 1 , all aflibercept groups were noninferior to the rbz monthly groups , with the average bcva of all 3 within 0.5 letters of the rbz group . between year 1 and year 2 , all the groups were changed from a fixed regimen to a variable regimen requiring at least quarterly dosing ( capped prn ) [ retreatment if loss of 5 letters , new or persistent fluid on oct , increase in crt of 100 microns , or new haemorrhage / cnv ] . they found no difference between the groups , although there was a small decrease in bcva letters gained during the 2nd year , in keeping with previous studies . the study showed that two monthly aflibercept gave equivalent va results to monthly rbz over 2 years , whilst needing 5 fewer injections . it did not however compare the groups to fixed - dosing rbz or aflibercept over the full 2 years , which may have given better bcva results . with regard to future treatments , there are numerous new drugs for amd going through phase ii studies . among them is fovista , a compound which inhibits platelet - derived growth factor from binding to pericytes . a recent phase 2 study showed that 6 monthly fovista injections in combination with monthly rbz were 60% more effective than rbz alone . also of note is potential topical anti - vegf agents , which would eliminate many of the risks or burden of regular intravitreal injections . panoptica are currently testing pan-90806 , a topical small molecule vegf antagonist in wet amd . sailor ( safety assessment of intravitreous lucentis for amd ) investigated rbz in a large population of 4300 . this study showed rbz was safe and well tolerated in a large population and a low risk of thromboembolism . horizon ( an open - label extension trial of ranibizumab for choroidal neovascularization secondary to age - related macular degeneration ) was a 2 year study investigating the long term safety and efficacy in 853 patients completing the controlled treatment phase of 1 of the 3 2-yr clinical trials ( anchor , marina & focus ) . incidence of serious ocular and non - ocular adverse events over the 2 yrs were low and consistent with previous phase 3 studies . sana ( systemic avastin for neovascular amd ) examined the short term safety of systemic bvz in 9 patients with subfoveal cnv and bcva letter scores of 70 to 20 and crt > 300 microns . safety was measured by , change in va scores , oct measurement , fluorescein angiography , and indocyanine green angiography . the study proposed that bvz was effective and safe for all patients over 24 months whilst acknowledging the study size . abc ( avastin ( bvz ) for choroidal neovascular age - related macular degeneration ) was a double masked randomised controlled trial with two parallel treatment groups ivt bvz versus pdt ( for classic ) or pagaptanib ( for occult ) . the proportion of patients gaining / losing > 15 letters at 1 yr demonstrated bvz being more effective than standard care with low rates of adverse events . retinal vein occlusion ( rvo ) is a common cause of visual loss in the united kingdom and is the second most common retinal vascular disease after diabetic retinopathy [ 47 , 48 ] . rvos can be divided into two categories depending on the site of obstruction : central rvo ( crvo ) when occlusion involved the whole central retinal vein and branch rvo ( brvo ) when the occlusion involves only one branch of the central retinal vein . brvo occurs 2 - 3 times more commonly than crvo [ 50 , 51 ] . in the beaver dam eye study the 15-year cumulative incidence of crvo and brvo was 0.5 and 1.8% , respectively . the pathogenesis of rvo is multifactorial with thrombus formation being the primary cause but other possible causes are external compression or disease of the vein wall , for example , vasculitis . crvo is caused by external compression of the central retinal vein , which shares a common fibrous sleeve with the vein . brvo and crvo typically occur in middle aged and elderly patients ( i.e. , over age of 50 years ) with equal sex distribution . crvo is classically characterised by disc oedema , increased dilatation and tortuosity of all retinal veins , widespread of deep and superficial haemorrhages , cotton wool spots , retinal oedema , and capillary nonperfusion . in less severe forms brvos have similar features except that they are confined to a portion of the fundus . eyes with initial visual acuity of 6/12 ( 20/40 ) or better have a better prognosis for retaining good vision than those with worse vision . in brvos 50% of untreated eyes retain vision of 6/12 or better whilst 25% will have vision < 6/60 . up to 34% of nonischaemic crvo convert to ischaemic forms within 3 years . vegf is produced by retinal pigmented epithelial cells , endothelial cells , muller cells , and other ocular tissues in response to retinal hypoxia and binds to specific receptors on endothelial cells acting as a proangiogenetic factor . this in turn leads to neovascularization and vascular hyperpermeability with subsequent breakdown of the blood - retina barrier and macular oedema . until recently the standard of care for macular oedema secondary to brvo was macular grid laser macular laser photocoagulation is not recommended for the treatment of macular oedema in crvo because it does not guarantee a significant improvement in visual acuity . panretinal photocoagulation of the ischemic retina is indicated both in brvo and in crvo if iris , retinal , or disc neovascularization is present . currently available anti - vegf agents ( ranibizumab , aflibercept , and bevacizumab ) have been applied successfully in reducing macular oedema due to rvo . in may 2013 nice recommended ranibizumab ( lucentis , novartis ) as a possible treatment for some people who have sight problems because of macular oedema caused by retinal vein occlusion . indications include macular oedema in crvo or in brvo only if previous grid laser has failed or laser is not suitable due to the extent of haemorrhage . two randomized , double - masked , multicenter phase 3 trials , the cruise ( central retinal vein occlusion ) and bravo ( branch retinal vein occlusion ) studies , evaluated the efficacy of ranibizumab , compared with a sham procedure , for treating visual impairment caused by macular oedema secondary to brvo and to crvo , respectively [ 56 , 57 ] . the bravo ( n = 397 ) and cruise ( n = 392 ) trials were both 3-armed rcts carried out at multiple centres in the usa . patients were eligible if they had foveal - involved macular oedema from a crvo or brvo occurring within 12 months of study entry , bcva of 20/40 to 20/320 ( in crvo ) and to 20/400 ( in brvo ) , and centre subfield thickness ( cst ) 250 m . patients were randomised equally to sham injection , monthly intraocular ranibizumab 0.3 mg , or monthly intraocular ranibizumab 0.5 mg . patients entered a 6-month treatment phase during which monthly injections were given . in the treatment phase of bravo , patients in both the sham injection and ranibizumab groups could receive grid laser photocoagulation for rescue treatment from 3 months . in both bravo and cruise , the treatment phase was followed by a 6-month observation phase during which all groups could receive ranibizumab as needed . patients in the observation phase of bravo ( but not cruise ) could receive grid laser photocoagulation for rescue treatment for 3 months ( i.e. , at month 9 of the study ) . the final treatment in both bravo and cruise was given at month 11 , with a final study visit at month 12 . patients who completed the 12-month bravo and cruise trials could enter an open - label extension study ( horizon ) . for patients enrolled in the cruise study baseline characteristics were well balanced among the three groups ; the mean age was 68 years , mean bcva was 20/100 , the mean time from diagnosis of crvo was 3.3 months , and the mean center point thickness ( cpt ) was 685 m . at 6 months , the primary endpoint , mean change from baseline bcva letter score , was 12.7 and 14.9 in the 0.3 mg and 0.5 mg ranibizumab groups and 0.8 in the sham group ( p < 0.0001 ) . the percentage of patients who gained 15 letters in bcva was 46.2% ( 0.3 mg ) and 47.7% ( 0.5 mg ) in the ranibizumab groups and 16.9% in the sham group ( p < 0.0001 ) . the percentage of patients with a snellen equivalent bcva of 20/40 or better was 43.9% ( 0.3 mg ) and 46.9% ( 0.5 mg ) compared with 20.8% in the sham group ( p < 0.0001 ) . the percentage of patients with a snellen equivalent bcva of 20/200 or worse was 15.2% ( 0.3 mg ) and 11.5% ( 0.5 mg ) compared with 27.7% in the sham group ( p < 0.005 ) . based upon the nei vfq-25 survey , patients who received ranibizumab felt they had greater improvement ( improvement from baseline in nei vfq score : 7.1 , 0.3 mg ; 6.2 , 0.5 mg ; 2.8 , sham ) . there was greater reduction of macular edema in the ranibizumab groups because cpt was reduced by 433.7 m ( 0.3 mg ) and 452.3 m ( 0.5 mg ) compared to 167.7 m in the sham group . the percentage of patients with cpt 250 m at 6 months was 75.0% ( 0.3 mg ) , 76.9% ( 0.5 mg ) , and 23.1% ( sham , p < 0.0001 ) . this study demonstrated that six sessions of monthly injections of 0.3 mg or 0.5 mg reduced macular oedema and provided substantial visual benefit in patients with crvo . baseline characteristics for those involved in the bravo study were well balanced among the three groups ; mean bcva was 20/80 , the mean time from diagnosis of brvo was 3.5 months , and the mean cpt was 520 m . starting at month 3 , patients were eligible for grid laser treatment if hemorrhages had cleared sufficiently to allow safe application of laser and the following criteria were met : snellen equivalent bcva 20/40 or mean cst 250 m , and compared with the visit 3 months before the current visit , the patient had a gain of < 5 letters in bcva or a decrease of < 50 m in mean cst . if rescue laser was not given at month 3 , the same criteria were applied at month 4 , and if rescue laser was not given at month 4 , the criteria were applied at month 5 . at month 6 , the primary endpoint , mean change from baseline bcva letter score , was 16.6 and 18.3 in the 0.3 mg and 0.5 mg ranibizumab groups and 7.3 in the sham group ( p < 0.0001 ) . the percentage of patients who gained 15 letters in bcva was 55.2% ( 0.3 mg ) and 61.1% ( 0.5 mg ) in the ranibizumab groups and 28.8% in the sham group ( p < 0.0001 ) . the percentage of patients with a snellen equivalent bcva of 20/40 or better was 67.9% ( 0.3 mg ) and 64.9% ( 0.5 mg ) compared with 41.7% in the sham group ( p < 0.0001 ) . the percentage of patients with a snellen equivalent bcva of 20/200 or worse was 1.5% ( 0.3 mg ) and 0.8% ( 0.5 mg ) compared with 9.1% in the sham group ( p < 0.01 ) . based upon the nei vfq-25 survey , patients who received ranibizumab felt they had greater improvement ( improvement from baseline in nei vfq score : 9.3 , 0.3 mg ; 10.4 , 0.5 mg ; 5.4 , sham ) . there was greater reduction of macular oedema in the ranibizumab groups because cpt was reduced by 337.3 m ( 0.3 mg ) and 345.2 m ( 0.5 mg ) compared to 157.7 m in the sham group . the percentage of patients with cpt 250 m at month 6 was 91% ( 0.3 mg ) , 84.7% ( 0.5 mg ) , and 45.5% ( sham , p < 0.0001 ) . more patients in the sham group ( 54.5% ) received rescue grid laser therapy than in the 0.3 mg ( 18.7% ) or 0.5 mg ( 19.8% ) ranibizumab groups . after the primary endpoint in the cruise and bravo trials , patients were evaluated every month and if study eye snellen equivalent bcva was 20/40 or mean cst was 250 m , they received an injection of ranibizumab ; patients in the ranibizumab groups received their assigned dose and patients in the sham group received 0.5 mg . in patients with crvo , the mean number of ranibizumab injections during the observation period was 3.9 , 3.6 , and 4.2 in the 0.3 mg , 0.5 mg , and sham/0.5 mg groups ; and the percentage of patients that did not receive any injections during the observation period was 7.0 , 6.7 , and 4.3 , respectively . at month 12 in the ranibizumab groups , the improvement from baseline in etdrs letter score was 13.9 , very similar to the month 6 results , indicating that vision is well maintained when injections are given only if there is recurrent or residual macular oedema . patients in the sham group showed substantial improvement during the observation period when they were able to receive ranibizumab ; improvement from baseline in letter score was 0.8 at month 6 and 7.3 at month 12 . the percentage of patients who had an improvement from baseline bcva letter score 15 at month 12 was 47.0% ( 0.3 mg ) and 50.8% ( 0.5 mg ) in the ranibizumab groups , almost identical to the month 6 results . in the sham group , 33.1% of patients improved from baseline 15 in letter score at month 12 compared to 16.9% at month 6 . at month 12 , 43% of patients in the two ranibizumab groups had a snellen equivalent bcva of 20/40 compared to 35% in the sham/0.5 mg group . in patients with brvo , the mean number of ranibizumab injections during the observation period was 2.9 , 2.8 , and 3.8 in the 0.3 mg , 0.5 mg , and sham/0.5 mg groups ; and the percentage of patients that did not receive any injections during the observation period was 17.2 , 20.0 , and 6.5 , respectively . at month 12 in the ranibizumab groups , the improvement from baseline in etdrs letter score was 16.4 ( 0.3 mg ) and 18.3 ( 0.5 mg ) , very similar to the month 6 results , indicating that vision is well maintained when injections are given only if there is recurrent or residual macular oedema . patients in the sham group showed substantial improvement during the observation period when they were able to receive ranibizumab ; improvement from baseline in letter score was 7.3 at month 6 and 12.1 at month 12 . the percentage of patients who had an improvement from baseline bcva letter score 15 at month 12 was 55.2% ( 0.3 mg ) and 61.1% ( 0.5 mg ) in the ranibizumab groups , almost identical to the month 6 results . in the sham group , 43.9% of patients improved from baseline 15 in letter score at month 12 compared to 28.8% at month 6 . at month 12 , 67.9% ( 0.3 mg ) and 64.4% ( 0.5 mg ) of patients in the ranibizumab groups had a snellen equivalent bcva of 20/40 compared to 56.8% in the sham/0.5 mg group . thus , in both cruise and bravo , patients in the sham groups showed a substantial improvement in vision during the second 6 months when they were able to receive ranibizumab as needed , but their vision at month 12 was not as good as that in patients in the ranibizumab groups . for patients who entered the open - label extension study ( horizon ) , 67% of patients from bravo and 60% of patients from cruise completed month 12 of horizon . the primary outcome for the horizon extension study was mean change from horizon baseline in bcva score up to 24 months . from the bravo trial baseline , patients receiving sham ( plus ranibizumab ) and those receiving 0.5 mg ranibizumab had mean gains in bcva score of 15.6 letters and 17.5 letters , respectively . from the cruise trial baseline , patients receiving sham ( plus ranibizumab ) and those receiving 0.5 mg ranibizumab had mean gains in bcva score of 7.6 and 12.0 letters , respectively ( no confidence intervals reported ) . adverse events were reported at 6 months and 12 months in both bravo and cruise trials and for a further 12 months ' follow - up in the horizon extension study . in bravo , at 6 months there were 7 ocular adverse events ( 5.4% ) in the ranibizumab group compared with 17 ( 13% ) in the sham group , excluding occurrences of raised intraocular pressure . nonocular serious adverse events ( potentially related to vascular endothelial growth factor [ vegf ] inhibition ) at 6 months were higher in the ranibizumab group ( 5 events [ 3.8% ] ) than in the sham group ( 1 event [ 0.8% ] ) . in cruise , at 6 months there were 13 ocular adverse events ( 10.1% ) in the ranibizumab group compared with 25 ( 19.4% ) in the sham group , excluding occurrences of raised intraocular pressure . in cruise , nonocular serious adverse events ( potentially related to vegf inhibition ) were similar in both the ranibizumab and sham groups ( 3 [ 2.3% ] and 2 [ 1.6% ] , resp . ) the most common adverse event reported in bravo and cruise at 12 months was cataract , with 8 ( 6.2% ) and 9 ( 7% ) instances associated with ranibizumab treatment , respectively ; in the sham ( plus ranibizumab ) group , 3 ( 2.6% ) and 2 ( 1.8% ) instances of cataract were reported for the treatment period of 6 to 12 months . instances of raised intraocular pressure were reported in both bravo and cruise at 6 months but were academic in confidence and therefore not reported here . in the horizon extension study , the incidence of any adverse event in the sham ( plus ranibizumab ) and ranibizumab groups was 2.2% and 5.8% , respectively , for the patients ( with brvo ) recruited from bravo and 5.2% and 3% , respectively , for the patients ( with crvo ) recruited from cruise . in february 2014 nice recommended aflibercept ( eylea , bayer ) as a possible treatment for people with sight problems caused by macular oedema from central retinal vein occlusion . aflibercept for crvo was studied in particular in the copernicus and galileo trials , both phase iii clinical trials for crvo - related macular oedema . copernicus ( vascular endothelial growth factor trap - eye for macular edema secondary to central retinal vein occlusion ) was a randomised , double - blind , multicentre trial conducted in 6 non - european countries . galileo was a randomised , double - blind , multicentre trial conducted in 10 european and asian - pacific countries . in both trials the included patients had been diagnosed less than 9 months before the start of the trial and they had not received previous treatment for crvo . the trials had identical designs and criteria for the first 6 months : both were randomized , multicentre , and double - masked , and both included patients with central retinal thickness ( crt ) of 250 m and bcva of 20/40 and 20/320 . from week 0 to week 24 , patients in the intervention group received intravitreal aflibercept injection ( iai ) every 4 weeks and patients in the comparator group received a sham injection every 4 weeks . the primary end point was the proportion of those patients who gained three or more lines , and the key secondary end points were total change in bcva and crt . the primary end points were assessed at 6 months and demonstrated that 23.3% of the sham - treated patients and 49.1% of the iai patients in copernicus , with 15 and 62% of respective patients in galileo , experienced a gain of three lines or more . mean change in va for copernicus at 6 months was four letters for the sham - treated group and + 17.3 letters for the iai - treated group . mean change in va for galileo at 6 months was + 3.3 letters for the sham - treated group and + 18.0 letters for the iai - treated group . in addition , at 6 months , the proportion of patients for copernicus without retinal edema was 15.3% in the sham - treated eyes and 74.5% in the iai - treated eyes . these studies also demonstrated a rapid response in visual and anatomical outcome that was more significant if treated within 2 months of the diagnosis . early treatment with iai results in better visual and anatomical results than delaying treatment , and these results were regardless of perfusion status of the retina at the time of treatment . the copernicus trial was extended to 100 weeks and allowed all patients to then be eligible for iai 2q4 pro re nata ( prn ) treatment protocol . the galileo trial was to be extended to 76 weeks had the iai group become the iai 2q4 prn group , but the sham - treatment group continued to be treated with sham until 52 weeks . after 52 weeks , the sham group was able to be treated with iai 2q4 prn . the retreatment criteria were an increase in crt of > 50 m from the lowest previous measurement ; new or persistent cystic retinal changes or subretinal fluid or diffuse edema 250 m in the central subfield ; a loss of five letters from previous bcva if associated with any increase in crt ; or a loss of five letters between the current and most recent visit . between 6 months and 1 year , for the copernicus trial , the injection group received additional 2.7 injections while the sham / crossover group received 3.9 injections . copernicus results at 100 weeks demonstrated sustained improvements in all parameters for the treatment group when converting to prn treatment at 6 months and an additional improvement for sham treatment that was converted to prn treatment at 6 months . aflibercept was effective at reducing edema and improving vision , even when a delay in treatment takes place but outlines the likely irreversible visual damage that limits the visual recovery when treatment is delayed . high myopia is classified as greater than 6 dioptres refractive error and commonly associated with an axial length of more than 26 mm [ 6163 ] . systemic associations of high myopia include prematurity and syndromes such as down , ehlers - danlos , knobloch , marfan , noonan , pierre - robin , and stickler syndrome [ 61 , 64 ] . pathological myopia , also called degenerative myopia , is characterised by excessive elongation of the globe and associated pathological changes at the posterior pole such as tessellated fundus , posterior staphyloma , and myopic conus [ 6062 , 64 ] . it has a prevalence of 0.93.1% and studies have identified it as the primary cause for blindness in 7% and 1227% of the population in europe and asia , respectively [ 60 , 65 ] . choroidal neovascularisation ( cnv ) is one of the complications of pathological myopia and occurs in 5.211.3% of patients with high myopia [ 6063 ] . clinically , myopic cnv may be associated with underlying myopic abnormalities such as focal chorioretinal atrophy or ruptures in the rpe - bruch 's membrane - choriocapillaris complex , also called myopic cnv is characterised by the formation of abnormal blood vessels in the retina or under the retinal pigment epithelium ( rpe ) , potentially penetrating bruch 's membrane into the subretinal space . on slit - lamp biomicroscopy , a flat greyish membrane is observed , occasionally with a hyperpigmented border if chronic or recurrent . as the cnv regresses , it leaves a fibrous pigmented scar called fuchs ' or forster - fuchs ' spot as well as surrounding chorioretinal atrophy in later stages . fundus fluorescein angiography ( ffa ) is used in diagnosis in the acute stage , showing a myopic cnv lights up as a highly reflective area above the rpe , occasionally with minimal subretinal fluid . the pathogenesis of myopic cnv is not yet fully understood but is thought to involve an imbalance of proangiogenic and antiangiogenic factors brought on by the mechanical stresses of a progressively elongating retina . genetic studies have also shown possible association of single nucleotide polymorphisms in the complement factor i ( cfi ) gene on chromosome 4 that encodes a protein involved in the alternative complement pathway [ 62 , 66 ] . alternatively , myopic cnv may occur de novo in patients with no previous signs of pathological myopia . of note , in patients with preexisting myopic cnv , one study showed that 35% of fellow eyes go on to develop cnv within 8 years [ 63 , 66 ] . poor prognostic indicators include lower baseline visual acuity , age above 40 years , extensive chorioretinal atrophy , subfoveal location of the cnv , and lesion size above 400 m [ 60 , 61 , 63 , 66 ] . prior to the advent of antivascular endothelial growth factor ( anti - vegf ) therapy , management of myopic cnv was largely based on laser photocoagulation for extrafoveal cnv and verteporfin photodynamic therapy for subfoveal cnv [ 61 , 63 , 66 ] . neither has been able to show any consistent long - term visual benefit but anti - vegf agents have recently been introduced with promising results . ranibizumab ( lucentis ) is the only anti - vegf therapy licensed for the treatment of myopic cnv and has shown great potential for visual gain and preventing irreversible retinal damage in phases ii and iii clinical trials repair and radiance ( a randomized controlled study of ranibizumab in patients with choroidal neovascularization secondary to pathologic myopia ) , respectively [ 4 , 66 ] . bevacizumab ( avastin ) has not been approved for intraocular use and therefore lacks both an established safety and efficacy profile . myrror , a phase iii clinical trial , is currently ongoing to establish the role of aflibercept ( eylea ) in the treatment of myopic cnv . this algorithm involves a single initial intravitreal injection of an anti - vegf agent , followed by subsequent as needed injections . follow - up is advised as monthly for two months and then 3-monthly in the first year . at follow - up , symptoms and signs that warrant another anti - vegf injection include reduced visual acuity , visual symptoms ( e.g. , metamorphopsia ) , or signs of disease activity on ffa or oct . following the first year of monitoring , follow - up further research and monitoring are yet to establish long - term outcomes and best management strategy . since their introduction into clinical practice , the list of possible uses for anti - vegf therapy in ophthalmology has steadily expanded . anti - vegf agents have been utilized on an off - license basis for a vast array of other ocular pathologies ranging from external eye disease to ocular oncology and glaucoma . indeed , a literature search in 2008 revealed 51 different ophthalmic diseases that had been treated with bevacizumab . the development of choroidal neovascular membranes may complicate any disease process which results in a defect in bruch 's membrane and can pose a threat to vision through leakage of fluid , haemorrhage , and subretinal fibrosis . anti - vegf agents are now widely used in the treatment of cnvm regardless of aetiology . in november 2013 , nice approved ranibizumab for the treatment of choroidal neovascularisation associated with pathological myopia in the uk . this decision was based upon evidence largely extracted from the radiance ( randomized controlled study of ranibizumab in patients with choroidal neovascularisation secondary to pathologic myopia ) trial which compared ranibizumab with verteporfin photodynamic therapy ( vpdt ) . this study showed superiority of ranibizumab over vpdt in terms of improvements in bcva at month three , with improvements being sustained up to month twelve through further injections as required based on visual acuity or disease activity criteria [ 65 , 68 ] . other studies have shown a similar efficacy of ranibizumab and bevacizumab in the treatment of myopic cnv [ 69 , 70 ] . intravitreal anti - vegf agents have also been successfully utilized in the treatment of cnv related to angioid streaks , uveitis , and trauma [ 7173 ] . intravitreal injection of an anti - vegf agent is now commonly performed as a pretreatment prior to pars plana vitrectomy for severe proliferative diabetic retinopathy . several studies have demonstrated that a single dose of bevacizumab delivered in the weeks before surgery facilitates the dissection of fibrovascular membranes and reduces the likelihood of intraoperative and postoperative bleeding [ 74 , 75 ] . development or progression of tractional retinal detachment has however been reported following intravitreal injection of bevacizumab as an adjunct to pars plana vitrectomy which may be attributable to the contraction of fibrovascular membranes induced by the drug . regression and a decrease in vascular permeability of new vessels in uncomplicated proliferative diabetic retinopathy have also been demonstrated following the intravitreal injection of bevacizumab . reduction in the drive for vegf production through pan - retinal laser photocoagulation however remains the first line treatment in the uk although anti - vegf may have a role as an adjunctive therapy , particularly in cases of persistent vitreous haemorrhage [ 78 , 79 ] . retinopathy of prematurity ( rop ) in its most severe form results in tractional retinal detachment secondary to fibrovascular proliferation and remains a leading cause of childhood blindness across the world . there has been much interest recently in the use of anti - vegf agents to reduce the angiogenic drive that underlies the pathology of rop . in their prospective randomized trial reported on behalf of the beat - rop cooperative group a significant benefit of anti - vegf in the treatment of stage 3 + zone 1 disease . they also found that the development of peripheral retinal vessels continued after bevacizumab but not after laser therapy which may have a beneficial effect on conservation of visual field . although relatively large , the beat - rop study was not adequately powered to determine the safety of anti - vegf in the developing child and some concerns remain regarding the potentially unknown long - term local and systemic side effects of these medications . central serous retinopathy ( csr ) is an idiopathic condition characterized by the accumulation of subretinal fluid at the macula . although the majority of cases resolve spontaneously within six months , in some the fluid persists which can result in ongoing reduction in visual acuity and metamorphopsia . intravitreal bevacizumab has been advocated by some as a possible treatment option in such cases of persistent csr . several small case series have reported significant reductions in central macular thickness and improvements in visual acuity in csr patients treated with intravitreal bevacizumab [ 81 , 82 ] . regression of choroidal metastases has been reported following treatment with both systemic and intravitreal bevacizumab [ 8385 ] . intravitreal bevacizumab has also been used by mason iii et al . in the treatment of radiation - induced macular although a reduction in central retinal thickness was demonstrated amongst these patients , the effect was short - lived and associated with only marginal improvements in visual acuity . intravitreal bevacizumab does not seem to halt the progression of choroidal melanoma in patients who have inadvertently received this drug on account of an initial misdiagnosis of cnvm .

ocular angiogenesis and macular oedema are major causes of sight loss across the world . aberrant neovascularisation , which may arise secondary to numerous disease processes , can result in reduced vision as a result of oedema , haemorrhage , and scarring . the development of antivascular endothelial growth factor ( anti - vegf ) agents has revolutionised the treatment of retinal vasogenic conditions . these drugs are now commonly employed for the treatment of a plethora of ocular pathologies including choroidal neovascularisation , diabetic macular oedema , and retinal vein occlusion to name a few . in this paper , we will explore the current use of anti - vegf in a variety of retinal diseases and the impact that these medications have had on visual outcome for patients .

1. Introduction 2. DMO 3. AMD 4. RVO 5. Myopic Choroidal Neovascularisation 6. Other Anti-VEGF Indications

however , the development of antivascular endothelial growth factor ( anti - vegf ) has revolutionised the treatment of a plethora of ocular angiogenic disease processes . it has become the favoured therapy for conditions such as choroidal neovascularisation , diabetic macula oedema , vein occlusions , myopic choroidal neovascularisation , and retinopathy of prematurity to name a few . it could be argued that the evolution of anti - vegf therapy can be traced back to 1948 , where michaelson hypothesised that a diffusible , hypoxia - induced , angiogenic factor x was responsible for iris and retinal neovascularisation associated with ischaemic retinopathies . isolated an endothelial mitogen from pituitary follicular cells leading to it being branded , vascular endothelial growth factor ( vegf ) . discovered a tumour - derived factor named vascular permeability factor ( vpf ) , which was responsible for inducing vascular permeability . in a further study , where the retinas of primates were rendered ischaemic by laser photocoagulation of the veins , neovascularisation of the iris developed suggesting the presence of a diffusible molecule . furthermore , elevated levels of vegf in ocular fluids from patients with active neovascular ocular disease were found compared with ocular fluids with no vascularisation . in 1997 , genetech initiated phase 1 trials for the development of an anti - vegf molecule named avastin ( bevacizumab ) . subsequent successful results from phases 2 and 3 trials led to fda approval in february 2004 for the treatment of colon cancer in combination with chemotherapy . ranibizumab ( lucentis ) was determined effective by two pivotal trials : the marina ( minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular amd ) and anchor ( anti - vegf antibody for the treatment of predominantly classic choroidal neovascularisation in amd ) trials . a recent anti - vegf strategy , developed by regeneron , consisted of a chimeric fusion protein that acted as a decoy receptor to sequester vegf and thereby block its biological effects . in this paper , we will explore the current indications of anti - vegf in a variety of ocular angiogenic conditions that has changed the visual outcomes of patients . diabetic macular oedema ( dmo or dme ) is the leading cause of visual impairment in patients aged 20 to 74 and represents a significant burden of disease with the increasing incidence and prevalence of diabetes worldwide [ 13 , 14 ] . the development of dmo occurs as a result of vascular endothelial damage with breakdown of the blood - retinal barrier . hypoxia caused by microvascular disease stimulates the release of vascular endothelial growth factor - a ( vegf - a ) which is a major contributor to this vascular permeability and angiogenesis . it has a european marketing authorisation for the treatment of visual impairment due to macular oedema in adults and has been recommended by the national institute for health and care excellence ( nice ) in april 2013 as a treatment option if the eye has a central retinal thickness of 400 micrometres ( m ) or more at the start of treatment . the four randomised controlled trials ( rcts ) submitted to nice as evidence for clinical effectiveness included restore ( ranibizumab monotherapy or combined with laser versus laser monotherapy for diabetic macular edema ) , diabetic retinopathy clinical research network protocol i ( drcr.net ) , resolve ( safety and efficacy of ranibizumab in diabetic macular edema ) , and read-2 ( ranibizumab for edema of the macula in diabetes ) [ 1721 ] . all patients were evaluated monthly for the need for macular laser according to protocol - specified criteria including central foveal thickness 250 m . as such , they have evaluated the efficacy of ivb for the treatment of dmo . it is likely that the use of ivb is limited in nhs patients mainly as a result of nice guidance in favour of an alternative therapy . aflibercept ( iva , eyelea ; regeneron / bayer healthcare ) is a recent addition to the anti - vegf class . mean improvements in bcva in the iva groups at week 52 were 11.0 , 13.1 , 9.7 , and 12.0 letters for different dosing regimens ( 0.5 mg every 4 weeks , 2 mg every 4 weeks , 2 mg every 8 weeks after 3 initial monthly doses , and 2 mg as needed after 3 initial monthly doses , resp . ) versus 1.3 letters for the laser group ( p 0.0001 versus laser ) . iva has very recently been approved by the european commission for the treatment of visual impairment as a result of dmo . after the first 12 months of treatment , the treatment interval may be extended based on visual and anatomic outcomes . overall , there is high quality evidence that anti - vegf drugs provide benefit compared with other therapeutic options for dmo . this concluded that anti - vegf pharmacotherapy , delivered by intravitreal injection , is reasonably safe and effective in the treatment of dme . a statement by the royal college of ophthalmologists in their published diabetic retinopathy guidelines confirms that anti - vegf injections are considered the new gold standard of therapy for eyes with centre - involving macular oedema and reduced vision [ 30 , 31 ] . recently , the targeted therapy of vegf has revolutionised the treatment of neovascular amd . patients had to be over 50 , bcva 20/40 to 20/320 , and with a lesion size of less than 5400 microns . patients had to be over 50 , bcva 20/40 to 20/320 , and with a lesion of less than 12 disc diameters . it found that although average va improved in the treatment groups for the first 3 months , there was a gradual decline in va ( approximately 2 letters ) from months 4 to 24 when on quarterly dosing . average letters gained at 1 year were 8.3 letters in the monthly group , 4.9 letters in the 0.3 mg group , and 3.8 letters in the 0.5 mg group . in 2004 , intravenous bevacizumab was approved for the treatment of metastatic colon cancer , and this leads to a number of ophthalmologists using the drug off - label as an intravitreal injection . it did not however compare the groups to fixed - dosing rbz or aflibercept over the full 2 years , which may have given better bcva results . also of note is potential topical anti - vegf agents , which would eliminate many of the risks or burden of regular intravitreal injections . this study showed rbz was safe and well tolerated in a large population and a low risk of thromboembolism . retinal vein occlusion ( rvo ) is a common cause of visual loss in the united kingdom and is the second most common retinal vascular disease after diabetic retinopathy [ 47 , 48 ] . rvos can be divided into two categories depending on the site of obstruction : central rvo ( crvo ) when occlusion involved the whole central retinal vein and branch rvo ( brvo ) when the occlusion involves only one branch of the central retinal vein . crvo is caused by external compression of the central retinal vein , which shares a common fibrous sleeve with the vein . crvo is classically characterised by disc oedema , increased dilatation and tortuosity of all retinal veins , widespread of deep and superficial haemorrhages , cotton wool spots , retinal oedema , and capillary nonperfusion . vegf is produced by retinal pigmented epithelial cells , endothelial cells , muller cells , and other ocular tissues in response to retinal hypoxia and binds to specific receptors on endothelial cells acting as a proangiogenetic factor . this in turn leads to neovascularization and vascular hyperpermeability with subsequent breakdown of the blood - retina barrier and macular oedema . until recently the standard of care for macular oedema secondary to brvo was macular grid laser macular laser photocoagulation is not recommended for the treatment of macular oedema in crvo because it does not guarantee a significant improvement in visual acuity . currently available anti - vegf agents ( ranibizumab , aflibercept , and bevacizumab ) have been applied successfully in reducing macular oedema due to rvo . in may 2013 nice recommended ranibizumab ( lucentis , novartis ) as a possible treatment for some people who have sight problems because of macular oedema caused by retinal vein occlusion . two randomized , double - masked , multicenter phase 3 trials , the cruise ( central retinal vein occlusion ) and bravo ( branch retinal vein occlusion ) studies , evaluated the efficacy of ranibizumab , compared with a sham procedure , for treating visual impairment caused by macular oedema secondary to brvo and to crvo , respectively [ 56 , 57 ] . patients were eligible if they had foveal - involved macular oedema from a crvo or brvo occurring within 12 months of study entry , bcva of 20/40 to 20/320 ( in crvo ) and to 20/400 ( in brvo ) , and centre subfield thickness ( cst ) 250 m . in both bravo and cruise , the treatment phase was followed by a 6-month observation phase during which all groups could receive ranibizumab as needed . for patients enrolled in the cruise study baseline characteristics were well balanced among the three groups ; the mean age was 68 years , mean bcva was 20/100 , the mean time from diagnosis of crvo was 3.3 months , and the mean center point thickness ( cpt ) was 685 m . baseline characteristics for those involved in the bravo study were well balanced among the three groups ; mean bcva was 20/80 , the mean time from diagnosis of brvo was 3.5 months , and the mean cpt was 520 m . starting at month 3 , patients were eligible for grid laser treatment if hemorrhages had cleared sufficiently to allow safe application of laser and the following criteria were met : snellen equivalent bcva 20/40 or mean cst 250 m , and compared with the visit 3 months before the current visit , the patient had a gain of < 5 letters in bcva or a decrease of < 50 m in mean cst . in patients with crvo , the mean number of ranibizumab injections during the observation period was 3.9 , 3.6 , and 4.2 in the 0.3 mg , 0.5 mg , and sham/0.5 mg groups ; and the percentage of patients that did not receive any injections during the observation period was 7.0 , 6.7 , and 4.3 , respectively . in patients with brvo , the mean number of ranibizumab injections during the observation period was 2.9 , 2.8 , and 3.8 in the 0.3 mg , 0.5 mg , and sham/0.5 mg groups ; and the percentage of patients that did not receive any injections during the observation period was 17.2 , 20.0 , and 6.5 , respectively . for patients who entered the open - label extension study ( horizon ) , 67% of patients from bravo and 60% of patients from cruise completed month 12 of horizon . the primary outcome for the horizon extension study was mean change from horizon baseline in bcva score up to 24 months . nonocular serious adverse events ( potentially related to vascular endothelial growth factor [ vegf ] inhibition ) at 6 months were higher in the ranibizumab group ( 5 events [ 3.8% ] ) than in the sham group ( 1 event [ 0.8% ] ) . the most common adverse event reported in bravo and cruise at 12 months was cataract , with 8 ( 6.2% ) and 9 ( 7% ) instances associated with ranibizumab treatment , respectively ; in the sham ( plus ranibizumab ) group , 3 ( 2.6% ) and 2 ( 1.8% ) instances of cataract were reported for the treatment period of 6 to 12 months . in february 2014 nice recommended aflibercept ( eylea , bayer ) as a possible treatment for people with sight problems caused by macular oedema from central retinal vein occlusion . copernicus ( vascular endothelial growth factor trap - eye for macular edema secondary to central retinal vein occlusion ) was a randomised , double - blind , multicentre trial conducted in 6 non - european countries . the trials had identical designs and criteria for the first 6 months : both were randomized , multicentre , and double - masked , and both included patients with central retinal thickness ( crt ) of 250 m and bcva of 20/40 and 20/320 . the primary end point was the proportion of those patients who gained three or more lines , and the key secondary end points were total change in bcva and crt . between 6 months and 1 year , for the copernicus trial , the injection group received additional 2.7 injections while the sham / crossover group received 3.9 injections . copernicus results at 100 weeks demonstrated sustained improvements in all parameters for the treatment group when converting to prn treatment at 6 months and an additional improvement for sham treatment that was converted to prn treatment at 6 months . fundus fluorescein angiography ( ffa ) is used in diagnosis in the acute stage , showing a myopic cnv lights up as a highly reflective area above the rpe , occasionally with minimal subretinal fluid . the pathogenesis of myopic cnv is not yet fully understood but is thought to involve an imbalance of proangiogenic and antiangiogenic factors brought on by the mechanical stresses of a progressively elongating retina . poor prognostic indicators include lower baseline visual acuity , age above 40 years , extensive chorioretinal atrophy , subfoveal location of the cnv , and lesion size above 400 m [ 60 , 61 , 63 , 66 ] . prior to the advent of antivascular endothelial growth factor ( anti - vegf ) therapy , management of myopic cnv was largely based on laser photocoagulation for extrafoveal cnv and verteporfin photodynamic therapy for subfoveal cnv [ 61 , 63 , 66 ] . neither has been able to show any consistent long - term visual benefit but anti - vegf agents have recently been introduced with promising results . ranibizumab ( lucentis ) is the only anti - vegf therapy licensed for the treatment of myopic cnv and has shown great potential for visual gain and preventing irreversible retinal damage in phases ii and iii clinical trials repair and radiance ( a randomized controlled study of ranibizumab in patients with choroidal neovascularization secondary to pathologic myopia ) , respectively [ 4 , 66 ] . myrror , a phase iii clinical trial , is currently ongoing to establish the role of aflibercept ( eylea ) in the treatment of myopic cnv . this algorithm involves a single initial intravitreal injection of an anti - vegf agent , followed by subsequent as needed injections . at follow - up , symptoms and signs that warrant another anti - vegf injection include reduced visual acuity , visual symptoms ( e.g. since their introduction into clinical practice , the list of possible uses for anti - vegf therapy in ophthalmology has steadily expanded . anti - vegf agents have been utilized on an off - license basis for a vast array of other ocular pathologies ranging from external eye disease to ocular oncology and glaucoma . the development of choroidal neovascular membranes may complicate any disease process which results in a defect in bruch 's membrane and can pose a threat to vision through leakage of fluid , haemorrhage , and subretinal fibrosis . anti - vegf agents are now widely used in the treatment of cnvm regardless of aetiology . in november 2013 , nice approved ranibizumab for the treatment of choroidal neovascularisation associated with pathological myopia in the uk . this decision was based upon evidence largely extracted from the radiance ( randomized controlled study of ranibizumab in patients with choroidal neovascularisation secondary to pathologic myopia ) trial which compared ranibizumab with verteporfin photodynamic therapy ( vpdt ) . other studies have shown a similar efficacy of ranibizumab and bevacizumab in the treatment of myopic cnv [ 69 , 70 ] . intravitreal anti - vegf agents have also been successfully utilized in the treatment of cnv related to angioid streaks , uveitis , and trauma [ 7173 ] . intravitreal injection of an anti - vegf agent is now commonly performed as a pretreatment prior to pars plana vitrectomy for severe proliferative diabetic retinopathy . development or progression of tractional retinal detachment has however been reported following intravitreal injection of bevacizumab as an adjunct to pars plana vitrectomy which may be attributable to the contraction of fibrovascular membranes induced by the drug . reduction in the drive for vegf production through pan - retinal laser photocoagulation however remains the first line treatment in the uk although anti - vegf may have a role as an adjunctive therapy , particularly in cases of persistent vitreous haemorrhage [ 78 , 79 ] . retinopathy of prematurity ( rop ) in its most severe form results in tractional retinal detachment secondary to fibrovascular proliferation and remains a leading cause of childhood blindness across the world . there has been much interest recently in the use of anti - vegf agents to reduce the angiogenic drive that underlies the pathology of rop . in their prospective randomized trial reported on behalf of the beat - rop cooperative group a significant benefit of anti - vegf in the treatment of stage 3 + zone 1 disease . they also found that the development of peripheral retinal vessels continued after bevacizumab but not after laser therapy which may have a beneficial effect on conservation of visual field . although relatively large , the beat - rop study was not adequately powered to determine the safety of anti - vegf in the developing child and some concerns remain regarding the potentially unknown long - term local and systemic side effects of these medications . although the majority of cases resolve spontaneously within six months , in some the fluid persists which can result in ongoing reduction in visual acuity and metamorphopsia . in the treatment of radiation - induced macular although a reduction in central retinal thickness was demonstrated amongst these patients , the effect was short - lived and associated with only marginal improvements in visual acuity .

based on world health organization statistics , falls are the second leading cause of unintentional injury - related deaths worldwide.1 around 424,000 people globally die every year from unintentional falls , second only to road traffic deaths . injuries sustained from falls are unfortunately quite common in the elderly population ; for instance , in the united kingdom 57% of all injury - related hospitalizations in the elderly are related to falls.2 multifocal glasses in the form of bifocals have been available since 1784 . benjamin franklin converted his glasses into bifocals , allowing him to see which french politician was speaking and at the same time eat his meal.3 multifocal glasses are convenient for activities such as cooking , shopping , and driving . however , multifocal glasses are a known contributor to falls in elderly people.4 when multifocal glasses are worn while negotiating steps , in outdoor environments and around the home , they increase the chance of a fall . given that 52% of elderly people wear multifocal glasses , a large proportion of falls may be preventable.5 why and how multifocal glasses increase falls is not completely understood . the reading portion may affect depth perception and contrast sensitivity;4 additionally , the clear corridor of vision is smaller in progressives due to the large amounts of astigmatism at the edges.6 of particular interest is the prismatic displacement effect of multifocal glasses on correcting postural stability . displacement of the image of an object when viewed through a prism and the illusion of false projection is due to the amount of prismatic displacement it creates . the amount a light ray will deviate when it passes through a prism depends on the apical angle , the index of refraction of the material ( and the material surrounding it ) , the wavelength of the light , and the angle from which the light approaches the prism.7 although the prismatic displacement effect of multifocal glasses is alluded to as a potential cause of falls,4,811 to our knowledge no one has tested this effect . the aim of this study was to 1 ) map the prismatic displacement of a progressive lens , and 2 ) test whether the displacement of a progressive addition lens ( pal ) impairs reaction time and accuracy . a case crossover trial was conducted in the school of physiotherapy balance clinic at the university of otago , dunedin , new zealand . participants reaction times and accuracy were compared while wearing both pals and single ( distance ) vision glasses . initial work illustrated the largest areas of prismatic displacement in a plano , + 4 d powered pal , which was mapped using the nikon projection focimeter ( nikon corporation , tokyo , japan ) . a + 4 d lens was chosen so that the area(s ) of maximal prismatic displacement was more apparent . a plastic mask , 40 mm in diameter , with a grid of holes 3 mm apart ( each hole 1 mm in diameter and 2 mm deep ) , vertically and horizontally ( seven by eleven holes , respectively ) was placed on top of the pal . figure 1 shows the area of the pal that was mapped and the prismatic displacement measurements that were recorded in diopters , and then converted to millimeters of displacement ( diopter 666/100= mm of displacement ) . of note , the measure 666 is the distance in millimeters from the participant to the target they reached for in this study and the value 100 is a factor to convert the displacement to millimeters . both the right and left pals were measured and the right pal was inverted and averaged with the left pals results . the measurements showed that pals had larger displacements at certain points . at the center of the lens the largest mean difference was 8.15 mm ( 95% confidence interval [ ci ] 6.549.75 ) of vertical displacement . at positions at the periphery of the glasses the difference was small , around 2.23 mm ( 95% ci 0.374.09 ) of horizontal displacement ( at 200 mm below center ) and 1.83 mm ( 95% ci 0.523.14 ) of vertical displacement ( at 100 mm below center ) . there was no difference in horizontal displacement at the center and vertical displacement at 300 mm below center between the two glasses ( p=0.271 and p=0.258 ) , respectively ( figures 2 and 3 ) . participants were residents 75-years - old or older from the dunedin and mosgiel communities who had acquired a new or updated prescription for a pal within the last 12 months . exclusion criteria were : 1 ) poorer than 6/12 vision in one eye or 6/12 with binocular vision when corrected with glasses;12 2 ) ocular / retinal problems ( ie , age - related macular degeneration , cataracts , diabetic retinopathy , glaucoma , field defects , and ocular - motor disorders ) ; 3 ) > 2 diopter difference of spherical anisometropia ; 4 ) > 4 diopter of oblique astigmatism ; 5 ) deformities of the arms , neck , or head ; 6 ) inability to stand ; 7 ) suffering from dizziness or a recent injury or medical condition that affected coordination and ability to grasp ; 8) not being able to understand the study requirements ; and 9 ) a mini mental state examination ( mmse)13 score lower than 26 . baseline information was collected from the participant s optometrist s notes and a home visit conducted by the investigator . information was recorded about demographics , living arrangement , medical conditions , medication use , and visual acuity plus physical functioning . the order of the tests of the two types of glasses was randomized with the investigator blinded to the order ; the position of the bar / black line at the maximal or minimal prismatic displacement was randomized . a statistician not involved with the study prepared a computer - generated randomization schedule and opaque envelopes with instructions for the order of glasses to be worn . the schedule was made available on testing day and explained to the participant by a person independent of the study , to preserve blinding of the investigator . numbered tags were placed on the frames of each pair of glasses to allow the participant to wear the glasses in the correct order . each pair of glasses were adjusted and measured so that the eye was 20 mm away from the vertex of the lens . the investigator restated the need for the participant to refrain from describing the glasses as progressives or single distance as it is imperative that the investigator did not know the order during and after the test . it was not possible for an inexperienced observer to tell if the glasses were pals or single vision glasses . because of this the investigator remained blind to the subsequent analysis of the order of glasses . the participants acquired their single vision glasses on testing day so that they did not have time to adapt to this pair of glasses . each new prescription of single vision glasses was identical and matched to the distance part of the individual s pal prescription and frame style . where this was not possible due to a lack of supply , the make , model , size , and style of frame was matched as closely as possible to the participant s chosen frame . the single vision glasses were not matched for scratch resistant lens coating and/or transitional tints , due to budget constraints . the powerlab 4sp data acquisition system and the lab - chart pro version 7 program14 ( adinstruments , dunedin , new zealand ) were used to measure reaction time and accuracy . accuracy measurements were recorded using the labchart program mentioned and a series of nine wire switches under a foam pad ( see figure 4 ) . the labchart program could measure which switch or switches were pushed first and therefore the distance from the black line ( see figure 5 ) . the participant s finger movements when pushing a black line on a foam pad were also recorded by a video camera behind the participant . this was done to ensure only their extraocular eye movements were used to visualize the bar / black line and light emitting diode ( led ) . participants were asked to sit comfortably on a chair with their arms by their side in a neutral position and stare at an led placed 1 meter in front of them . they were instructed to reach out with their dominant hand and grab the whole bar or push the black line with their index finger when the led turned on , as quickly as possible . enough strength to grip the bar or push the black line the bar / black line was placed horizontally and vertically in front and to the dominant hand side of the participant , at a distance of 666 mm . the bar and the black line were attached to a walking frame ( mobilis quad [ cubro ltd , tauranga , new zealand ] , see figure 6 ) . the areas of visual fixation through the glasses were based on the focimeter results from the preliminary investigation , which showed the areas of maximal and minimal prismatic displacement . the coordinates ( 500 , 300 ) , ( 100 , 100 ) , ( 100 , 200 ) , ( 900 , 100 ) , ( 900 , 200 ) , and ( 500 , 0 ) from figures 2 and 3 indicate where participants fixated through their glasses on the bar / black line . the bar / black line was placed at the horizontal and vertical center of the lens ( 500 , 300 ) . the bar / black line was placed vertically only at coordinate ( 100 , 100 ) or ( 900 , 100 ) depending on the participant s dominant hand ; this position was 35 peripherally and 200 mm below center . the bar was placed horizontally at coordinate ( 100 , 200 ) or ( 900 , 200 ) , once again depending on the participant s dominant hand ; this position was 35 peripherally and 100 mm below center . finally , the black line was placed horizontally at coordinate ( 500 , 0 ) , which was 300 mm below center . the main outcome was reaction time when grasping the bar and reaction time plus accuracy when pushing the black line at different points of fixation through the glasses . each test was repeated three times and the fastest time plus its associated accuracy were used in the analyses . data were analyzed using the statistical package for the social sciences version 18 ( ibm corporation , armonk , ny , usa ) and presented as mean ( standard deviation [ sd ] ) , mean difference ( 95% confidence interval [ ci ] ) , or number ( percentage ) . to test whether measures of reaction time differed between the two types of glasses , repeated measures analysis of variance and covariance were used , controlling for length of time since updating their glasses , prismatic displacement , and order of glasses . accuracy measurements were compared for the two types of glasses using the chi - squared mcnemar test . the labchart measurements were verified from the video recordings , to confirm whether the participant pushed the black line . initial work illustrated the largest areas of prismatic displacement in a plano , + 4 d powered pal , which was mapped using the nikon projection focimeter ( nikon corporation , tokyo , japan ) . a + 4 d lens was chosen so that the area(s ) of maximal prismatic displacement was more apparent . a plastic mask , 40 mm in diameter , with a grid of holes 3 mm apart ( each hole 1 mm in diameter and 2 mm deep ) , vertically and horizontally ( seven by eleven holes , respectively ) was placed on top of the pal . figure 1 shows the area of the pal that was mapped and the prismatic displacement measurements that were recorded in diopters , and then converted to millimeters of displacement ( diopter 666/100= mm of displacement ) . of note , the measure 666 is the distance in millimeters from the participant to the target they reached for in this study and the value 100 is a factor to convert the displacement to millimeters . both the right and left pals were measured and the right pal was inverted and averaged with the left pals results . the measurements showed that pals had larger displacements at certain points . at the center of the lens the largest mean difference was 8.15 mm ( 95% confidence interval [ ci ] 6.549.75 ) of vertical displacement . at positions at the periphery of the glasses the difference was small , around 2.23 mm ( 95% ci 0.374.09 ) of horizontal displacement ( at 200 mm below center ) and 1.83 mm ( 95% ci 0.523.14 ) of vertical displacement ( at 100 mm below center ) . there was no difference in horizontal displacement at the center and vertical displacement at 300 mm below center between the two glasses ( p=0.271 and p=0.258 ) , respectively ( figures 2 and 3 ) . participants were residents 75-years - old or older from the dunedin and mosgiel communities who had acquired a new or updated prescription for a pal within the last 12 months . exclusion criteria were : 1 ) poorer than 6/12 vision in one eye or 6/12 with binocular vision when corrected with glasses;12 2 ) ocular / retinal problems ( ie , age - related macular degeneration , cataracts , diabetic retinopathy , glaucoma , field defects , and ocular - motor disorders ) ; 3 ) > 2 diopter difference of spherical anisometropia ; 4 ) > 4 diopter of oblique astigmatism ; 5 ) deformities of the arms , neck , or head ; 6 ) inability to stand ; 7 ) suffering from dizziness or a recent injury or medical condition that affected coordination and ability to grasp ; 8) not being able to understand the study requirements ; and 9 ) a mini mental state examination ( mmse)13 score lower than 26 . baseline information was collected from the participant s optometrist s notes and a home visit conducted by the investigator . information was recorded about demographics , living arrangement , medical conditions , medication use , and visual acuity plus physical functioning . the order of the tests of the two types of glasses was randomized with the investigator blinded to the order ; the position of the bar / black line at the maximal or minimal prismatic displacement was randomized . a statistician not involved with the study prepared a computer - generated randomization schedule and opaque envelopes with instructions for the order of glasses to be worn . the schedule was made available on testing day and explained to the participant by a person independent of the study , to preserve blinding of the investigator . numbered tags were placed on the frames of each pair of glasses to allow the participant to wear the glasses in the correct order . each pair of glasses were adjusted and measured so that the eye was 20 mm away from the vertex of the lens . the investigator restated the need for the participant to refrain from describing the glasses as progressives or single distance as it is imperative that the investigator did not know the order during and after the test . it was not possible for an inexperienced observer to tell if the glasses were pals or single vision glasses . because of this the investigator remained blind to the subsequent analysis of the order of glasses . the participants acquired their single vision glasses on testing day so that they did not have time to adapt to this pair of glasses . each new prescription of single vision glasses was identical and matched to the distance part of the individual s pal prescription and frame style . where this was not possible due to a lack of supply , the make , model , size , and style of frame was matched as closely as possible to the participant s chosen frame . the single vision glasses were not matched for scratch resistant lens coating and/or transitional tints , due to budget constraints . the powerlab 4sp data acquisition system and the lab - chart pro version 7 program14 ( adinstruments , dunedin , new zealand ) were used to measure reaction time and accuracy . accuracy measurements were recorded using the labchart program mentioned and a series of nine wire switches under a foam pad ( see figure 4 ) . the labchart program could measure which switch or switches were pushed first and therefore the distance from the black line ( see figure 5 ) . the participant s finger movements when pushing a black line on a foam pad were also recorded by a video camera behind the participant . this was done to ensure only their extraocular eye movements were used to visualize the bar / black line and light emitting diode ( led ) . participants were asked to sit comfortably on a chair with their arms by their side in a neutral position and stare at an led placed 1 meter in front of them . they were instructed to reach out with their dominant hand and grab the whole bar or push the black line with their index finger when the led turned on , as quickly as possible . enough strength to grip the bar or push the black line the bar / black line was placed horizontally and vertically in front and to the dominant hand side of the participant , at a distance of 666 mm . the bar and the black line were attached to a walking frame ( mobilis quad [ cubro ltd , tauranga , new zealand ] , see figure 6 ) . the areas of visual fixation through the glasses were based on the focimeter results from the preliminary investigation , which showed the areas of maximal and minimal prismatic displacement . the coordinates ( 500 , 300 ) , ( 100 , 100 ) , ( 100 , 200 ) , ( 900 , 100 ) , ( 900 , 200 ) , and ( 500 , 0 ) from figures 2 and 3 indicate where participants fixated through their glasses on the bar / black line . the bar / black line was placed at the horizontal and vertical center of the lens ( 500 , 300 ) . the bar / black line was placed vertically only at coordinate ( 100 , 100 ) or ( 900 , 100 ) depending on the participant s dominant hand ; this position was 35 peripherally and 200 mm below center . the bar was placed horizontally at coordinate ( 100 , 200 ) or ( 900 , 200 ) , once again depending on the participant s dominant hand ; this position was 35 peripherally and 100 mm below center . finally , the black line was placed horizontally at coordinate ( 500 , 0 ) , which was 300 mm below center . the main outcome was reaction time when grasping the bar and reaction time plus accuracy when pushing the black line at different points of fixation through the glasses . each test was repeated three times and the fastest time plus its associated accuracy were used in the analyses . data were analyzed using the statistical package for the social sciences version 18 ( ibm corporation , armonk , ny , usa ) and presented as mean ( standard deviation [ sd ] ) , mean difference ( 95% confidence interval [ ci ] ) , or number ( percentage ) . to test whether measures of reaction time differed between the two types of glasses , repeated measures analysis of variance and covariance were used , controlling for length of time since updating their glasses , prismatic displacement , and order of glasses . accuracy measurements were compared for the two types of glasses using the chi - squared mcnemar test . the labchart measurements were verified from the video recordings , to confirm whether the participant pushed the black line . thirty - one participants were recruited for the study ( see figure 7 ) and their characteristics are summarized in table 1 . when wearing pals , participants had a significantly faster reaction time compared to when they were wearing single vision glasses . this improved reaction time was noted when localizing the bar / line through two areas : 1 ) at the center of the lens , when grasping a horizontal bar , pals were faster by 101 ms , p=0.011 ( repeated measures for order effect , time to update their pals , and prismatic displacement ) ; 2 ) at 300 mm below center , when pushing a horizontal black line , pals were faster by 80 ms , p=0.007 . this study found no statistically significant differences for reaction time for pushing a black line and grasping a bar at the other positions tested , fixating through the two different glasses ( see table 2 ) . figures 8 and 9 represent graphical interpretations of the reaction time measurements and illustrate the significant differences between the two glasses in the central visual axis . table 3 and figure 10 illustrate accuracy at the specific points that participants localized through the lens . differences between measurements did not reach statistical significance ; however , participants showed a tendency to be less accurate when wearing pals compared with single vision glasses at the edges of the glasses . at the center of the lens , pals showed a tendency to be more accurate when the black line was placed vertically ; accuracy between the two glasses was similar when the black line was placed horizontally ( see table 3 and figure 10 ) . the video tape recordings of participants pushing the foam pad with a black line on it endorsed the findings of the switches using the labchart and powerlab data acquisition systems . it has been shown that elderly people who can grasp a support stick at a distance further away from them have a lower incidence of falls.18 the ability to reach out and grab a support with accuracy and without hesitation is important in a fall . to our knowledge this is the first study to look at the prismatic displacement effect on reaction time and accuracy when an elderly person extends their arm to touch a target . in our study , participants were faster at grasping a bar and pushing a black line placed in the central visual axis when wearing pals compared with single vision glasses . this primary finding is consistent with the study by lord et al on falls among long - term multifocal wearers.4 one of the secondary measures of lord et al s trial was reaction time with a light as a stimulus and participants wearing their preferred eyewear . the study showed that participants who wore multifocal glasses had a tendency to react faster compared with non - multifocal glasses wearers ; mean ( sd ) time to react was 276 ( 57 ) and 288 ( 58 ) ms , respectively . results from our study showed that reaction time tested in the majority of areas of fixation and all of the accuracy measurements were non - significant between the two types of glasses , which is consistent with the visible trial . the visible trial suggests that swapping multifocal glasses with single vision distance glasses had no overall effect on fall- prevention ( incidence rate ratio [ irr ] 0.92 [ 95% ci 0.731.16]).8 additionally , preplanned subgroup analysis showed that by replacing multifocal with single vision glasses there was a significant increase in falls in older frail people compared with controls who continued wearing their multifocal glasses , especially in an outdoor ( unfamiliar ) environment ( irr 1.56 ; 95% ci 1.112.19).8 however , the visible trial did illustrate through the same subgroup analysis that active older people benefit from replacing their multifocal glasses with single vision glasses ( irr 0.60 ; 0.420.87 ) , specifically when in an outdoor environment ( irr 0.61 ; 95% ci 0.420.87).8 anecdotal evidence suggests that when first prescribed multifocal glasses , the person experiences a significant undulating effect , which decreases with time . the sensation of inappropriate motion ( oscillopsia ) seems to be most present around the initial time of adaptation.19 furthermore , it has been shown that people can adjust to glasses that invert the image seen ; once the glasses are removed , they simply adjust back within 30 minutes.20 it is suggested that this is due to the brain accommodating the change , through recalibration of the proprioception receptors in the upper and lower limbs and extra retinal eye movements ( egocentric and oculocentric visual directions ) , rather than a correction of the vision . with that in mind , the participants in this trial may have adapted to their pals , having worn them for a mean of 228 days ( sd 146 days ) , and were thus able to react more quickly than with a new pair of single vision glasses . in changing glasses and thus reversing the prismatic effect , this effect was not specifically tested in our study and it is not unreasonable to postulate that the effect of replacing pals with single vision glasses may work in reverse , but this would need further study . the findings from the study by cumming et al suggest caution should be taken with a sudden change in older people s vision.21 in this study the intervention was aimed at improving vision ( eg , arranging for new eyeglasses ) and this increased rather than decreased the number of falls by participants over a 12 month period . the majority of the intervention participants that required visual improvement acquired a new pair of glasses ( 92 of 146 , 63% ) . furthermore , most participants in the intervention arm had their glasses changed 13 months before the trial started ( 209 of 309 , 68% ) ; perhaps a more gradual process of updating is required to allow for adjustment to the change in vision . the increase in fall rate seen in the intervention arm was most prominent within the first 3 months of updating their glasses . however , the length of time needed to adapt to multifocal glasses has not been defined . when an older person walks into an unfamiliar environment , he or she unconsciously remembers the potential support objects in space in that room , spatial memory known as stored central visual spatial information;22 it is possible that this information is stored incorrectly when replacing glasses with different prismatic displacements . therefore , when a trip , slip , or loss of balance does occur and the older person needs to rely on that stored information , they may miss the support object . according to our study , this would be true only for the vertical displacement in the center and the horizontal bar placed at this position for reaction time . our results found a difference in reaction time when the black line was placed at a position of 300 mm down from center , but this can not be explained by the prismatic displacement effect as there was no statistically significant vertical displacement between the two glasses at this point ( p=0.258 ) . alternatively the result at this position could be explained by the reading portion of pals because the black line was placed 666 mm away from the participants . this is the optimal focus that is , around 600 mm for the reading power of multifocal glasses.4 the mean focal distance of participants pal reading portion in this study was 395 mm ( range , 500333 mm ) . therefore , the reading portion brought the line closer to the participant and the resolution may be clearer , enabling the participant to push the line more quickly . there are alternative explanations for why multifocal glasses have an effect on falls in the elderly population . timmis et al s work suggests that participants wearing single vision glasses had more control when stepping down from a block height when compared with individuals who were well adapted to multifocal lenses.9 the study compared kinematic mechanics in 20 older people and found that those participants wearing single vision glasses had reduced vertical center of mass velocity , increased single - limb support time , and reduced peak ankle and knee angular velocities . this suggested that landing occurred in a more controlled manner when wearing single vision glasses . another study by elliott et al involved ten elderly participants and their stepping kinematics onto a raised surface when wearing a positive or negative blurring.23 the study found that stepping movements were refected by the magnification effect of the lens on the position and size of the step . the step looked closer with a positive lens , which influenced the participant s trailing foot to be placed further away and increased their leading vertical toe clearance . with a negative lens the reverse was true . they recommend that partial changes in correction should be prescribed and that updating glasses should therefore occur more frequently . replacing multifocal glasses with single vision glasses could be harmful for other reasons . in the visible trial , secondary measures were recorded of non - fall - related injuries such as lacerations , lifting or twisting injuries , burns or scalds , eye injuries , collisions , and pedestrian injuries.8 they found that 26% of participants replacing multifocal glasses with single vision glasses had a non - fall related injury , compared with 17% of the control group ( p=0.01 ) . this effect could be due to the convenience of multifocal glasses when carrying out everyday tasks such as cooking , driving , shopping , or similar activities . the study population were healthy , active participants , which may not enable generalizability to the older population at greatest risk of a fall . the technique used to map the prismatic displacement was limited to the mask used to map the section of the lens . there was a balance between mapping the largest prismatic displacement at the edge of the glasses , while testing had to occur within the spectacle frame . the general trend was for the prismatic displacement to increase the further out from the center of the lens tested . therefore , if the targets used to test accuracy and reaction time were placed in a more peripheral position , they could have potentially shown a larger effect compared with the areas of fixation that were tested . however , this was not possible due to the frames of the participants glasses . although the frames of the new single vision glasses were matched to the participant s pals , there could have been a slight differences in tilt , vertex , adjustment , and size of the new pair of glasses . another limitation with this study is that a light stimulus was used instead of a trip , slip , or loss of balance . therefore , there was no heightened awareness through a fight - or - flight response . a one - off trial which involves a perturbation rather than a light stimulus would possibly be a better method to simulate a fall.22,24 our study expands on the paper by lord et al4 and examines the influence on response to a loss of balance when replacing multifocal glasses with single vision ( distance ) glasses . to our knowledge , this is the first study to test prismatic displacement and fall risk in older people . in conclusion , this study suggests that updating glasses should possibly occur more often in the elderly population to avoid abrupt changes in prismatic displacement .

backgroundmultifocal glasses ( bifocals , trifocals , and progressives ) increase the risk of falling in elderly people , but how they do so is unclear . to explain why glasses with progressive addition lenses increase the risk of falls and whether this can be attributed to false projection , this study aimed to 1 ) map the prismatic displacement of a progressive lens , and 2 ) test whether this displacement impaired reaction time and accuracy.methodsthe reaction times of healthy 75-year - olds ( 31 participants ) were measured when grasping for a bar and touching a black line . participants performed each test twice , wearing their progressives and new , matched single vision ( distance ) glasses in random order . the line and bar targets were positioned according to the maximum and minimum prismatic displacement effect through the progressive lens , mapped using a focimeter.resultsprogressive spectacle lenses have large areas of prismatic displacement in the central visual axis and edges . reaction time was faster for progressives compared with single vision glasses with a centrally - placed horizontal grab bar ( mean difference 101 ms , p=0.011 [ repeated measures analysis ] ) and a horizontal black line placed 300 mm below center ( mean difference 80 ms , p=0.007 ) . there was no difference in accuracy between the two types of glasses.conclusionolder people appear to adapt to the false projection of progressives in the central visual axis . this adaptation means that swapping to new glasses or a large change in prescription may lead to a fall . frequently updating glasses may be more beneficial .

Introduction Methods Preliminary investigation Study design Single vision glasses Outcomes Statistical analysis Results Discussion Conclusion

however , multifocal glasses are a known contributor to falls in elderly people.4 when multifocal glasses are worn while negotiating steps , in outdoor environments and around the home , they increase the chance of a fall . given that 52% of elderly people wear multifocal glasses , a large proportion of falls may be preventable.5 why and how multifocal glasses increase falls is not completely understood . the reading portion may affect depth perception and contrast sensitivity;4 additionally , the clear corridor of vision is smaller in progressives due to the large amounts of astigmatism at the edges.6 of particular interest is the prismatic displacement effect of multifocal glasses on correcting postural stability . displacement of the image of an object when viewed through a prism and the illusion of false projection is due to the amount of prismatic displacement it creates . the amount a light ray will deviate when it passes through a prism depends on the apical angle , the index of refraction of the material ( and the material surrounding it ) , the wavelength of the light , and the angle from which the light approaches the prism.7 although the prismatic displacement effect of multifocal glasses is alluded to as a potential cause of falls,4,811 to our knowledge no one has tested this effect . the aim of this study was to 1 ) map the prismatic displacement of a progressive lens , and 2 ) test whether the displacement of a progressive addition lens ( pal ) impairs reaction time and accuracy . participants reaction times and accuracy were compared while wearing both pals and single ( distance ) vision glasses . initial work illustrated the largest areas of prismatic displacement in a plano , + 4 d powered pal , which was mapped using the nikon projection focimeter ( nikon corporation , tokyo , japan ) . a plastic mask , 40 mm in diameter , with a grid of holes 3 mm apart ( each hole 1 mm in diameter and 2 mm deep ) , vertically and horizontally ( seven by eleven holes , respectively ) was placed on top of the pal . figure 1 shows the area of the pal that was mapped and the prismatic displacement measurements that were recorded in diopters , and then converted to millimeters of displacement ( diopter 666/100= mm of displacement ) . of note , the measure 666 is the distance in millimeters from the participant to the target they reached for in this study and the value 100 is a factor to convert the displacement to millimeters . at positions at the periphery of the glasses the difference was small , around 2.23 mm ( 95% ci 0.374.09 ) of horizontal displacement ( at 200 mm below center ) and 1.83 mm ( 95% ci 0.523.14 ) of vertical displacement ( at 100 mm below center ) . there was no difference in horizontal displacement at the center and vertical displacement at 300 mm below center between the two glasses ( p=0.271 and p=0.258 ) , respectively ( figures 2 and 3 ) . exclusion criteria were : 1 ) poorer than 6/12 vision in one eye or 6/12 with binocular vision when corrected with glasses;12 2 ) ocular / retinal problems ( ie , age - related macular degeneration , cataracts , diabetic retinopathy , glaucoma , field defects , and ocular - motor disorders ) ; 3 ) > 2 diopter difference of spherical anisometropia ; 4 ) > 4 diopter of oblique astigmatism ; 5 ) deformities of the arms , neck , or head ; 6 ) inability to stand ; 7 ) suffering from dizziness or a recent injury or medical condition that affected coordination and ability to grasp ; 8) not being able to understand the study requirements ; and 9 ) a mini mental state examination ( mmse)13 score lower than 26 . the order of the tests of the two types of glasses was randomized with the investigator blinded to the order ; the position of the bar / black line at the maximal or minimal prismatic displacement was randomized . numbered tags were placed on the frames of each pair of glasses to allow the participant to wear the glasses in the correct order . because of this the investigator remained blind to the subsequent analysis of the order of glasses . the participants acquired their single vision glasses on testing day so that they did not have time to adapt to this pair of glasses . each new prescription of single vision glasses was identical and matched to the distance part of the individual s pal prescription and frame style . where this was not possible due to a lack of supply , the make , model , size , and style of frame was matched as closely as possible to the participant s chosen frame . the single vision glasses were not matched for scratch resistant lens coating and/or transitional tints , due to budget constraints . the powerlab 4sp data acquisition system and the lab - chart pro version 7 program14 ( adinstruments , dunedin , new zealand ) were used to measure reaction time and accuracy . this was done to ensure only their extraocular eye movements were used to visualize the bar / black line and light emitting diode ( led ) . the bar and the black line were attached to a walking frame ( mobilis quad [ cubro ltd , tauranga , new zealand ] , see figure 6 ) . the areas of visual fixation through the glasses were based on the focimeter results from the preliminary investigation , which showed the areas of maximal and minimal prismatic displacement . the bar / black line was placed vertically only at coordinate ( 100 , 100 ) or ( 900 , 100 ) depending on the participant s dominant hand ; this position was 35 peripherally and 200 mm below center . finally , the black line was placed horizontally at coordinate ( 500 , 0 ) , which was 300 mm below center . the main outcome was reaction time when grasping the bar and reaction time plus accuracy when pushing the black line at different points of fixation through the glasses . data were analyzed using the statistical package for the social sciences version 18 ( ibm corporation , armonk , ny , usa ) and presented as mean ( standard deviation [ sd ] ) , mean difference ( 95% confidence interval [ ci ] ) , or number ( percentage ) . to test whether measures of reaction time differed between the two types of glasses , repeated measures analysis of variance and covariance were used , controlling for length of time since updating their glasses , prismatic displacement , and order of glasses . accuracy measurements were compared for the two types of glasses using the chi - squared mcnemar test . initial work illustrated the largest areas of prismatic displacement in a plano , + 4 d powered pal , which was mapped using the nikon projection focimeter ( nikon corporation , tokyo , japan ) . a plastic mask , 40 mm in diameter , with a grid of holes 3 mm apart ( each hole 1 mm in diameter and 2 mm deep ) , vertically and horizontally ( seven by eleven holes , respectively ) was placed on top of the pal . figure 1 shows the area of the pal that was mapped and the prismatic displacement measurements that were recorded in diopters , and then converted to millimeters of displacement ( diopter 666/100= mm of displacement ) . of note , the measure 666 is the distance in millimeters from the participant to the target they reached for in this study and the value 100 is a factor to convert the displacement to millimeters . at positions at the periphery of the glasses the difference was small , around 2.23 mm ( 95% ci 0.374.09 ) of horizontal displacement ( at 200 mm below center ) and 1.83 mm ( 95% ci 0.523.14 ) of vertical displacement ( at 100 mm below center ) . there was no difference in horizontal displacement at the center and vertical displacement at 300 mm below center between the two glasses ( p=0.271 and p=0.258 ) , respectively ( figures 2 and 3 ) . exclusion criteria were : 1 ) poorer than 6/12 vision in one eye or 6/12 with binocular vision when corrected with glasses;12 2 ) ocular / retinal problems ( ie , age - related macular degeneration , cataracts , diabetic retinopathy , glaucoma , field defects , and ocular - motor disorders ) ; 3 ) > 2 diopter difference of spherical anisometropia ; 4 ) > 4 diopter of oblique astigmatism ; 5 ) deformities of the arms , neck , or head ; 6 ) inability to stand ; 7 ) suffering from dizziness or a recent injury or medical condition that affected coordination and ability to grasp ; 8) not being able to understand the study requirements ; and 9 ) a mini mental state examination ( mmse)13 score lower than 26 . the order of the tests of the two types of glasses was randomized with the investigator blinded to the order ; the position of the bar / black line at the maximal or minimal prismatic displacement was randomized . numbered tags were placed on the frames of each pair of glasses to allow the participant to wear the glasses in the correct order . it was not possible for an inexperienced observer to tell if the glasses were pals or single vision glasses . the participants acquired their single vision glasses on testing day so that they did not have time to adapt to this pair of glasses . each new prescription of single vision glasses was identical and matched to the distance part of the individual s pal prescription and frame style . where this was not possible due to a lack of supply , the make , model , size , and style of frame was matched as closely as possible to the participant s chosen frame . the single vision glasses were not matched for scratch resistant lens coating and/or transitional tints , due to budget constraints . the powerlab 4sp data acquisition system and the lab - chart pro version 7 program14 ( adinstruments , dunedin , new zealand ) were used to measure reaction time and accuracy . the participant s finger movements when pushing a black line on a foam pad were also recorded by a video camera behind the participant . the bar and the black line were attached to a walking frame ( mobilis quad [ cubro ltd , tauranga , new zealand ] , see figure 6 ) . the areas of visual fixation through the glasses were based on the focimeter results from the preliminary investigation , which showed the areas of maximal and minimal prismatic displacement . the bar / black line was placed vertically only at coordinate ( 100 , 100 ) or ( 900 , 100 ) depending on the participant s dominant hand ; this position was 35 peripherally and 200 mm below center . finally , the black line was placed horizontally at coordinate ( 500 , 0 ) , which was 300 mm below center . the main outcome was reaction time when grasping the bar and reaction time plus accuracy when pushing the black line at different points of fixation through the glasses . data were analyzed using the statistical package for the social sciences version 18 ( ibm corporation , armonk , ny , usa ) and presented as mean ( standard deviation [ sd ] ) , mean difference ( 95% confidence interval [ ci ] ) , or number ( percentage ) . to test whether measures of reaction time differed between the two types of glasses , repeated measures analysis of variance and covariance were used , controlling for length of time since updating their glasses , prismatic displacement , and order of glasses . accuracy measurements were compared for the two types of glasses using the chi - squared mcnemar test . when wearing pals , participants had a significantly faster reaction time compared to when they were wearing single vision glasses . this improved reaction time was noted when localizing the bar / line through two areas : 1 ) at the center of the lens , when grasping a horizontal bar , pals were faster by 101 ms , p=0.011 ( repeated measures for order effect , time to update their pals , and prismatic displacement ) ; 2 ) at 300 mm below center , when pushing a horizontal black line , pals were faster by 80 ms , p=0.007 . this study found no statistically significant differences for reaction time for pushing a black line and grasping a bar at the other positions tested , fixating through the two different glasses ( see table 2 ) . figures 8 and 9 represent graphical interpretations of the reaction time measurements and illustrate the significant differences between the two glasses in the central visual axis . differences between measurements did not reach statistical significance ; however , participants showed a tendency to be less accurate when wearing pals compared with single vision glasses at the edges of the glasses . at the center of the lens , pals showed a tendency to be more accurate when the black line was placed vertically ; accuracy between the two glasses was similar when the black line was placed horizontally ( see table 3 and figure 10 ) . the video tape recordings of participants pushing the foam pad with a black line on it endorsed the findings of the switches using the labchart and powerlab data acquisition systems . it has been shown that elderly people who can grasp a support stick at a distance further away from them have a lower incidence of falls.18 the ability to reach out and grab a support with accuracy and without hesitation is important in a fall . to our knowledge this is the first study to look at the prismatic displacement effect on reaction time and accuracy when an elderly person extends their arm to touch a target . in our study , participants were faster at grasping a bar and pushing a black line placed in the central visual axis when wearing pals compared with single vision glasses . this primary finding is consistent with the study by lord et al on falls among long - term multifocal wearers.4 one of the secondary measures of lord et al s trial was reaction time with a light as a stimulus and participants wearing their preferred eyewear . the study showed that participants who wore multifocal glasses had a tendency to react faster compared with non - multifocal glasses wearers ; mean ( sd ) time to react was 276 ( 57 ) and 288 ( 58 ) ms , respectively . results from our study showed that reaction time tested in the majority of areas of fixation and all of the accuracy measurements were non - significant between the two types of glasses , which is consistent with the visible trial . the visible trial suggests that swapping multifocal glasses with single vision distance glasses had no overall effect on fall- prevention ( incidence rate ratio [ irr ] 0.92 [ 95% ci 0.731.16]).8 additionally , preplanned subgroup analysis showed that by replacing multifocal with single vision glasses there was a significant increase in falls in older frail people compared with controls who continued wearing their multifocal glasses , especially in an outdoor ( unfamiliar ) environment ( irr 1.56 ; 95% ci 1.112.19).8 however , the visible trial did illustrate through the same subgroup analysis that active older people benefit from replacing their multifocal glasses with single vision glasses ( irr 0.60 ; 0.420.87 ) , specifically when in an outdoor environment ( irr 0.61 ; 95% ci 0.420.87).8 anecdotal evidence suggests that when first prescribed multifocal glasses , the person experiences a significant undulating effect , which decreases with time . the sensation of inappropriate motion ( oscillopsia ) seems to be most present around the initial time of adaptation.19 furthermore , it has been shown that people can adjust to glasses that invert the image seen ; once the glasses are removed , they simply adjust back within 30 minutes.20 it is suggested that this is due to the brain accommodating the change , through recalibration of the proprioception receptors in the upper and lower limbs and extra retinal eye movements ( egocentric and oculocentric visual directions ) , rather than a correction of the vision . with that in mind , the participants in this trial may have adapted to their pals , having worn them for a mean of 228 days ( sd 146 days ) , and were thus able to react more quickly than with a new pair of single vision glasses . in changing glasses and thus reversing the prismatic effect , this effect was not specifically tested in our study and it is not unreasonable to postulate that the effect of replacing pals with single vision glasses may work in reverse , but this would need further study . the findings from the study by cumming et al suggest caution should be taken with a sudden change in older people s vision.21 in this study the intervention was aimed at improving vision ( eg , arranging for new eyeglasses ) and this increased rather than decreased the number of falls by participants over a 12 month period . furthermore , most participants in the intervention arm had their glasses changed 13 months before the trial started ( 209 of 309 , 68% ) ; perhaps a more gradual process of updating is required to allow for adjustment to the change in vision . however , the length of time needed to adapt to multifocal glasses has not been defined . according to our study , this would be true only for the vertical displacement in the center and the horizontal bar placed at this position for reaction time . our results found a difference in reaction time when the black line was placed at a position of 300 mm down from center , but this can not be explained by the prismatic displacement effect as there was no statistically significant vertical displacement between the two glasses at this point ( p=0.258 ) . therefore , the reading portion brought the line closer to the participant and the resolution may be clearer , enabling the participant to push the line more quickly . timmis et al s work suggests that participants wearing single vision glasses had more control when stepping down from a block height when compared with individuals who were well adapted to multifocal lenses.9 the study compared kinematic mechanics in 20 older people and found that those participants wearing single vision glasses had reduced vertical center of mass velocity , increased single - limb support time , and reduced peak ankle and knee angular velocities . the step looked closer with a positive lens , which influenced the participant s trailing foot to be placed further away and increased their leading vertical toe clearance . replacing multifocal glasses with single vision glasses could be harmful for other reasons . in the visible trial , secondary measures were recorded of non - fall - related injuries such as lacerations , lifting or twisting injuries , burns or scalds , eye injuries , collisions , and pedestrian injuries.8 they found that 26% of participants replacing multifocal glasses with single vision glasses had a non - fall related injury , compared with 17% of the control group ( p=0.01 ) . the study population were healthy , active participants , which may not enable generalizability to the older population at greatest risk of a fall . the technique used to map the prismatic displacement was limited to the mask used to map the section of the lens . there was a balance between mapping the largest prismatic displacement at the edge of the glasses , while testing had to occur within the spectacle frame . the general trend was for the prismatic displacement to increase the further out from the center of the lens tested . therefore , if the targets used to test accuracy and reaction time were placed in a more peripheral position , they could have potentially shown a larger effect compared with the areas of fixation that were tested . however , this was not possible due to the frames of the participants glasses . although the frames of the new single vision glasses were matched to the participant s pals , there could have been a slight differences in tilt , vertex , adjustment , and size of the new pair of glasses . another limitation with this study is that a light stimulus was used instead of a trip , slip , or loss of balance . therefore , there was no heightened awareness through a fight - or - flight response . a one - off trial which involves a perturbation rather than a light stimulus would possibly be a better method to simulate a fall.22,24 our study expands on the paper by lord et al4 and examines the influence on response to a loss of balance when replacing multifocal glasses with single vision ( distance ) glasses . in conclusion , this study suggests that updating glasses should possibly occur more often in the elderly population to avoid abrupt changes in prismatic displacement .

because of the high cost and time requirements associated with traditional high - throughput screens , many researchers now use computational methods to prefilter candidate ligands prior to experimental testing . a number of ligand - based computational techniques for identifying likely binders have been utilized . these include 2d screening with fingerprints , shape - based screening , and pharmacophore matching , which identify potential actives by comparing their atomic connectivities , three - dimensional shapes , and three - dimensional pharmacophores to those of known ligands , respectively . when structural information about a macromolecular drug target is known ( e.g. , from x - ray crystallography or nmr ) , computer docking programs are often used to identify candidate ligands . these programs position three - dimensional models of small molecules into models of target binding pockets ; associated scoring functions subsequently predict the binding affinities of these posed candidate ligands . while certainly useful as an enrichment tool , docking has not yet reached its full potential . in part , the inaccuracies inherent in this technique stem from factors that are independent of the scoring function itself . for example , most docking programs do not account for full receptor flexibility , despite the fact that flexibility plays a critical role in modern theories of small - molecule binding ( e.g. , induced - fit and population - shift models ) . indeed , efforts to account for receptor flexibility have proven effective and have led to the identification of a number of experimentally validated ligands . similarly , most docking programs do not account for binding - pocket water molecules , which can in some cases play critical roles in mediating receptor ligand interactions . docking program would fail to identify true ligands when presented with sterically incompatible binding - pocket conformations and/or pockets devoid of crucial water molecules . however , some of the inaccuracies associated with computer docking are intrinsic to the scoring functions themselves . in recent years , much work has been directed toward improving these functions without sacrificing speed . some of our own recent efforts have focused on training neural networks to rapidly predict the binding energies of protein ligand complexes leading to the creation of two neural - network - based scoring functions , nnscore 1.0 and nnscore 2.0 . neural networks are computer models that mimic , albeit inadequately , the microscopic architecture and organization of the brain . biological neurons and synapses are simulated in silico as neurodes and connections . data to be analyzed is encoded on an input layer of neurodes , triggering a cascade of signals that propagates through the network . both the organization and number of the neurodes as well as the weights ( i.e. , strengths ) assigned to each neurode the cascade eventually reaches an output layer of artificial neurons , where an analysis of the original input signal is ultimately encoded . in the nnscore implementations , the strengths of the connections between neurodes were varied until the networks could reliably predict binding affinity when given descriptors of a ligand , these descriptors included the number of protein ligand close contacts , categorized by autodock atom types ; the electrostatic energy of those close contacts ; the number of ligand atoms of each atom type ; and the number of ligand rotatable bonds . for nnscore 2.0 , the input additionally included the descriptors provided by the binana algorithm ( counts of the number of hydrophobic , , hydrogen - bond , and salt - bridge interactions ) , as well as the components of the vina scoring function ( steric , hydrophobic , hydrogen - bond , and ligand - rotatable - bond terms ) . while some efforts have been made to demonstrate the favorable performance of these neural - network scoring functions , these efforts focused on a limited number of systems , and the neural - network functions were not directly compared to top - tier proprietary docking programs like schrdinger s glide . in the current work , we use autodock vina and glide to dock the diverse compounds of the nci diversity set iii , a popular compound library available through the national cancer institute ( nci ) , into the 40 protein receptors of the directory of useful decoys ( dud ) . additionally , vina- and glide - docked poses are reevaluated using nnscore 1.0 and 2.0 . the mean screening performance obtained when candidate ligands are docked with vina and rescored with nnscore 1.0 is not statistically different than the mean performance obtained when docking and scoring with glide . this is particularly noteworthy given that glide , while state of the art , is expensive and has a restrictive token system . in contrast , autodock vina and nnscore 1.0 are both free and open source . additionally , we note a correlation between certain chemical properties and the associated docking scores , suggesting systematic bias . for both vina and nnscore , docking scores tended to correlate with small - molecule chemical properties like size and polarizability , regardless of the target receptor . compensating in part for these potential biases improves virtual - screen performance . creating composite scoring functions suited to a specific receptor can improve performance further still . though the mean screening performances of glide and ( vina + nnscore 1.0 ) over all 40 dud receptors are not statistically different , our results do confirm what has been found by others : the best scoring function to use for a specific pharmacological target , be it vina , nnscore , or glide , is highly system dependent . positive controls ( known inhibitors ) , when available , should be included in virtual screens to ensure that the docking protocol chosen is suited to the system at hand . however , when positive controls are not available , we recommend ( vina + nnscore 1.0 ) as a potential alternative to proprietary docking programs like schrdinger s glide . the 40 protein receptors of the dud were downloaded from the dud website ( http://dud.docking.org/ ) . all ligands and water molecules were removed . hydrogen atoms were added to the protein structures ( neutral ph ) and hydrogen bonds were optimized using schrdinger maestro s protein preparation wizard . these processed models were then converted to the autodock pdbqt format with mgltools 1.5.4 for use in the vina and nnscore screens . models of ligands known to bind to the 40 dud receptors were likewise downloaded from the dud website . additionally , the compounds of the nci diversity set iii were obtained from the website of the nci / nih developmental therapeutics program ( http://dtp.nci.nih.gov/ ) . all these small molecules were processed with schrdinger s ligprep module to generate models with appropriate tautomeric , isomeric , and ionization states for ph values ranging from 5.0 to 9.0 . these models were ultimately converted to the pdbqt format using mgltools 1.5.4 for use in the vina and nnscore screens , and to the sdf format for use in the glide screens . a few molecular models could not be generated ; 1560 nci models were ultimately used in the virtual screens . for vina docking , all ligands were docked into regions ( boxes ) centered on the respective receptor binding pockets . edge lengths ranged from 35.9 to 50.0 , and box volumes ranged from 56,365.8 to 88,845.5 . for the vina analysis , the best - scoring pose as judged by the vina docking score was used . for the nnscore analysis , all vina poses were reevaluated with nnscore 1.0 and nnscore 2.0 , and the best - scoring vina pose as judged by nnscore was considered for subsequent analysis . in the case of nnscore 1.0 , the original programmers provided 24 neural networks that were particularly adept at identifying potent ligands when given descriptors of crystallographic ligand receptor complexes . the final nn1 scores for each docking were derived by averaging the output of these 24 networks . for glide docking , the docking grid was defined by two cubes centered on the dud - specified active sites . the inner cube , with 14 edges , imposed restrictions on the location of the ligand center , and the outer cube , with 45 edges , imposed restrictions on the location of all ligand atoms . for each system , both the positive controls and the compounds of the nci diversity set iii were first docked with glide htvs . to test more advanced glide docking protocols , the top 50% of these compounds were subsequently docked with glide sp , and the top 25% of the remaining compounds were docked with glide xp . glide - htvs- and glide - xp - docked models were also rescored with nnscore 1.0 and 2.0 . the default parameters were again used for both . in all screens , where there were multiple models of a distinct compound due to alternate tautomeric , stereoisomeric , or protonation states , the best - scoring model was selected , and all others were discarded . receiver operating characteristic ( roc ) curves can be generated when performing virtual screens of compound libraries that include known binders . following docking and scoring , a moving cutoff is then employed that sweeps from the best predicted binder to the worst . at each cutoff , the list of compounds is partitioned . the compounds above the cutoff are tentatively considered to be binders , and the compounds below are considered to be nonbinders . if all compounds not known to be binders are considered decoys , true and false positive rates can then be calculated for each partition . the roc curve is generated by plotting all ( false positive rate , true positive rate ) points . roc curves assess the performance of a virtual screen from the best predicted binder to the worst . in practice , however , computational chemists are typically interested in the top - ranked compounds . one useful way to assess the top - performing ligands is to determine the true positive rate for a given fixed false positive rate . in the current work , we identify the true positive rate when the false positive rate is fixed at 5% . when required to facilitate the optimization of this metric , the roc curve was smoothed using a linear interpolation as implemented in numpy / scipy . the roc curves generated using the multi - tiered ( htvs - sp - xp ) glide protocol against dihydrofolate reductase and epidermal growth factor receptor were not complete enough to calculate the early - performance metric . to maintain equal sample sizes for the anova and t - test analyses , all screens against these two receptors were discarded when appropriate , regardless of the docking scoring protocol used . mean and median early - performance metrics over all 40 dud receptors were calculated using all available screen results . first , we perform a systematic comparison of common autodock vina , nnscore , and glide protocols . in this study , a protocol refers to the two - step process used to computationally identify potential ligands . first , a compound model must be placed within a model of the binding pocket . these two steps are called docking and scoring , respectively , and are often combined into a single algorithm . vina and glide , for example , both dock and score potential ligands ; nnscore 1.0 and 2.0 only score ( or rescore ) . nnscore-1.0 and vina nnscore-2.0 ( henceforth abbreviated vina - nn1 and vina - nn2 , respectively ) and several popular protocols that are based exclusively on either vina or glide . we show that while the performance of these docking schemes is highly receptor dependent , the mean screening performances of the vina - nn1 and glide protocols are not statistically different . second , we demonstrate that there are biases in most of the docking protocols studied , as has been demonstrated for other scoring functions . correcting for these biases improves the performance of the vina - nn1 protocol further . in order to compare multiple docking protocols , it is useful to perform a series of mock virtual screens that draw from compound libraries containing both known ligands ( actives ) and presumed decoy molecules . as the actives are known a priori , screen performance can be assessed by examining the ability of a given docking protocol to accurately separate out actives from decoys . the performance of a given protocol is often receptor specific ; consequently , it is prudent to perform multiple screens into many diverse receptors when attempting to assess global utility . the directory of useful decoys ( dud ) , an excellent resource for facilitating these assessments , contains 40 diverse protein receptors and 2950 known actives . for each active , the dud contains 36 topologically distinct presumed decoys that are by design chemically similar to the known inhibitors , as judged by metrics like molecular weight , clogp , and the number of hydrogen - bonding groups . in the current work , we use the dud receptors and known active compounds to assess several docking protocols ; however , rather than using the dud decoy molecules , we instead used 1560 models of compounds from the nci diversity set iii ( presumed decoys ) , a set of publically available , diverse , drug - like molecules provided by the national cancer institute free of charge . without wishing to in any way disparage the dud decoy set , which is certainly useful in many contexts , it is important to understand why we opted to use the nci compounds as decoys instead . factors that influence molecular binding can be divided into two general categories : those that are ligand specific ( i.e. , independent of the receptor ) and those that are binding specific ( i.e. , dependent on specific receptor ligand interactions ) . the number of ligand rotatable bonds is a good example of a ligand - specific factor , as the immobility of highly flexible ligands that generally occurs upon binding is thought to be entropically unfavorable , independent of the receptor . in contrast , receptor ligand complementarity of hydrogen - bond donors and acceptors is a good example of a binding - specific factor , as it depends specifically on interactions between the ligand and the receptor . in predicting ligand binding the dud decoys were specifically selected so as to be chemically similar to known actives ; they consequently may lack the chemical heterogeneity that one would see in a set of compounds selected with diversity in mind ( e.g. , the nci diversity set ) . on one hand , it is certainly possible that some scoring functions may be inappropriately biased in their assessment of ligand - specific factors . what if , for example , a scoring function inappropriately assigns better docking scores to compounds with larger dipole moments independent of the receptor , and coincidentally , the actives being screened tend to have larger dipole moments than the decoys ? the idea of controlling for this inappropriate bias by intentionally selecting decoy molecules with dipole moments similar to those of the actives certainly has its appeal . on the other hand , insufficient chemical heterogeneity in the decoys may unfairly bias the evaluation of scoring functions that rely on valid assessments of ligand - specific factors . what if , for example , a scoring function correctly considers the number of ligand rotatable bonds in assessing the likelihood of binding but the actives and decoys all have the same number of rotatable bonds ? such a scoring function would be inappropriately penalized because its ability to utilize information about ligand rotatable bonds would be underexploited . indeed , these types of concerns have lead others to use modified versions of the dud decoy set . of note , vina includes one ligand - specific term in its scoring function ( number of rotatable bonds ) , and the nnscore functions include additional ligand - specific terms related to the number of ligand atom types . consequently , while we believe convincing arguments can be made in favor of using the dud decoys , in the current work , we opted to use the nci compounds as decoys instead . a separate issue related to decoy selection must also be addressed . high - throughput screens typically have hit rates that range from 0.1% to 1.0% ; it is therefore reasonable to assume that for each dud protein , the nci set contains between 1 and 16 decoys that are in fact actives . a similar assumption underlies the set of dud decoys , which have likewise not been explicitly tested to rule out binding . possible inaccuracies in comparison metrics introduced by these kinds of assumptions are at least in part ameliorated by the fact that all the docking scoring protocols being compared are subject to the same assumption . furthermore , the nci set used in the current project may well have fewer true binders than the widely used dud set , given that the dud decoys were , as mentioned above , carefully chosen to be chemically similar to the dud actives . having selected the receptors , actives , and decoys , we next turn to the question of how best to evaluate virtual - screening performance . among the many methods that have been considered , receiver operating characteristic ( roc ) curves are appealing because they are independent of the ratio of actives vs inactives and have desirable statistical properties . the area under the roc curve ( roc - auc ) is thought to correspond to the probability that a known binder picked at random will rank higher than a known nonbinder picked at random . to compare docking scoring protocols using the roc - auc metric , we docked nci decoys and dud actives into the 40 dud receptors using autodock vina and glide htvs , a state of the art , fast docking algorithm designed specifically for screening large libraries . the vina - docked poses were then rescored with nnscore 1.0 and nnscore 2.0 . the more rigorous glide sp and glide xp docking protocols were not used at this juncture because , while impressively precise , they are not as well suited for use in high - throughput virtual screens . given that an average of 1634 compounds had to be docked into each of the 40 dud receptors ( 74 dud actives and 1560 nci decoys ) , 65,350 individual dockings were required to test each docking protocol . we note that others have similarly eschewed an exclusive use of glide sp / xp for projects requiring comparable numbers of individual dockings . as has been shown previously , our results demonstrate that the ideal docking protocol for a given project is highly system dependent . for example , when the screens were assessed by the roc - auc metric , vina nn2 performed best for docking into epidermal growth factor receptor and platelet - derived growth factor receptor kinase . htvs performed best for docking into adenosine deaminase and ampc -lactamase ( table 1 ) . given that nn2 considers features of molecular binding that nn1 neglects , it is curious that for many individual receptors vina one common criticism of neural networks is that , unlike some other machine - learning techniques , they are essentially black boxes ; it is difficult to impossible to determine precisely how they come to their ultimate conclusions . though speculative , we suspect two factors explain the favorable performance of vina nn1 . first , the additional features of molecular binding that nn2 explicitly considers may not provide additional information over what nn1 can infer implicitly . for example , in estimating binding affinity , nn2 explicitly considers the number stacking interactions ; however , nn1 might be able to implicitly infer stacking by considering the number of receptor and ligand aromatic carbon atoms that are in close proximity . nn1 is trained to return a binary response : good binder or poor binder . in contrast , nn2 is trained to return a range of scores roughly equivalent to pki or pic50 values . it may be that binary classification is more effective than continuous classification in this case . future versions of nnscore currently in development will return to the binary - classification paradigm . setting the specific details of virtual screens against individual proteins aside , the best way of assessing the global utility of a docking scoring function is to consider its performance over multiple diverse receptors . when the average area under the roc curve calculated over all 40 dud receptors was considered , vina vina and htvs htvs . to determine whether or not this difference was statistically significant , we used a technique called analysis of variance ( anova ) . anova asserts the null hypothesis that the means of multiple samples are equal ( i.e. , that multiple samples are drawn from populations with the same mean ) . in this sense , anova is similar to the t - test , which is limited to two samples . when assessing multiple samples , one might be tempted to simply perform multiple t - tests between all sample pairs ; however , each t - test carries with it the risk of incorrectly rejecting the null hypothesis ( i.e. , committing a type i error by rejecting the conclusion that two samples have statistically equal means when in fact they are statistically equal ) . as more and more t - tests anova avoids the problem by considering multiple samples in conjunction rather than pairwise . both anova and the t - test allow one to assess the degree of statistical significance via a p value . the p value in this case represents the probability that the multiple samples could have means that differ to the degree observed or greater , given that the null hypothesis is true ( i.e. , given that the samples are drawn from populations with equal means ) . if p < 0.05 , the null hypothesis is rejected . htvs protocols were not statistically different ( p = 0.16 , not quite the 0.05 required to reject the null hypothesis ) . clearly , known inhibitors , when available , should be included in a virtual screen and used to determine which docking scoring protocol is best suited to the specific system at hand . in the absence of any information about known binders , however , we recommend docking with vina and rescoring with nnscore 1.0 , as that protocol did have the highest roc - auc mean and median performances . though the roc - auc metric is frequently used to evaluate virtual - screening performance , some have criticized its use because it assesses that performance by considering all screened compounds from the best predicted binder to the worst . in practice , computational chemists are most interested in the top - ranked compounds , the ones that will be subsequently submitted for experimental validation . it is therefore the initial portion of the roc curve , some argue , that ought to be of primary interest . a number of performance metrics have been proposed to address this issue ( e.g. , the bedroc metric derived from a modified roc curve that weights top - ranked compounds ) . in this scheme , one analyzes a roc curve to determine the true positive rate for a fixed false positive rate ( 5% in the current work ) . we used the metric of early performance to compare the vina- , nnscore- , and htvs - based protocols to a common multi - tiered glide protocol ( htvs sp xp ) that has been used extensively in the literature ( see , for example , refs ( 52 ) , ( 55 ) , ( 56 ) , and ( 57 ) ) . the top 50% best ligands as judged by the htvs the top 25% of the glide - sp compounds were then redocked with glide xp . these xp - docked poses were additionally rescored with nnscore 1.0 and nnscore 2.0 to facilitate comparison . admittedly , the analysis herein described required that only 1634 compounds be docked into each dud receptor on average , suggesting that glide - sp or -xp docking alone might have been feasible . however , as mentioned above , because we considered all 40 dud receptors simultaneously , 65,350 dockings would have ultimately been required had the multi - tiered approach been abandoned . others have similarly turned to multi - tiered glide protocols for projects requiring comparable numbers of individual dockings . many compounds were filtered out in the initial htvs and sp steps of the htvs consequently , it was not possible to calculate a complete roc curve for the htvs however , by assuming that compounds filtered out in the preliminary htvs and sp steps truly were poor binders ( the reasoning implicit in the multi - tiered approach ) , it was possible to generate the arguably paramount initial portion of the roc curve . while individual results were system dependent , vina nn1 and vina vina as judged by the average early - performance metric ( table 2 ) . surprisingly , the mean performances of the htvs htvs , htvs sp xp , and vina in contrast , t - tests comparing the mean performance of the htvs sp xp protocol to that of the vina vina and vina nn2 protocols led us to reject the null hypothesis of equivalence ( t - test , p = 0.002 and 0.049 , respectively ) . sp xp performed better , on average , than vina vina and vina nn2 . to further compare the glide xp and nnscore scoring functions , we reevaluated the htvs nevertheless , anova demonstrated that the mean performances of the htvs sp xp , htvs sp xp nn1 , and htvs sp xp nn2 protocols were not statistically different ( p = 0.88 ) . nn1 were likewise not statistically different ( t - test , p = 0.70 ) . in hopes of further improving virtual - screening accuracy , we next sought to specifically characterize scoring - function biases . schrdinger s qikprop program was used to analyze the screened compounds ( known inhibitors and putative decoys ) . a statistical correlation between certain chemical properties and the average rank of each compound across all 40 dud receptors was noted . nn2 scores tended to correlate with properties associated with molecular size ( molecular weight , total solvent accessible surface area , volume , number of ring atoms , and number of heteroatoms ) and polarizability ( table 3 ) . it is interesting that both vina and the neural - network scoring functions demonstrated similar trends , even though they evaluate ligand binding using very different methodologies . others have identified similar biases in the flexx and gold docking programs . the htvs htvs and htvs sp xp protocols did not exhibit these biases to the same extent ( table 3 ) . for the interested reader , we provide a real - world example of how scoring - function biases can affect screening results in the supporting information . for the htvs xp protocol , only ligands that were scored using glide xp in at least 10 out of 40 receptors were considered . in the end , this amounted to about 200 ligands . these potentially artifactual correlations between ligand properties and docking scores may result in part from a general neglect of penalty terms that ought to be associated with binding ( e.g. , ligand desolvation , trapping binding - site waters , etc . ) . we do not mean to imply that vina and nnscore neglect penalty terms entirely . the vina scoring function , for example , has three steric - interaction terms . additionally , nnscore may be able to implicitly account for some penalty terms as well ; information about energetically unfavorable buried polar groups , for example , could potentially be extracted from the pairwise receptor ligand atom - type information that nnscore considers . nevertheless , both vina and nnscore are likely blind to many important penalty phenomena . indeed , glide - based protocols may perform well relative to many other scoring functions because they better account for these penalties , as evidenced by the fact that htvs and xp scores are not strongly correlated with molecular weight ( table 3 ) . future versions of nnscore currently being developed will consider three - dimensional descriptions of ligand receptor binding and so may be even more effective than current implementations . on the other hand , one must consider the possibility that at least a portion of these scoring - function biases in fact represent accurate characterizations of small - molecule binding . to this end , we tested whether or not correlations exist between the molecular properties and experimentally measured binding affinities of known ligands independent of receptor . all the binding data deposited in the binding moad database as of march 2013 was considered . in total , 2081 entries representing 1598 unique compounds had both known structures and precise or approximate ki measurements ( i.e. , measurements described as = or interestingly , both molecular weight and polarizabiliy were correlated with the experimentally measured pki values ; these molecular properties are plotted as a function of average pki ( independent of receptor ) in figure 1a and b , respectively . linear regression suggested that the relationship between molecular weight and pki was mw = 39.2710 ( pki ) + 122.1835 , with a r value of 0.23 . a t - test on the slope coefficient yielded a p - value of 0.0 , leading us to reject the null hypothesis that there is no true relationship between pki and molecular weight ( i.e. , that the true slope coefficient is 0 ) . similarly , the regression equation describing the relationship between polarizability and pki was pol = 4.3326 ( pki ) + 7.3163 , with a r value of 0.30 . a t - test on the slope coefficient of this regression similarly produced a p - value of 0.0 ; the hypothesis that polarizability and pki are not correlated was thus similarly rejected . subjectively , the notion that ligand binding may be in part dependent on factors that are entirely ligand centric has some appeal . for example , while it is certainly true that in the absence of ligand binding - site complementarity bigger is not necessarily better , larger molecules may well have more interacting moieties on average that serve to enhance potency if complementarity is assumed . on the other hand , it may be that the noted correlations between molecular properties and experimentally measured pki values are more reflective of traditional and perhaps flawed approaches to medicinal chemistry than of actual physical phenomena . for example , during the drug - optimization process , molecules do tend to increase in size . regardless , our goal ought to be to compensate for true biases while maintaining correlations that reflect actual physical phenomena . to compensate for potential scoring - function biases , we considered 15 relevant qikprop properties for each nci decoy and known dud inhibitor : accpthb ( estimated number of hydrogen - bond acceptors ) , ciqplogs ( predicted aqueous solubility ) , dipole ( computed ligand dipole moment ) , donorhb ( estimated number of hydrogen - bond donors ) , sasa ( total solvent accessible surface area , ) , fisa ( hydrophilic component of the sasa , ) , fosa ( hydrophobic component of the sasa , ) , mol_mw ( molecular weight , daltons ) , nonhatm ( number of heavy atoms ) , pisa ( component of the sasa , ) , qplogpc16 ( predicted hexadecane / gas partition coefficient ) , qplogpo / w ( predicted octanol / water partition coefficient ) , qppolrz ( predicted polarizability , ) , rotor ( number of rotatable bonds ) , and volume ( total solvent - accessible volume , ) . nn1 score , pi is the i chemical property ( listed above ) , and ci is a coefficient associated with that property . a stepwise selection method was used to identify the composite scoring functions that best improved screen performance . first , training sets were generated for each dud receptor by randomly selecting 75% of the associated known actives and merging them with the set of all nci decoys . all 15 coefficients were initially set to 0.0 so that the composite and vina was then used to adjust the coefficients so as to maximize the average screen performance over all 40 training sets . once training was complete , the resulting composite scoring function was then evaluated by calculating the average early - performance metric over the 40 testing sets , now without adjusting the coefficients . the above protocol was repeated ; each time , a different coefficient was fixed at 0.0 so that the associated chemical property was essentially ignored . the single chemical property that when discounted was associated with the smallest drop in the average screen performance over the 40 testing sets was identified . a new set of 14 chemical properties was generated by excluding this chemical property . in total , this elimination step was repeated 15 times until no additional chemical properties remained ( figure 2a ) . screen performance when composite scoring functions were used . in each graph , the leftmost data point describes the average performance over all 40 dud receptors when a composite function that incorporates the vina a general - purpose scoring function was identified by considering the minimum number of chemical properties required to maintain optimal early performance ( figure 2a ) . specifically , when the compounds were ranked by ( nn1 + 0.1093 donorhb + 0.0011 fosa + 0.0008 pisa ) , the average early - performance metric over all 40 receptors was 0.48 , substantially improved over the 0.35 obtained with vina nn1 alone . a t - test in fact required that we reject the notion that the mean performances of these two screens were statistically equal ( p = 0.04 ) ( table 2 , composite / general ) . given that we opted to use decoys that were not necessarily chemically similar to the dud actives , it was especially important to ensure that the above general - purpose scoring function was not overtrained . for example , consider the hypothetical possibility that the decoys and dud actives all have fewer than three and more than four hydrogen - bond donors , respectively . it would be possible to identify the actives using ligand - specific factors alone , independent of any important ligand receptor interactions . on the other hand , some ligand - specific factors ( e.g. , the number of rotatable bonds ) may well be legitimately useful for distinguishing actives from decoys . it is not unreasonable to expect some genuine enrichment when compounds are ranked by a scoring function comprised exclusively of ligand - specific terms ; nevertheless , one would expect that screen performance would improve further still when additional binding - specific terms ( e.g. , the nnscore ) are included . to ensure that actives were not being identified based on their chemical properties alone , we generated a second scoring function of the same form ( 0.1093 donorhb + 0.0603 fosa + 0.0458 pisa ) , comprised exclusively of the ligand - specific terms of the parent general - purpose function . the average early - performance metric over all 40 receptors when the ligands were ranked by this ligand - specific scoring function was 0.22 . a t - test required that we reject the null hypothesis that the mean performances of the ligand - specific scoring function and its parent function were statistically equivalent ( p = 0.00005 ) , suggesting that the general - purpose function achieved its enhanced performance by considering both ligand - specific and binding - specific factors . the implicit assumption behind the creation of any general - purpose scoring function is that a single function can perform optimally across any number of receptors ; however , given the demonstrated system dependence of scoring functions in general , this supposition is not likely to be correct . we therefore repeated the above scoring - function optimizations , now generating an independent composite scoring function for each receptor ( figure 2b ) . the average early - performance metric over the 40 receptors was substantially improved when receptor - specific scoring functions that included terms for fisa , mol_mw , and volume were employed ( 0.68 ) . a t - test required us to reject the null hypothesis that the mean early - performance metrics of this scoring function and the general - purpose scoring function described above were statistically equivalent ( p = 0.00073 ; table 2 , composite / individual ) . an analogous scoring function containing only ligand - specific terms was also generated , as above . again , we rejected the null hypothesis that the mean performances of the ligand - specific scoring function and its parent function with the additional nn1 term were statistically equivalent ( 0.53 vs 0.68 , p = 0.01 ) . it is not necessarily our purpose to provide specific composite functions for others to use in their virtual screens ; rather , we wish to demonstrate the general utility of deriving such functions when positive controls ( known inhibitors ) are available . a composite scoring function tailored to a specific receptor and designed to optimize the ranking of known inhibitors can potentially enhance virtual - screening performance . the performance of a docking scoring protocol is highly dependent on the specific receptor being studied . when possible , positive controls ( known binders ) should be included in the screen so many different protocols can be tested . however , in the absence of known ligands and when a free , open source , general - purpose solution is sought , docking with autodock vina and rescoring with nnscore 1.0 is an excellent option . we are hopeful that this work will help guide computational chemists in their efforts to best utilize computer - docking programs .

we compare established docking programs , autodock vina and schrdinger s glide , to the recently published nnscore scoring functions . as expected , the best protocol to use in a virtual - screening project is highly dependent on the target receptor being studied . however , the mean screening performance obtained when candidate ligands are docked with vina and rescored with nnscore 1.0 is not statistically different than the mean performance obtained when docking and scoring with glide . we further demonstrate that the vina and nnscore docking scores both correlate with chemical properties like small - molecule size and polarizability . compensating for these potential biases leads to improvements in virtual screen performance . composite nnscore - based scoring functions suited to a specific receptor further improve performance . we are hopeful that the current study will prove useful for those interested in computer - aided drug design .

Introduction Materials and Methods Results and Discussion Conclusion

these include 2d screening with fingerprints , shape - based screening , and pharmacophore matching , which identify potential actives by comparing their atomic connectivities , three - dimensional shapes , and three - dimensional pharmacophores to those of known ligands , respectively . , from x - ray crystallography or nmr ) , computer docking programs are often used to identify candidate ligands . these programs position three - dimensional models of small molecules into models of target binding pockets ; associated scoring functions subsequently predict the binding affinities of these posed candidate ligands . in part , the inaccuracies inherent in this technique stem from factors that are independent of the scoring function itself . for example , most docking programs do not account for full receptor flexibility , despite the fact that flexibility plays a critical role in modern theories of small - molecule binding ( e.g. however , some of the inaccuracies associated with computer docking are intrinsic to the scoring functions themselves . some of our own recent efforts have focused on training neural networks to rapidly predict the binding energies of protein ligand complexes leading to the creation of two neural - network - based scoring functions , nnscore 1.0 and nnscore 2.0 . neural networks are computer models that mimic , albeit inadequately , the microscopic architecture and organization of the brain . for nnscore 2.0 , the input additionally included the descriptors provided by the binana algorithm ( counts of the number of hydrophobic , , hydrogen - bond , and salt - bridge interactions ) , as well as the components of the vina scoring function ( steric , hydrophobic , hydrogen - bond , and ligand - rotatable - bond terms ) . while some efforts have been made to demonstrate the favorable performance of these neural - network scoring functions , these efforts focused on a limited number of systems , and the neural - network functions were not directly compared to top - tier proprietary docking programs like schrdinger s glide . in the current work , we use autodock vina and glide to dock the diverse compounds of the nci diversity set iii , a popular compound library available through the national cancer institute ( nci ) , into the 40 protein receptors of the directory of useful decoys ( dud ) . additionally , vina- and glide - docked poses are reevaluated using nnscore 1.0 and 2.0 . the mean screening performance obtained when candidate ligands are docked with vina and rescored with nnscore 1.0 is not statistically different than the mean performance obtained when docking and scoring with glide . in contrast , autodock vina and nnscore 1.0 are both free and open source . additionally , we note a correlation between certain chemical properties and the associated docking scores , suggesting systematic bias . for both vina and nnscore , docking scores tended to correlate with small - molecule chemical properties like size and polarizability , regardless of the target receptor . compensating in part for these potential biases improves virtual - screen performance . creating composite scoring functions suited to a specific receptor can improve performance further still . though the mean screening performances of glide and ( vina + nnscore 1.0 ) over all 40 dud receptors are not statistically different , our results do confirm what has been found by others : the best scoring function to use for a specific pharmacological target , be it vina , nnscore , or glide , is highly system dependent . positive controls ( known inhibitors ) , when available , should be included in virtual screens to ensure that the docking protocol chosen is suited to the system at hand . however , when positive controls are not available , we recommend ( vina + nnscore 1.0 ) as a potential alternative to proprietary docking programs like schrdinger s glide . these processed models were then converted to the autodock pdbqt format with mgltools 1.5.4 for use in the vina and nnscore screens . these models were ultimately converted to the pdbqt format using mgltools 1.5.4 for use in the vina and nnscore screens , and to the sdf format for use in the glide screens . for the vina analysis , the best - scoring pose as judged by the vina docking score was used . for the nnscore analysis , all vina poses were reevaluated with nnscore 1.0 and nnscore 2.0 , and the best - scoring vina pose as judged by nnscore was considered for subsequent analysis . in the case of nnscore 1.0 , the original programmers provided 24 neural networks that were particularly adept at identifying potent ligands when given descriptors of crystallographic ligand receptor complexes . for glide docking , the docking grid was defined by two cubes centered on the dud - specified active sites . for each system , both the positive controls and the compounds of the nci diversity set iii were first docked with glide htvs . to test more advanced glide docking protocols , the top 50% of these compounds were subsequently docked with glide sp , and the top 25% of the remaining compounds were docked with glide xp . glide - htvs- and glide - xp - docked models were also rescored with nnscore 1.0 and 2.0 . in all screens , where there were multiple models of a distinct compound due to alternate tautomeric , stereoisomeric , or protonation states , the best - scoring model was selected , and all others were discarded . following docking and scoring , a moving cutoff is then employed that sweeps from the best predicted binder to the worst . at each cutoff , the list of compounds is partitioned . roc curves assess the performance of a virtual screen from the best predicted binder to the worst . in practice , however , computational chemists are typically interested in the top - ranked compounds . first , we perform a systematic comparison of common autodock vina , nnscore , and glide protocols . these two steps are called docking and scoring , respectively , and are often combined into a single algorithm . vina and glide , for example , both dock and score potential ligands ; nnscore 1.0 and 2.0 only score ( or rescore ) . we show that while the performance of these docking schemes is highly receptor dependent , the mean screening performances of the vina - nn1 and glide protocols are not statistically different . second , we demonstrate that there are biases in most of the docking protocols studied , as has been demonstrated for other scoring functions . correcting for these biases improves the performance of the vina - nn1 protocol further . as the actives are known a priori , screen performance can be assessed by examining the ability of a given docking protocol to accurately separate out actives from decoys . for each active , the dud contains 36 topologically distinct presumed decoys that are by design chemically similar to the known inhibitors , as judged by metrics like molecular weight , clogp , and the number of hydrogen - bonding groups . in the current work , we use the dud receptors and known active compounds to assess several docking protocols ; however , rather than using the dud decoy molecules , we instead used 1560 models of compounds from the nci diversity set iii ( presumed decoys ) , a set of publically available , diverse , drug - like molecules provided by the national cancer institute free of charge . without wishing to in any way disparage the dud decoy set , which is certainly useful in many contexts , it is important to understand why we opted to use the nci compounds as decoys instead . , dependent on specific receptor ligand interactions ) . what if , for example , a scoring function inappropriately assigns better docking scores to compounds with larger dipole moments independent of the receptor , and coincidentally , the actives being screened tend to have larger dipole moments than the decoys ? on the other hand , insufficient chemical heterogeneity in the decoys may unfairly bias the evaluation of scoring functions that rely on valid assessments of ligand - specific factors . consequently , while we believe convincing arguments can be made in favor of using the dud decoys , in the current work , we opted to use the nci compounds as decoys instead . furthermore , the nci set used in the current project may well have fewer true binders than the widely used dud set , given that the dud decoys were , as mentioned above , carefully chosen to be chemically similar to the dud actives . having selected the receptors , actives , and decoys , we next turn to the question of how best to evaluate virtual - screening performance . to compare docking scoring protocols using the roc - auc metric , we docked nci decoys and dud actives into the 40 dud receptors using autodock vina and glide htvs , a state of the art , fast docking algorithm designed specifically for screening large libraries . the vina - docked poses were then rescored with nnscore 1.0 and nnscore 2.0 . as has been shown previously , our results demonstrate that the ideal docking protocol for a given project is highly system dependent . future versions of nnscore currently in development will return to the binary - classification paradigm . setting the specific details of virtual screens against individual proteins aside , the best way of assessing the global utility of a docking scoring function is to consider its performance over multiple diverse receptors . the p value in this case represents the probability that the multiple samples could have means that differ to the degree observed or greater , given that the null hypothesis is true ( i.e. htvs protocols were not statistically different ( p = 0.16 , not quite the 0.05 required to reject the null hypothesis ) . clearly , known inhibitors , when available , should be included in a virtual screen and used to determine which docking scoring protocol is best suited to the specific system at hand . in the absence of any information about known binders , however , we recommend docking with vina and rescoring with nnscore 1.0 , as that protocol did have the highest roc - auc mean and median performances . though the roc - auc metric is frequently used to evaluate virtual - screening performance , some have criticized its use because it assesses that performance by considering all screened compounds from the best predicted binder to the worst . in practice , computational chemists are most interested in the top - ranked compounds , the ones that will be subsequently submitted for experimental validation . we used the metric of early performance to compare the vina- , nnscore- , and htvs - based protocols to a common multi - tiered glide protocol ( htvs sp xp ) that has been used extensively in the literature ( see , for example , refs ( 52 ) , ( 55 ) , ( 56 ) , and ( 57 ) ) . these xp - docked poses were additionally rescored with nnscore 1.0 and nnscore 2.0 to facilitate comparison . admittedly , the analysis herein described required that only 1634 compounds be docked into each dud receptor on average , suggesting that glide - sp or -xp docking alone might have been feasible . surprisingly , the mean performances of the htvs htvs , htvs sp xp , and vina in contrast , t - tests comparing the mean performance of the htvs sp xp protocol to that of the vina vina and vina nn2 protocols led us to reject the null hypothesis of equivalence ( t - test , p = 0.002 and 0.049 , respectively ) . to further compare the glide xp and nnscore scoring functions , we reevaluated the htvs nevertheless , anova demonstrated that the mean performances of the htvs sp xp , htvs sp xp nn1 , and htvs sp xp nn2 protocols were not statistically different ( p = 0.88 ) . nn1 were likewise not statistically different ( t - test , p = 0.70 ) . in hopes of further improving virtual - screening accuracy , we next sought to specifically characterize scoring - function biases . schrdinger s qikprop program was used to analyze the screened compounds ( known inhibitors and putative decoys ) . nn2 scores tended to correlate with properties associated with molecular size ( molecular weight , total solvent accessible surface area , volume , number of ring atoms , and number of heteroatoms ) and polarizability ( table 3 ) . it is interesting that both vina and the neural - network scoring functions demonstrated similar trends , even though they evaluate ligand binding using very different methodologies . we do not mean to imply that vina and nnscore neglect penalty terms entirely . nevertheless , both vina and nnscore are likely blind to many important penalty phenomena . indeed , glide - based protocols may perform well relative to many other scoring functions because they better account for these penalties , as evidenced by the fact that htvs and xp scores are not strongly correlated with molecular weight ( table 3 ) . on the other hand , one must consider the possibility that at least a portion of these scoring - function biases in fact represent accurate characterizations of small - molecule binding . similarly , the regression equation describing the relationship between polarizability and pki was pol = 4.3326 ( pki ) + 7.3163 , with a r value of 0.30 . subjectively , the notion that ligand binding may be in part dependent on factors that are entirely ligand centric has some appeal . for example , while it is certainly true that in the absence of ligand binding - site complementarity bigger is not necessarily better , larger molecules may well have more interacting moieties on average that serve to enhance potency if complementarity is assumed . on the other hand , it may be that the noted correlations between molecular properties and experimentally measured pki values are more reflective of traditional and perhaps flawed approaches to medicinal chemistry than of actual physical phenomena . a stepwise selection method was used to identify the composite scoring functions that best improved screen performance . all 15 coefficients were initially set to 0.0 so that the composite and vina was then used to adjust the coefficients so as to maximize the average screen performance over all 40 training sets . the single chemical property that when discounted was associated with the smallest drop in the average screen performance over the 40 testing sets was identified . screen performance when composite scoring functions were used . in each graph , the leftmost data point describes the average performance over all 40 dud receptors when a composite function that incorporates the vina a general - purpose scoring function was identified by considering the minimum number of chemical properties required to maintain optimal early performance ( figure 2a ) . specifically , when the compounds were ranked by ( nn1 + 0.1093 donorhb + 0.0011 fosa + 0.0008 pisa ) , the average early - performance metric over all 40 receptors was 0.48 , substantially improved over the 0.35 obtained with vina nn1 alone . a t - test in fact required that we reject the notion that the mean performances of these two screens were statistically equal ( p = 0.04 ) ( table 2 , composite / general ) . given that we opted to use decoys that were not necessarily chemically similar to the dud actives , it was especially important to ensure that the above general - purpose scoring function was not overtrained . , the number of rotatable bonds ) may well be legitimately useful for distinguishing actives from decoys . it is not unreasonable to expect some genuine enrichment when compounds are ranked by a scoring function comprised exclusively of ligand - specific terms ; nevertheless , one would expect that screen performance would improve further still when additional binding - specific terms ( e.g. a t - test required that we reject the null hypothesis that the mean performances of the ligand - specific scoring function and its parent function were statistically equivalent ( p = 0.00005 ) , suggesting that the general - purpose function achieved its enhanced performance by considering both ligand - specific and binding - specific factors . the implicit assumption behind the creation of any general - purpose scoring function is that a single function can perform optimally across any number of receptors ; however , given the demonstrated system dependence of scoring functions in general , this supposition is not likely to be correct . a t - test required us to reject the null hypothesis that the mean early - performance metrics of this scoring function and the general - purpose scoring function described above were statistically equivalent ( p = 0.00073 ; table 2 , composite / individual ) . again , we rejected the null hypothesis that the mean performances of the ligand - specific scoring function and its parent function with the additional nn1 term were statistically equivalent ( 0.53 vs 0.68 , p = 0.01 ) . it is not necessarily our purpose to provide specific composite functions for others to use in their virtual screens ; rather , we wish to demonstrate the general utility of deriving such functions when positive controls ( known inhibitors ) are available . a composite scoring function tailored to a specific receptor and designed to optimize the ranking of known inhibitors can potentially enhance virtual - screening performance . the performance of a docking scoring protocol is highly dependent on the specific receptor being studied . however , in the absence of known ligands and when a free , open source , general - purpose solution is sought , docking with autodock vina and rescoring with nnscore 1.0 is an excellent option . we are hopeful that this work will help guide computational chemists in their efforts to best utilize computer - docking programs .

brachytherapy as a boost or monotherapy plays an important role in treating breast cancer [ 1 , 2 ] . in the recent years , accelerated partial breast irradiation ( apbi ) with its 4 - 5 day treatment course has been widely used worldwide to treat early stage breast cancer patients . different techniques are available for apbi with brachytherapy ( bt ) and external beam radiation therapy ( ebrt ) . brachytherapy techniques include interstitial implants , intracavitary devices with single / multilumen balloons , hybrid applicators , and irradiation with low energy x - ray [ 1 , 2 ] . external beam radiation therapy can be performed with 3d conformal radiotherapy ( 3d - crt ) , intensity modulated radiation therapy ( imrt ) , volume modulated arc therapy ( vmat ) , proton therapy , and intraoperative electron irradiation . among all apbi techniques , currently multicatheter interstitial brachytherapy ( mibt ) has the longest patient follow - up and has a level 1 evidence as a valid treatment alternative to whole breast irradiation after breast conserving surgery . recommendations for apbi patient selection and treatment workflow were published in europe and in the usa [ 6 , 7 , 8 ] . recently , the gec - estro ( groupe europen de curiethrapie european society for radiotherapy and oncology ) breast cancer working group has compiled guidelines for target definition and delineation for partial breast irradiation using mibt after breast conserving closed and open cavity surgery [ 9 , 10 ] . in classical breast brachytherapy , treatment planning was based on two x - ray films . using different reconstruction methods ( e.g. , orthogonal , variable angle , semi - orthogonal ) and a digitizer device , catheters were reconstructed in three dimensions ( 3d ) , but the anatomical information about the patients was very limited . the surgical clips approximately showed the position of the lumpectomy cavity , and the catheter fixation buttons indicated the skin surface in a number of points only . at that time , neither dose coverage nor conformality was possible to be determined , and it was assumed that the catheters are in the right positions . later , with the introduction of cross - sectional imaging , the real 3d dose planning became available . the reconstructed catheters geometry and the delineation of target volume and organs at risk on computed tomography ( ct ) slices made possible for conformal irradiation . but , at the beginnings , the post - implant ct imaging was used only for evaluating dose plans , and just a few quality indices were calculated [ 11 , 12 , 13 , 14 ] . these studies clearly demonstrated that the conventional fluoroscopy based implantations usually could not result in acceptable dose distributions regarding all dose parameters . reported significantly increased target coverage and dose homogeneity after implementing their ct - guided implantation technique . in a paper by major et al . , 91% target coverage and 0.33 for dose non - uniformity ratio was published evaluating 28 ct - based treatment plans . these values were significantly better than the corresponding data of the radiography - based implantation method . nowadays , based on the persuading early dosimetric results , ct - based treatment planning has become the gold standard for interstitial bt of breast cancer . the aim of this article is to review treatment planning issues of multicatheter interstitial brachytherapy and share our experience in forward and inverse planning methods . since even the most advanced optimization technique can not compensate for a wrong implant geometry , it is of the utmost importance to place the catheters in the right positions . in order to get an acceptable dose distribution , the planning target volume ( ptv ) has to be geometrically covered by the catheters . to achieve this , different imaging methods can be used . with ultrasound ( us ) guidance , the deepest needles can be inserted manually if the resection cavity is visible , and then the rest of catheters are placed with a template , which can ensure an even spacing between catheters . another approach is when the superficial needles are inserted as first under us guidance followed by the deeper needles with template guidance , but the artefacts from the shallow needles may cause visibility problem . if the needles are manually inserted using us imaging , the ptv contour and all entry and exit points have to be marked on the skin surface in order to plan the needles distribution . however , the best method is when a 3d imaging with full anatomical information about the resection cavity , ptv , and organs at risk ( oars ) is available before the insertions . then , the number and positions of the catheters can be determined using a 3d rendered image with a template placed around the breast ( figure 1 ) . if the intention of the treatment is delivering a boost dose , steel needles can be used with 1 - 3 fractions , but in monotherapy treatments with 7 - 10 fractions , the needles are replaced with plastic catheters for the sake of patient s convenience . 3d rendering of patient anatomy with a template on the right breast in needles - eye - view after pre - implant computed tomography imaging . the red colored planning target volume is projected into the template with the holes and the light blue dots show the planned positions of the catheters however , any imaging technique is used before the implantation , after the insertions of the catheters , a new ct imaging is needed for planning purpose . generally , the patient is positioned on the ct table in the same way as for ebrt making sure the same anatomical conditions at boost treatment as for external irradiation . generally , 3 mm slice thickness is acceptable for accurate catheter reconstruction and target delineation . organs at risk and resection cavity must be outlined , and the ptv should be created according to the available guidelines . using special markers in the catheters with good visibility but without artefact is a great help during catheter reconstruction ( figure 2 ) . nevertheless , without markers , the catheters can be also visualized merely by the inside air ( figure 2 ) , and with proper windowing , the reconstruction can be properly performed . in this case , it is important to see and identify the fixation button at the distal end of each catheter , since the first possible dwell position has to be related to it . the catheter reconstruction can be performed in axial , sagittal , coronal planes , or even in oblique planes , which have been created parallel with or orthogonal to the catheters ( extra coordinate system ecs ) . catheters should be numbered or uniquely labelled , so that each individual catheter can be identified . the active source positions inside the ptv can be defined manually or automatically with or without margin inside or outside the target surface , if needed . in classical bt planning using the paris dosimetry system ( pds ) , the basal dose points in the central plane play an important role . the central plane can be easily created using the ecs in the ct - based planning . the midpoints between catheters ( basal dose points ) can be placed manually by visual inspection , or automatically by a software if the catheters geometry is regular . after normalizing the dose distribution ( 100% ) to the mean central dose ( mcd in paris system ) , which is the mean dose in basal dose points , the isodose level for prescription can be selected using a special multiplication factor between 0.1 and 10 . for example , using the factor of 0.85 , the dose will be prescribed to the 85% isodose level corresponding to the pds . to calculate the treatment time , theoretically , the pds provides homogeneous dose distribution , but the target coverage and conformality may not be sufficient enough . to increase the coverage lower than 85% , isodose level can be selected for dose prescription , but the worsening homogeneity must be taken into consideration . in the contrary , if increasing the isodose level for prescription , the dose distributions will be more homogeneous , and the target coverage decreases simultaneously . another way to manage the target volume coverage and homogeneity of dose distribution is applying several dwell time optimization methods . a transversal and sagittal computed tomography slice with implanted catheters . in most of the catheters , special markers are inserted ( shown with white lines / dots ) , but some of them do not have any markers and only the inside air makes them visible ( shown with black lines / dots ) various dose optimizations are available in bt using a stepping source ( table 1 ) . the classical paris dosimetry system can be imitated by using uniform dwell times in all dwell positions , which means no optimization is applied . in that case , the dose distribution is always homogeneous , but the conformality will not be optimal . the target volume can be covered by the prescription isodose surface adequately , but relatively large volume of normal tissue will be unnecessarily irradiated by the prescription dose ( pd ) . this is the consequence of the extending active lengths in the catheters out of the target volume , which is required in the pds . however , the concepts of its implant geometry and dose prescription are still employed even if the dwell times are calculated with a forward optimization method . in most optimization methods , the 3d target volume is not used directly during optimization ; only in plan evaluation , the dvh parameters are related to the ptv . optimization methods in high - dose - rate brachytherapy stepping source dosimetry system inverse planning simulated annealing hybrid inverse planning optimization changing the individual dwell times or weight factors manually in a spreadsheet is not an effective method to shape the dose distribution . it is rather time consuming and recommended only for small local adjustments . after geometrical optimization another advantage is that there is no need to define dose points for optimization because the source dwell positions themselves will serve as reference dose points . the dwell time at any dwell position will be inversely proportional to the dose delivered by the other dwell positions . the consequence is the increased dwell times at the ends of the catheters , and in dwell positions in deviating catheters . if the catheters geometrically cover the target volume properly , the reference isodose surface will always include the ptv . since the dose distribution is related only to the catheters , if there is a geometric miss , a significant underdosed region may develop in the ptv . by selecting a lower isodose level for dose prescription , the target coverage can be slightly improved , but the dose to normal tissues may increase . at polynomial optimization , an objective function is minimized , which describes the difference between the calculated and required dose values in specified dose points . these points can be placed at a distance outwards of or in mid - positions between the catheters ( distance or volume dose points ) . using distance dose points , it is assumed that they are placed on the surface of the target volume . with volume dose points , we can optimize the doses in central points between the catheters in the whole volume and not only in the central plane . this method is called stepping source dosimetry system ( ssds ) and is the extension of the pds . regarding dosimetry , due to the stepping source , the dose can be prescribed for a proper isodose level similarly to the go . when the ptv is defined three - dimensionally , we can place dose points on its surface and can optimize and prescribe the dose ( 100% ) on them ( conformal dosimetry system ) . with this method , even if the target volume is irregular , very conformal dose distribution can be obtained if the catheters are evenly distributed inside the volume . since the dose homogeneity is not taken into account during optimization , the dose homogeneity will be unacceptable , since large high dose volumes develop inside the ptv . the graphical optimization ( gro ) is an attractive way to change the shape of an isodose curve locally or globally . on the screen with the cursor , we can select an isodose line , and with the drag and drop method , the isodose line can be shifted into the desired position . if the local adjustment option is selected , only the nearby dose distribution will be changed , while with the global option , the dose distribution will change isotropically in 3d . the coverage and conformality can be easily improved with gro , but it must not be forgotten that at the same time , the homogeneity will deteriorate . in many cases , the go followed by gro results in acceptable dose distribution , since the go can provide the homogeneity , and with the gro , the coverage can be further increased . the key point in inverse optimization is that all dosimetric requirements ( dose coverage , dose homogeneity , and organs at risk protection ) are simultaneously and automatically taken into account in the planning process . in the first step , the clinical objectives are translated into minimum or maximum dose limits and importance factors , and then , the source dwell times are calculated with minimizing objective functions , describing the differences between the calculated and required doses . the prerequisites for inverse optimization are as follows : reconstructed catheters geometry , delineated ptv and oars , defined active source dwell positions , dose objectives for ptv , oar - s and normal tissue . the two most common inverse algorithms are the hipo ( hybrid inverse planning optimization ) and ipsa ( inverse planning simulated annealing ) [ 22 , 23 , 24 ] . the former uses deterministic , while the latter works with stochastic method . hipo is implemented in the oncentra brachy version 4.3 planning system ( nucletron , an elekta company , elekta ab , stockholm , sweden ) , where the target coverage and dose homogeneity are controlled by minimum and maximum dose values in the ptv , respectively . in a clinical case , the minimum value in the ptv is 100% corresponding to the pd , while the maximum value is selected 150% of the pd . during optimization , furthermore , with a weight factor , the importance of the given requirement can be taken into account . regarding protection of normal tissue and oars , maximum doses can be specified for them , which are also taken into consideration during optimization . the min value and max value refer to relative doses , while the min weight and max weight mean relative importance factors in classical bt , the plan evaluation was limited , having dose values in reference points and looking at the 2d dose distributions related to catheters / applicators . the use of these evaluation methods is still recommended , but additional tools are now available to assess the quality of the implant . visual inspection of dose distribution on ct slices including sagittal and coronal reconstructions is still mandatory , however , the large number of ct slices and outlined organs make the evaluation quite subjective . for an objective assessment , implant related parameters can always be calculated , even if the target volume is not defined . they characterize the size of the irradiated volume and homogeneity of the dose distribution . in optimal implant , the dnr is minimal and the dhi is maximal , since they are complementary indices , namely dhi = 1 dnr . when 3d ptv is defined , additional parameters can be calculated . additionally , the dhi can be determined for the target , when volumes in the ptv only are taken into account . in the relative homogeneity index ( hi ) , volume receiving a dose between 100% and 150% of the pd is related to the volume of the ptv . although only small differences occur between the various dhis , its exact definition always must be given when used for reporting dose homogeneity and for intercomparisons of different plans . the overdose volume index ( oi ) characterizes the dose homogeneity with volume irradiated by 2 x pd , related to volume of ptv . in an ideal implant the coverage index ( ci ) is the fraction of the ptv receiving at least the pd . the conformal index ( coin ) considers the coverage of the ptv by the pd ( ptvpd / vptv ) , and also the unwanted irradiation of normal tissues outside the ptv ( ptvpd / vpd ) . ptvpd is the volume inside the ptv irradiated by the pd , and the vpd is the volume receiving at least the pd . the definition of coin can be extended to critical structures also , but this formalism is rarely used in breast bt , since the oars nearly always receive less dose than the pd . the dose distribution is most conformal when the coin is maximal and is as close to 1 as possible . the external volume index ( ei ) is also related to conformality ; it is the ratio of the normal tissue volume outside the ptv receiving at least the pd , to the volume of the ptv . dose that irradiates certain part of the ptv is also used , e.g. d10 means the relative dose that irradiates 10% of the ptv . the most common dose - volume parameters used in interstitial brachytherapy pd prescribed dose , ptvpd volume in ptv received at least the pd depending on the type of the oar , the mean dose , volume irradiated by a given relative ( v5 ) , absolute dose ( v5gy ) , or dose irradiating a small volume ( eg . d0.1 cm , d2 cm ) are generally reported . in several studies , however , recently in ct - based planning , volumetric doses are already recommended to be used , e.g. d0.01 cm or d0.1 cm . the rationale behind is that the point dose is clinically meaningless , and the dose to a volume may be better correlated with the side effects . in the bt community , no general agreement exists regarding the acceptable criteria for a good breast implant . in the gec - estro randomized study , the coverage index ( ci ) had to be larger than 0.9 , i.e. at least 90% of the ptv had to receive the pd . there was a requirement for dose uniformity , namely the dnr < 0.35 . in the study , only skin was regarded as oar , and the maximum surface dose had to be less than 70% of the pd . for reporting purpose , several additional parameters had to be recorded , but fulfilment of condition was not set up for them . in the nsabp b-39/rtog 0413 protocol for multi - catheter breast bt , the dhi for implant should be 0.75 , which corresponds to dnr 0.25 . the volume of tissue receiving 150% and 200% of the pd should be less than or equal to 70 cm and 20 cm , respectively . regarding target coverage , the dose 90% of the pd must cover 90% of the ptv . for uninvolved normal breast , the limitation is that < 60% of the whole breast volume should receive 50% of the pd , and the skin surface dose should not exceed the pd . the dose to other organs including the heart , lung , and ribs is generally minimal , so no thresholds are used , however , recording few parameters for reporting is recommended . in a paper by darby et al . , it was found that the risk of major cardiac events increased linearly by 7.4% per gray of mean heart dose , therefore , to characterize the dose to heart reporting the mean dose is recommended . in multicatheter breast bt , chest wall pain or rib fracture are rare events , but the doses should be kept as low as possible . initially , ct imaging was used only for evaluation purpose after standard fluoroscopy - guided implantation [ 12 , 13 , 14 , 15 , 29 ] , but afterwards , the implantations themselves were performed using 3d ct information [ 15 , 16 , 30 , 31 , 32 , 33 ] . these and other studies confirmed that with introduction of image - guided implantation techniques , the treatment plans improved considerably compared to the traditional fluoroscopy - guided implantation and planning [ 11 , 34 ] . reported that the percentage of patients satisfying with their dosimetric goals of target coverage and dose homogeneity increased from 42 to 93% , when ct - guided technique was used instead of fluoroscopic guided free - handed catheter insertion technique . in their study , not only the coverage but also the dhi was better with the ct planning . compared two different pre - planning methods ( 2d vs. 3d ) . in the first group of patients , the catheter positions were planned with x - ray fluoroscopy imaging ( 2d ) , while in the second group , the treatments were pre - planned with cone beam ct imaging ( 3d ) . for dvh - based plan evaluations , the target coverage ( v100 ) was significantly higher for 3d pre - planning ( 91.7% vs. 86.1% ) , and the dose distributions were more homogeneous ( dhi : 0.60 vs. 0.53 ) . the transition from classical x - ray film based planning to 3d planning was carried out step - by - step in many institutions , and the 3d dosimetry induced lots of challenges for radiation oncologists and medical physicists . furthermore , a learning curve can be observed during this process as it was well demonstrated by cholewka et al . and reported by wiercinska et al . . the quality of dose distributions in interstitial bt can be improved with the use of different dose optimization methods and image based 3d information for planning the geometry of the catheter positions [ 12 , 13 , 14 , 37 ] . geometrically optimized implants are much more conformal than the non - optimized ones ( pds ) with much less dose to normal tissues . with conformal dose planning , the target volume coverage can be significantly increased compared to the traditional dosimetry systems , but the dose homogeneity worsens [ 13 , 14 , 16 , 18 ] . geometrical optimization followed by graphical optimization can provide acceptable dose plans in most of the clinical cases [ 18 , 32 ] . in classical breast bt , the dosimetry was relied on reference dose points , which were related to the catheters geometry . the dose points were placed either in the center points between catheters or at specified distances from the catheters in outer directions . in the former case , the prescription isodose was generally selected as 85% of the mean central dose like in the pds , while with the latter technique , the dose was normalized to the mean dose in the dose points . in both cases , the target volume could only be approximated roughly . from this follows that the plan evaluation confined to the evaluation on dose uniformity in the implant volume and dose values in points . anatomy - based inverse optimization algorithms are already available in brachytherapy systems for clinical use [ 22 , 23 , 24 , 38 ] . during the optimization process , the source dwell times for active source dwell positions are calculated with the aim of fulfilling thresholds for dose - volume parameters . then , an objective or cost function measuring how well the limits are fulfilled is minimized . since the calculation is very time - consuming , in order to get the results in a reliable time , a powerful computer is needed for the clinical use . the requirements for target coverage , dose homogeneity , and sparing of oars are conflicting and to find the proper parameters and weight factors is a challenge for the physicist . this is demonstrated in figure 4 , where dose homogeneity characterized by the dnr and target coverage denoted by v100 are shown in the function of weight factor of maximum dose value ( max weight ) . the calculations were performed in the oncentra brachy version 4.3 planning system , and the oars were not taken into account . we note , that the weight factor for maximum dose ( max weight and max value in figure 3 ) controls the homogeneity , and the weight factor for minimum dose ( min weight and min value in figure 3 ) influences coverage . figure 4a shows that by increasing the weight factor for maximum dose , the dnr is becoming less meaning better homogeneity . however , at the same time , the v100 is also decreasing and the target coverage is worsening . to select the proper weight factor , it requires clinical judgement , balancing between dose homogeneity and target coverage . a weight factor in the range between 20 - 40 seems to be an appropriate choice with low dnr and concurrently high v100 . from the figure 4 , the role of minimum dose factor ( min weight ) can be observed . by using a higher minimum dose factor , the coverage is better ( red line in figure 4b ) , but simultaneously , the dose will be less homogeneous ( red line in figure 4a ) . at first sight , the inverse planning seems to be an easy task , but it requires some experience to find those input parameters , which will result in clinically acceptable dose distribution . in figure 5a , qualitative comparison between dose distributions of forward and inverse planning is presented in a ct slice . regarding target coverage , the difference is negligible , but the high dose volume surrounded by the 150% is less with the hipo ( figure 5b ) resulting in better dose homogeneity . table 3 shows a quantitative comparison between dosimetric parameters calculated for fifteen treatment plans made with geometrical optimization , followed by graphical optimization ( geom+graph ) and hipo . at the same target coverage ( 91% ) , the hipo plans were superior over the forward plans regarding all other parameters . the better conformality ( coin : 0.77 vs. 0.71 ) resulted in 29% less high dose volume ( v100 ) in the ipsilateral non - target breast . the dose homogeneity improved ( dnr : 0.35 vs. 0.37 ) , and dose to oars was less by percentage values in the range of 1 - 15% . based on our initial experience and quantitative data presented here , we can claim that hipo is an optimization tool that can produce clinically sound treatment plans at a significantly reduced overall planning and optimization time . dosimetrical comparison of treatment plans made by forward planning using geometrical optimization followed by graphical optimization ( geom+graph ) and inverse optimization ( hipo ) . the percentage difference is related to geom+graph plans dependence of dose homogeneity ( a ) and target coverage ( b ) on the weight factor for maximum dose ( max weight ) in the planning target volume ( ptv ) . min weight is a weight factor , which controls the target coverage through minimum dose at the periphery of the ptv representative relative dose distributions on an axial computed tomography ( ct ) slice in treatment plan made with a ) forward planning ( geom+graph ) , and with b ) inverse planning ( hipo ) . more homogeneous dose distribution can be observed in the hipo plan nowadays , quantitative dose - volume histogram based evaluation of treatment plans is proposed . however , there is no consensus on which parameters should be reported . for the ptv , the minimum requirement is to report the ci or v100 , dhi or dnr , and the coin . in our department in line with the gec - estro study , we intend to have at least 90% ptv coverage by the prescribed dose ( v100 ) , while in the nsabp b-39/rtog 0413 protocol , the 90% of the prescribed dose should cover at least 90% of the ptv . with the latter constraint , conformality is rarely investigated in breast bt , and there is no consensus on what degree of conformality is acceptable or desirable . . obtained a conformality number of 0.48 at a typical breast implant with geometrical optimization . kolotas et al . reported coin value of 0.76 for 42 breast implants , while baltas et al . aimed to achieve a coin value above 0.64 by using their ct - based planning system . in a study by major et al , the mean coin was 0.68 ( range , 0.510.82 ) calculated for 49 patients . similar coins were reported by cholewka et al . and gurran et al . with the values of 0.69 ( range , 0.37 - 0.84 ) and 0.67 ( range , 0.59 - 0.87 ) , respectively . reporting the dose to oars is particularly not uniform at all . from this follows that the dose - volume limits can be very different for the various parameters . moreover , no common guidelines are available on how to define the skin dose or the skin as an organ . berger et al . investigated dvh parameters for skin dose , and they recommended a more sophisticated way to report organ dose than using point doses only . similarly , lasota et al . used different skin models to estimate the dose to skin , and they concluded that large differences exist between models using various skin contouring protocols . recommend using d0.2 cm for a 2-mm thick skin layer as a surrogate for dose to skin area of 1 cm . in a paper of lettmaier et al . , dose to 1 cm and 0.1 cm of skin volume with 5 mm thickness were reported . gifford et al . has found that large differences in skin volume definition leads to confusion and can result in significant dose differences , making the dosimetric comparisons and correlations with toxicities difficult or impossible . traditionally , the dose to skin was characterized with the maximum surface dose , which is a point dose . in a recent paper by major et al . , doses to the most exposed small volumes in oars are reported . to make the intercomparisons between point doses and volumetric doses , a correlation analysis should be made . figure 6 presents the correlation between the dose to small volumes and maximum point dose for skin and ribs calculated for thirty - four breast implant patients . the skin was outlined as a 5-mm shell inside the body , and the maximum skin dose was calculated on the outer surface , while the maximum dose in ribs was calculated inside the organ . regarding the skin , the d0.01 cm is not a good substitute for dmax ( r = 0.8513 ) , because the dmax is always on the outer surface of the skin volume , while the high dose volumes ( e.g. 0.01 cm ) develop in the inner part of the skin closer to the source dwell positions and the corresponding doses can be different . for the ribs , an excellent correlation ( r = 0.9977 ) can be observed between d0.01 cm and dmax , since these two values are nearly always identical . the dmax is always inside the most exposed volume of 0.01 cm . for larger volumes ( 0.1 cm , 1 cm ) correlation between the dose to the most exposed small volumes and maximum point dose for the a ) skin , and b ) ribs similarly to ebrt , inverse planning is expected to be used in bt in daily clinical practice very soon , especially in multicatheter breast implants . however , the protocols with the dose - volume constraints used during the optimization still should be defined . for a plan evaluation , the use of volumetric dose parameters is already recommended , and will be obligatory in the future in order to find a correlation between dosimetry and clinical outcome . however , a consensus is needed on which parameters should be reported , and which can have real clinical significance .

in the last decades , treatment planning for multicatheter interstitial breast brachytherapy has evolved considerably from fluoroscopy - based 2d to anatomy - based 3d planning . to plan the right positions of the catheters , ultrasound or computed tomography ( ct ) imaging can be used , but the treatment plan is always based on postimplant ct images . with ct imaging , the 3d target volume can be defined more precisely and delineation of the organs at risk volumes is also possible . consequently , parameters calculated from dose - volume histogram can be used for quantitative plan evaluation . the catheter reconstruction is also easier and faster on ct images compared to x - ray films . in high dose rate brachytherapy , using a stepping source , a number of forward dose optimization methods ( manual , geometrical , on dose points , graphical ) are available to shape the dose distribution to the target volume , and these influence dose homogeneities to different extent . currently , inverse optimization algorithms offer new possibilities to improve dose distributions further considering the requirements for dose coverage , dose homogeneity , and dose to organs at risk simultaneously and automatically . in this article , the evolvement of treatment planning for interstitial breast implants is reviewed , different forward optimization methods are discussed , and dose - volume parameters used for quantitative plan evaluation are described . finally , some questions of the inverse optimization method are investigated and initial experiences of the authors are presented .

Purpose Material and methods Discussion Future directions

brachytherapy techniques include interstitial implants , intracavitary devices with single / multilumen balloons , hybrid applicators , and irradiation with low energy x - ray [ 1 , 2 ] . external beam radiation therapy can be performed with 3d conformal radiotherapy ( 3d - crt ) , intensity modulated radiation therapy ( imrt ) , volume modulated arc therapy ( vmat ) , proton therapy , and intraoperative electron irradiation . in classical breast brachytherapy , treatment planning was based on two x - ray films . the surgical clips approximately showed the position of the lumpectomy cavity , and the catheter fixation buttons indicated the skin surface in a number of points only . at that time , neither dose coverage nor conformality was possible to be determined , and it was assumed that the catheters are in the right positions . later , with the introduction of cross - sectional imaging , the real 3d dose planning became available . the reconstructed catheters geometry and the delineation of target volume and organs at risk on computed tomography ( ct ) slices made possible for conformal irradiation . but , at the beginnings , the post - implant ct imaging was used only for evaluating dose plans , and just a few quality indices were calculated [ 11 , 12 , 13 , 14 ] . reported significantly increased target coverage and dose homogeneity after implementing their ct - guided implantation technique . these values were significantly better than the corresponding data of the radiography - based implantation method . nowadays , based on the persuading early dosimetric results , ct - based treatment planning has become the gold standard for interstitial bt of breast cancer . the aim of this article is to review treatment planning issues of multicatheter interstitial brachytherapy and share our experience in forward and inverse planning methods . since even the most advanced optimization technique can not compensate for a wrong implant geometry , it is of the utmost importance to place the catheters in the right positions . in order to get an acceptable dose distribution , the planning target volume ( ptv ) has to be geometrically covered by the catheters . to achieve this , different imaging methods can be used . with ultrasound ( us ) guidance , the deepest needles can be inserted manually if the resection cavity is visible , and then the rest of catheters are placed with a template , which can ensure an even spacing between catheters . if the needles are manually inserted using us imaging , the ptv contour and all entry and exit points have to be marked on the skin surface in order to plan the needles distribution . however , the best method is when a 3d imaging with full anatomical information about the resection cavity , ptv , and organs at risk ( oars ) is available before the insertions . then , the number and positions of the catheters can be determined using a 3d rendered image with a template placed around the breast ( figure 1 ) . if the intention of the treatment is delivering a boost dose , steel needles can be used with 1 - 3 fractions , but in monotherapy treatments with 7 - 10 fractions , the needles are replaced with plastic catheters for the sake of patient s convenience . 3d rendering of patient anatomy with a template on the right breast in needles - eye - view after pre - implant computed tomography imaging . the red colored planning target volume is projected into the template with the holes and the light blue dots show the planned positions of the catheters however , any imaging technique is used before the implantation , after the insertions of the catheters , a new ct imaging is needed for planning purpose . organs at risk and resection cavity must be outlined , and the ptv should be created according to the available guidelines . using special markers in the catheters with good visibility but without artefact is a great help during catheter reconstruction ( figure 2 ) . nevertheless , without markers , the catheters can be also visualized merely by the inside air ( figure 2 ) , and with proper windowing , the reconstruction can be properly performed . the catheter reconstruction can be performed in axial , sagittal , coronal planes , or even in oblique planes , which have been created parallel with or orthogonal to the catheters ( extra coordinate system ecs ) . the active source positions inside the ptv can be defined manually or automatically with or without margin inside or outside the target surface , if needed . in classical bt planning using the paris dosimetry system ( pds ) , the basal dose points in the central plane play an important role . the central plane can be easily created using the ecs in the ct - based planning . the midpoints between catheters ( basal dose points ) can be placed manually by visual inspection , or automatically by a software if the catheters geometry is regular . after normalizing the dose distribution ( 100% ) to the mean central dose ( mcd in paris system ) , which is the mean dose in basal dose points , the isodose level for prescription can be selected using a special multiplication factor between 0.1 and 10 . for example , using the factor of 0.85 , the dose will be prescribed to the 85% isodose level corresponding to the pds . to calculate the treatment time , theoretically , the pds provides homogeneous dose distribution , but the target coverage and conformality may not be sufficient enough . to increase the coverage lower than 85% , isodose level can be selected for dose prescription , but the worsening homogeneity must be taken into consideration . in the contrary , if increasing the isodose level for prescription , the dose distributions will be more homogeneous , and the target coverage decreases simultaneously . another way to manage the target volume coverage and homogeneity of dose distribution is applying several dwell time optimization methods . in most of the catheters , special markers are inserted ( shown with white lines / dots ) , but some of them do not have any markers and only the inside air makes them visible ( shown with black lines / dots ) various dose optimizations are available in bt using a stepping source ( table 1 ) . in that case , the dose distribution is always homogeneous , but the conformality will not be optimal . the target volume can be covered by the prescription isodose surface adequately , but relatively large volume of normal tissue will be unnecessarily irradiated by the prescription dose ( pd ) . this is the consequence of the extending active lengths in the catheters out of the target volume , which is required in the pds . however , the concepts of its implant geometry and dose prescription are still employed even if the dwell times are calculated with a forward optimization method . in most optimization methods , the 3d target volume is not used directly during optimization ; only in plan evaluation , the dvh parameters are related to the ptv . optimization methods in high - dose - rate brachytherapy stepping source dosimetry system inverse planning simulated annealing hybrid inverse planning optimization changing the individual dwell times or weight factors manually in a spreadsheet is not an effective method to shape the dose distribution . the consequence is the increased dwell times at the ends of the catheters , and in dwell positions in deviating catheters . if the catheters geometrically cover the target volume properly , the reference isodose surface will always include the ptv . since the dose distribution is related only to the catheters , if there is a geometric miss , a significant underdosed region may develop in the ptv . by selecting a lower isodose level for dose prescription , the target coverage can be slightly improved , but the dose to normal tissues may increase . these points can be placed at a distance outwards of or in mid - positions between the catheters ( distance or volume dose points ) . using distance dose points , it is assumed that they are placed on the surface of the target volume . with volume dose points , we can optimize the doses in central points between the catheters in the whole volume and not only in the central plane . this method is called stepping source dosimetry system ( ssds ) and is the extension of the pds . regarding dosimetry , due to the stepping source , the dose can be prescribed for a proper isodose level similarly to the go . with this method , even if the target volume is irregular , very conformal dose distribution can be obtained if the catheters are evenly distributed inside the volume . since the dose homogeneity is not taken into account during optimization , the dose homogeneity will be unacceptable , since large high dose volumes develop inside the ptv . on the screen with the cursor , we can select an isodose line , and with the drag and drop method , the isodose line can be shifted into the desired position . if the local adjustment option is selected , only the nearby dose distribution will be changed , while with the global option , the dose distribution will change isotropically in 3d . the coverage and conformality can be easily improved with gro , but it must not be forgotten that at the same time , the homogeneity will deteriorate . in many cases , the go followed by gro results in acceptable dose distribution , since the go can provide the homogeneity , and with the gro , the coverage can be further increased . the key point in inverse optimization is that all dosimetric requirements ( dose coverage , dose homogeneity , and organs at risk protection ) are simultaneously and automatically taken into account in the planning process . in the first step , the clinical objectives are translated into minimum or maximum dose limits and importance factors , and then , the source dwell times are calculated with minimizing objective functions , describing the differences between the calculated and required doses . hipo is implemented in the oncentra brachy version 4.3 planning system ( nucletron , an elekta company , elekta ab , stockholm , sweden ) , where the target coverage and dose homogeneity are controlled by minimum and maximum dose values in the ptv , respectively . in a clinical case , the minimum value in the ptv is 100% corresponding to the pd , while the maximum value is selected 150% of the pd . during optimization , furthermore , with a weight factor , the importance of the given requirement can be taken into account . the min value and max value refer to relative doses , while the min weight and max weight mean relative importance factors in classical bt , the plan evaluation was limited , having dose values in reference points and looking at the 2d dose distributions related to catheters / applicators . the use of these evaluation methods is still recommended , but additional tools are now available to assess the quality of the implant . visual inspection of dose distribution on ct slices including sagittal and coronal reconstructions is still mandatory , however , the large number of ct slices and outlined organs make the evaluation quite subjective . they characterize the size of the irradiated volume and homogeneity of the dose distribution . additionally , the dhi can be determined for the target , when volumes in the ptv only are taken into account . in the relative homogeneity index ( hi ) , volume receiving a dose between 100% and 150% of the pd is related to the volume of the ptv . although only small differences occur between the various dhis , its exact definition always must be given when used for reporting dose homogeneity and for intercomparisons of different plans . the overdose volume index ( oi ) characterizes the dose homogeneity with volume irradiated by 2 x pd , related to volume of ptv . the external volume index ( ei ) is also related to conformality ; it is the ratio of the normal tissue volume outside the ptv receiving at least the pd , to the volume of the ptv . dose that irradiates certain part of the ptv is also used , e.g. the most common dose - volume parameters used in interstitial brachytherapy pd prescribed dose , ptvpd volume in ptv received at least the pd depending on the type of the oar , the mean dose , volume irradiated by a given relative ( v5 ) , absolute dose ( v5gy ) , or dose irradiating a small volume ( eg . in several studies , however , recently in ct - based planning , volumetric doses are already recommended to be used , e.g. the rationale behind is that the point dose is clinically meaningless , and the dose to a volume may be better correlated with the side effects . in the study , only skin was regarded as oar , and the maximum surface dose had to be less than 70% of the pd . regarding target coverage , the dose 90% of the pd must cover 90% of the ptv . for uninvolved normal breast , the limitation is that < 60% of the whole breast volume should receive 50% of the pd , and the skin surface dose should not exceed the pd . the dose to other organs including the heart , lung , and ribs is generally minimal , so no thresholds are used , however , recording few parameters for reporting is recommended . , it was found that the risk of major cardiac events increased linearly by 7.4% per gray of mean heart dose , therefore , to characterize the dose to heart reporting the mean dose is recommended . initially , ct imaging was used only for evaluation purpose after standard fluoroscopy - guided implantation [ 12 , 13 , 14 , 15 , 29 ] , but afterwards , the implantations themselves were performed using 3d ct information [ 15 , 16 , 30 , 31 , 32 , 33 ] . these and other studies confirmed that with introduction of image - guided implantation techniques , the treatment plans improved considerably compared to the traditional fluoroscopy - guided implantation and planning [ 11 , 34 ] . in the first group of patients , the catheter positions were planned with x - ray fluoroscopy imaging ( 2d ) , while in the second group , the treatments were pre - planned with cone beam ct imaging ( 3d ) . for dvh - based plan evaluations , the target coverage ( v100 ) was significantly higher for 3d pre - planning ( 91.7% vs. 86.1% ) , and the dose distributions were more homogeneous ( dhi : 0.60 vs. 0.53 ) . the transition from classical x - ray film based planning to 3d planning was carried out step - by - step in many institutions , and the 3d dosimetry induced lots of challenges for radiation oncologists and medical physicists . the quality of dose distributions in interstitial bt can be improved with the use of different dose optimization methods and image based 3d information for planning the geometry of the catheter positions [ 12 , 13 , 14 , 37 ] . with conformal dose planning , the target volume coverage can be significantly increased compared to the traditional dosimetry systems , but the dose homogeneity worsens [ 13 , 14 , 16 , 18 ] . in classical breast bt , the dosimetry was relied on reference dose points , which were related to the catheters geometry . the dose points were placed either in the center points between catheters or at specified distances from the catheters in outer directions . in the former case , the prescription isodose was generally selected as 85% of the mean central dose like in the pds , while with the latter technique , the dose was normalized to the mean dose in the dose points . in both cases , the target volume could only be approximated roughly . from this follows that the plan evaluation confined to the evaluation on dose uniformity in the implant volume and dose values in points . anatomy - based inverse optimization algorithms are already available in brachytherapy systems for clinical use [ 22 , 23 , 24 , 38 ] . during the optimization process , the source dwell times for active source dwell positions are calculated with the aim of fulfilling thresholds for dose - volume parameters . the requirements for target coverage , dose homogeneity , and sparing of oars are conflicting and to find the proper parameters and weight factors is a challenge for the physicist . however , at the same time , the v100 is also decreasing and the target coverage is worsening . by using a higher minimum dose factor , the coverage is better ( red line in figure 4b ) , but simultaneously , the dose will be less homogeneous ( red line in figure 4a ) . at first sight , the inverse planning seems to be an easy task , but it requires some experience to find those input parameters , which will result in clinically acceptable dose distribution . regarding target coverage , the difference is negligible , but the high dose volume surrounded by the 150% is less with the hipo ( figure 5b ) resulting in better dose homogeneity . the dose homogeneity improved ( dnr : 0.35 vs. 0.37 ) , and dose to oars was less by percentage values in the range of 1 - 15% . dosimetrical comparison of treatment plans made by forward planning using geometrical optimization followed by graphical optimization ( geom+graph ) and inverse optimization ( hipo ) . the percentage difference is related to geom+graph plans dependence of dose homogeneity ( a ) and target coverage ( b ) on the weight factor for maximum dose ( max weight ) in the planning target volume ( ptv ) . min weight is a weight factor , which controls the target coverage through minimum dose at the periphery of the ptv representative relative dose distributions on an axial computed tomography ( ct ) slice in treatment plan made with a ) forward planning ( geom+graph ) , and with b ) inverse planning ( hipo ) . more homogeneous dose distribution can be observed in the hipo plan nowadays , quantitative dose - volume histogram based evaluation of treatment plans is proposed . for the ptv , the minimum requirement is to report the ci or v100 , dhi or dnr , and the coin . in our department in line with the gec - estro study , we intend to have at least 90% ptv coverage by the prescribed dose ( v100 ) , while in the nsabp b-39/rtog 0413 protocol , the 90% of the prescribed dose should cover at least 90% of the ptv . from this follows that the dose - volume limits can be very different for the various parameters . used different skin models to estimate the dose to skin , and they concluded that large differences exist between models using various skin contouring protocols . traditionally , the dose to skin was characterized with the maximum surface dose , which is a point dose . regarding the skin , the d0.01 cm is not a good substitute for dmax ( r = 0.8513 ) , because the dmax is always on the outer surface of the skin volume , while the high dose volumes ( e.g. 0.01 cm ) develop in the inner part of the skin closer to the source dwell positions and the corresponding doses can be different . for larger volumes ( 0.1 cm , 1 cm ) correlation between the dose to the most exposed small volumes and maximum point dose for the a ) skin , and b ) ribs similarly to ebrt , inverse planning is expected to be used in bt in daily clinical practice very soon , especially in multicatheter breast implants . however , the protocols with the dose - volume constraints used during the optimization still should be defined . for a plan evaluation , the use of volumetric dose parameters is already recommended , and will be obligatory in the future in order to find a correlation between dosimetry and clinical outcome . however , a consensus is needed on which parameters should be reported , and which can have real clinical significance .

the colonic mucosa is constantly exposed to a wide range of luminal agents that have the potential for either mucosal damage , or mucosal protection . normal colonic transit time varies widely in humans but within physiological boundaries would be between 24 and 48 hours , in comparison to transit through the upper gi tract which occurs within a few hours [ 1 , 2 ] . therefore , there is a longer exposure time of the colonic mucosa to luminal agents than to the underlying tissues of other areas of the gut . in addition , due to the role of the colon in the salvage of unabsorbed nutrients and absorption of fluid , these luminal agents will be concentrated ( particularly in the distal bowel ) , resulting in further increases of mucosal exposure to these agents . while removal of water from the faecal bulk is likely to reduce the diffusion of agents from the majority of the faecal cross - section , direct contact will still occur between the mucus and the outer surfaces of the colonic luminal contents . the large bowel also plays host to approximately 10 bacteria and other micro - organisms and is thought to include over 500 bacterial species . as such , changes to the prevalent species within the microfloral population or to microfloral functioning and output within the bowel are likely to be intimately linked with colorectal health and disease . while digestion per se does not tend to occur in the colon , the colonic microflora acts to degrade dietary fibre and/or other dietary factors that escape digestion to produce further agents that could either harm or protect the underlying mucosa . the colonic mucus barrier is the first line of defence the underlying mucosa has against the myriad of damaging agents that occur within the colonic lumen . the colonic mucus barrier also acts to greatly reduce the shear stress caused by the passage of the luminal bolus along the colon . this barrier can also act as an energy source or as a niche for bacteria within the large bowel [ 9 , 10 ] . despite the relatively high potential for luminal exposure , previous studies in healthy humans suggest that bacteria do not routinely associate with the colonic mucosa and only occur at the luminal side of the intestinal mucus layer . the remainder of this paper will focus on current evidence for how the interplay between microflora and mucus may be a key factor in mucosal health and disease and will also highlight what areas of research may be considered in the future to further understanding of this topic . mucus acts to protect most mucosal surfaces in the gut , airways , and urinogenital tract . disulphide bridges between cysteine - rich areas towards the c- and n - termini of the mucin backbone act to endow mucus with its characteristic viscoelastic gel properties . the terminal sugars of these side chains are believed to play a crucial role in the adhesion of mucins to different bacterial cells ( e.g. , [ 12 , 13 ] ) . changes in both the muc gene product and glycosylation patterns are believed to be associated with the onset or development of colonic mucosal diseases , such as colorectal cancer and inflammatory bowel disease ( ibd ) . in humans , there are five polymeric , secretory mucin gene products that are currently known , muc2 , muc5ac , muc5b , muc6 , and muc19 . these genes for muc2 , muc5ac , muc5b , and muc6 are all expressed from the same chromosome locus ( 11p15.5 ) . throughout the small and large intestine are highly negatively charged , due to the presence of ester sulphate and terminal sialic acids . reduction of this negative charge in secreted mucins is generally believed to be associated with colorectal disease onset and progression [ 20 , 21 ] . following transcription , mucin gene products are firstly n - glycosylated and dimerise ( through cysteine - rich regions at the c - terminal of the mucin backbone ) in the rough endoplasmic reticulum . this n - glycosylation is also believed to be important in the subsequent transfer of mucins into the golgi apparatus . within this compartment , mucin granules are subsequently packaged tightly due to the presence of high levels of calcium ions . recent studies in this area have noted that the granules of the secreted gel - forming mucin muc5b ( isolated from saliva ) appeal to each contain somewhere in the region of 50100 sub - units of mucin , organised into 1015 isolated polymers , which are believed to represent the grouping of cysteine - rich c and n - terminal regions . granules rapidly expand from a diameter of approximately 350 nm to around 1000 nm , with the end products being polymeric chains of 48 mucin subunits . upon their release , mucin molecules become disassociated from the calcium ions and are believed to unfurl in the presence of the aqueous milieu . it has previously been suggested that the rheologically thick mucus secretion seen in cystic fibrosis is a result of incomplete hydration of mucin granules , possibly as a result of defective hco3 transport [ 28 , 29 ] . triggering of mucin synthesis or secretion , alongside goblet cell / epithelial proliferation and crypt lengthening , may be mediated by a spectrum of neurohumoral , local , and immune factors . total mucin output from the colon can be elevated as a result of increased mucin biosynthesis , exocytosis rates , and total goblet cell numbers . increased muc2 mrna was noted by quantitative rt - pcr analysis in human colon cancer cell lines in response to a single stimulation with il-4 ( approximate two - fold increase in comparison to baseline ) , il-13 , and tnf- ( c.2.5-fold increase in muc2 mrna ) via map kinase pathways . n - glycosylation of muc2 monomers appears to be necessary to drive further processing of mucin subunits , and is a required step prior to passing into the golgi apparatus . the expression of mrna of 3 out of 8 tested isoenzymes governing o - glycosylation of mucins ( polypeptide n - acetylgalactosaminyltransferases ) in a colon cancer cell line , was also noted to be upregulated by the th2 cytokine il-4 , resulting in increased incorporation of galnac into mucin o - glycans . within a random mutagenesis model of murine colitis , an increase in both the amounts of th1 and th2 cytokines secreted by cultured leukocytes and the increased leukocyte numbers within mesenteric lymph nodes were associated with the accumulation of an unglycosylated muc2 oligomers precursor in the golgi apparatus . within the same study , unglycosylated muc2 precursors were also noted to occur in human ulcerative colitis , even in noninflamed intestinal tissue . in il-10-deficient mice , total muc2 output and synthesis was reduced in germ - free animals . upon application of a commensal microflora , there was a significant reduction in mucin sulphation compared to sulphation in the germ - free animal . this evidence would therefore suggest a major role for th2 cytokines in the control of mucin synthesis . in studies on isolated colonic crypts from macroscopically normal tissues , it was noted that goblet cell exocytosis , as assessed by differential interference contrast microscopy , occurred during cholinergic and histamine - mediated stimulation . within these studies , prostaglandin e2 stimulation did not affect mucin exocytosis from goblet cells but appeared to drive fluid loss from columnar cells , which is likely to wash - out studies on total mucin output from an isolated , vascularly perfused rat colon have noted an increase in the number of cavitated goblet cells following stimulation with cholinergic agonists , prostaglandin e2 , and peptide yy . in further studies using this model , total mucin output ( as assessed by elisa ) was also increased in response to the agents used in the previous study as well as serotonin , vasoactive intestinal peptide ( vip ) , interleukin-1 , and no precursors . while it is not possible to isolate the effect of these factors on , for example , mucin biosynthesis or granular exocytosis within such studies , these data give a strong physiological indicator of drives for increased colonic mucin discharge . while mostly associated with goblet cell proliferation in the lung , recent studies have suggested a role for the ets transcription factor spdef in intestinal goblet cell proliferation [ 37 , 38 ] . similarly , il-9 has been linked to the lung inflammatory pathologies . in il-9-overexpressing mice , muc2 expression ( as well as other goblet cell - related genes ) knock - out mouse studies suggested a necessity for the presence of il-13 for this hypersecretion and goblet cell hyperplasia to occur . in vivo studies would suggest that the colonic mucus barrier is a functional bilayer [ 8 , 40 ] . the two layers are rheologically distinct . upon the application of shear stress , the outer layer of mucus rapidly moves from a gel state to a liquid state . this layer is therefore believed to act as a lubricant and is important in reducing colonic shear stress . as it is constantly removed , this outer layer may also act to return the material back into the centre of the lumen , thereby aiding in the reduction of mucosal exposure to such material . the inner , adherent mucus can not be removed by suction and is believed to act as a selective physical barrier to the contact of luminal factors with the underlying mucosa , while still allowing absorptive function to occur . due to the large hydration spheres of mucins in the hydrated mucus gel , it is likely that the mucus gel is imbued with a functional pore size anywhere in the region of 10500 nm . diffusion through these pores will be dependent on the charge of the secreted mucins and the properties of the particle crossing the mucus , as well as the thickness of the mucus layer . studies assessing the secretion dynamics of these mucus layers in an anaesthetised rat model would suggest that the outer , lubricative mucus layer equilibrates to maximal thicknesses of over 600 m , and the shear - resistant inner layer is maintained at approximately 100200 m in rats fed a standard diet [ 8 , 40 , 44 ] . it must be noted that within these studies , the mucus layer is measured in the absence of normal colonic contents . the colonic mucus barrier can act as either an energy source , or as a potential support media for growth to the colonic microflora . while a single bacterial species may not possess the necessary enzymes to cleave all the chemical linkages within the mucin structure , it has previously been hypothesised that the ability of each species to thrive as a whole may be dependent on the presence of upstream degradation of mucin by the colonic microbiome . previous faecal culture studies under anaerobic conditions in mucin - agar gels could suggest that enterobacteria and bacteroides species could be the most predominant within the colonic mucus . however , it must be noted that these studies used gastric mucin as a starting point which has different carbohydrate structures than colonic mucin , which would be expected to affect both bacterial adhesion and degradation . due to their proximity to the underlying mucosa , it is likely that the bacteria that populate the colonic mucus barrier will have the greatest effect on colonic mucosal responses , including mucus secretion , immunity , and inflammatory responses . however , very little is known about the types of bacteria that inhabit the mucus barrier . recently , a series of experiments outlined by johansson et al . , ( 2008 ) have moved the research in this area forwards . perhaps the major finding of this work was that within the normal mouse colonic mucus bilayer , bacteria were only found to occur within the outer , lubricative layer and did not occur within the inner adherent layer ( as evidenced by 16s ribosomal rna in situ hybridisation of histological sections ) . this would therefore suggest that under normal conditions , the adherent layer is impenetrable to colonic bacteria and that the outer layer could be a major habitat for commensal bacteria . while colonic mucus acts as both a barrier and a potential niche for the microflora to exploit , it also appears as if the presence of bacteria in the colon is a major drive of both mucus secretion and normal colonic morphology . this is highlighted by the classic histological observation that germ - free animals have a thinner colonic musculature , with shorter colonic crypts alongside a lack of goblet cells and thin mucus layer [ 5053 ] . in normal human development , the colonic microflora begins to develop from parturition due to indirect maternal inoculation . while germ - free conditions are unlikely in either human physiology or pathophysiology , large - scale changes to bacterial numbers or content could greatly affect mucosal protection . in the case of an already deficient mucus layer , increasing numbers of bacteria that are able to degrade proteins would be more likely to cause mucosal damage / infiltration . temporary reduction of colonic bacterial numbers ( e.g. , during antibiotic therapy ) would be unlikely to cause unwanted mucosal side effects , but in the long term could lead to a less protective mucus barrier . studies in knock - out mice have suggested that deletion of the murine muc2 orthologue muc2 results in the onset of spontaneous ( i.e. , not chemically induced ) colorectal cancer and colitis [ 54 , 55 ] . within studies on human colorectal adenoma progression , changes to mucin gene product expression and glycosylation patterns have been noted . the muc5ac gene product , which is normally secreted in the stomach , but is absent from normal colon , mucinous and nonmucinous carcinomas exhibit separate phenotypic changes to mucin gene expression . in the mucinous adenocarcinoma , there is an increased expression of both muc5ac and muc2 in comparison to the nonmucinous form . in the nonmucinous carcinoma , there is a reduction in total mucus output , accompanied by a shortening of the mucin oligosaccharide chains , particularly through the increased presence of two - residue long galnac -sialic acids ( sialyl - tn antigens ) . the change in mucin oligosaccharides is also characterised by a reduction in sulphation levels versus normal mucosa and reduced sialic acid content . the loss of these factors from oligosaccharides results in the reduction of negative charge from the secreted mucin . similar losses in sulphation ( as well as fucosylation ) have also been noted to occur in ulcerative colitis [ 21 , 60 , 61 ] . a reduction in total muc2 secretion also seems evident in active ulcerative colitis [ 62 , 63 ] . as with colorectal cancer , there appears to be an increased expression of muc5ac in ulcerative colitis [ 64 , 65 ] , which both animal and patient studies would suggest be linked to pre- or early neoplastic changes [ 66 , 67 ] . the above evidence highlights that changes to both the protein and carbohydrate portions of secreted mucins occur in the diseased state . such changes are likely to reduce the protective potential of the colonic mucus gel and may lead to an altering of the available microfloral niche within the secreted mucus , thereby potentially changing the bacterial population . recent preliminary metabolic profiling studies have suggested that an increased appearance of cysteine and proline occur in the faecal water extracts of individuals with colorectal cancer compared to controls . both of these amino acids are found in high amounts in mucins ( cysteine is found in globular terminal structures and is necessary for polymerisation whereas proline is found in high amounts in the glycosylated variable number of tandem repeat structures , where it is thought to act as a spacer between glycosylated residues ( serine or threonine ) that imbue the molecule with a greater degree of flexibility ) . the increased presence of these amino acids would be suggestive of elevated mucolysis , yet would more broadly predict an increase in protein degradation in the colorectal lumen . certain bacterial strains appear to have the ability to preferentially target human colonic mucins [ 69 , 70 ] . adhesion to mucins is believed to be driven by the interaction between external bacterial structures and mucin carbohydrate structures . proteomic analysis of mouse colonic mucus gels demonstrated that fc - gamma - binding protein was found covalently bound to isolated mucins . 16s ribosomal rna analysis has previously been used to assess global bacterial make - up of the human colonic microflora . in some cases , this technology has been used to assess the occurrence of bacteria within mucosal biopsies ( referred to as mucosa - associated bacteria but likely to be a mixture of any bacteria adhered to the mucosa and those associated with the outer and inner mucus layers ) . such studies would suggest that the faecal microflora is distinct from that found in mucosal biopsies , with differences occurring in the mucosal biopsy microflora along the length of the large bowel , with marked intra - individual variations being also noted . the mucosal biopsy microflora of ulcerative colitis moving from remission to relapse patients was noted to be considerably less stable over time than healthy control individuals in a small cohort study . a wider diversity of the colonic biopsy tm7-bacteria was also recently noted in crohn 's disease compared to ulcerative colitis patients or controls . it is possible that there is either a direct bacterial degradation of colorectal mucins by colonic bacteria in colorectal mucosal disease or that the presence of certain bacterial species or by - products alters the pathways of mucin biosynthesis and secretion . while the relative ratios of constituent bacterial species within the colonic microflora ( particularly the relative proportion of bifidobacteria and lactobacilli ) have been postulated to be of importance to colonic health and disease [ 77 , 78 ] , it should be noted that the overall enzymatic spectrum of the microflora or the by - products thereof may be of greater relevance to human health . a number of studies have suggested that there is an increase in the numbers of mucosa - associated bacteria , as reviewed by strober et al . , ( 2007 ) , although these levels did not necessarily appear to correlate well with mucosal inflammation . there is no consensus on large - scale changes of bacterial populations or obvious mucosal infections that occur within ibd or colorectal cancer . within the case of epithelial damage and/or infiltration , it is likely that bacterial contact with toll - like receptors could trigger inflammatory and immune responses , including increased mucin secretion . although the colonic mucosa is surrounded by a wide variety of potentially damaging agents , the physiological response could be described as a dampened inflammation or general tolerance . as previously discussed , an absence of colonic microflora tends to result in a reduction in the standard maturation of the colonic epithelium , as seen in atypical histology . as very few bacteria cross the colonic epithelia ( and possibly even the inner adherent mucus barrier ) outside of major mucosal trauma , it is unlikely that bacterial presence is having a direct effect on colonic physiology until end - stage mucosal infection . therefore , many of the effects of bacteria on mucus secretion may be elicited indirectly by bacterial by - products . previous evidence from experimental models ( see below ) would support this hypothesis although it must be noted that only a fraction of bacterial by - products have been tested for their potential to affect such processes . upon bacterial cell death , lipopolysaccharides ( lps ) are shed into the colonic lumen . in germ - free rats , the level of interleukin-8 ( il-8 ) and total mucin mrna levels have also been shown to be significantly elevated in a mucus - producing colonic cell culture study in response to lps stimulation . both increased mucin release and increased goblet cell numbers have been reported with direct instillation of low levels of butyrate ( 5 mm ) into a vascularly perfused rat colon model . higher levels of butyrate ( up to 100 mm ) actually lowered the level of mucin secretion whereas the same concentration range of acetate ( 5100 mm ) increased mucin secretion in a dose - dependent manner and propionate had no effect . previous studies on mucus secretion dynamics have suggested that low luminal concentrations of butyrate ( 7 mm ) resulted in an increased rate of mucus barrier secretion following removal , but also resulted in a decrease in the maximal mucus thickness attained ( see figure 2 ) . similar effects were noted in more recent studies where long - term administration of high butyrate concentrations ( 100 mm ) directly into the mouse colon over a 7-day period resulted in an upregulation of muc2 gene expression , but a reduction in the histologically assessed adherent mucus layer was noted . mechanistic studies have suggested that mucin output and upregulation of muc2 gene expression are dependent on cholinergic pathways and myofibroblast - derived prostaglandins , respectively . reactive oxygen species ( ross ) are a by - product of aerobic respiration that have been shown to occur to millimolar levels in human colonic luminal contents . ros have been shown to increase mucus secretion rates at low luminal concentrations ( 5 mm h2o2 in the presence of fe ) , but lead to mucus degradation and mucosal reddening at higher concentrations ( 25 and 50 mm h2o2 . these data would suggest that the presence of ros could be sensed by the colonic mucosa , with the low levels driving the secretion of a more protective mucus barrier . at higher levels , the degradation of mucus , and potential damage of the underlying mucosa , results in degradation of the mucus barrier that outweighs increased secretion . the colonic mucus bilayer acts to reduce shear stress and protect the underlying mucosa from damaging luminal entities while still allowing colonic salvage to occur . as such there is evidence that the muc gene products secreted are different in the normal state compared to colorectal pathophysiology , such as adenoma formation and ulcerative colitis . . however , the inner layer of colonic mucus appears to be generally impermeable to this resident microflora and maintains a physical barrier with an exclusion limit of 100 microns between the overlying bacteria and the underlying epithelium . the outer , lubricative mucus layer appears to act as a niche for bacterial population . 16s ribosomal rna analysis would suggest qualitative differences between the bacterial population that resides within the mucus and that occurring within the lumen ( approximated by faecal sampling ) . it is likely that the bacteria that reside within the mucus will have the greatest impact on the physiology and pathophysiology of the colonic mucosa . development of normal colonic morphology , including production of a functionally relevant mucus barrier , is largely driven by the presence of the resident colonic microflora . as bacteria rarely appear to interact directly with the colonic epithelium under normal physiological conditions , it appears likely that the diffusion of bacterial by - products across the colonic mucus barrier to the underlying mucosa acts as a major drive for mucosal maturation and hence affects the processes that govern mucus secretion ( mucin synthesis , mucin granule exocytosis , and goblet cell proliferation ) previous evidence notes particular roles for lps and scfa in driving mucus secretion . there is a need for further studies into how fluctuations in specific bacterial populations affect mucin synthesis and secretion , as well as how such populations adhere to or degrade mucus / mucins in a mixed culture . coassessment of faecal mucins and bacterial populations / bacterial by - products could be utilised as a noninvasive screening technique in human participants . this methodology may be an indirect route of testing ( a ) whether changes in the microflora drive changes to mucin secretion / degradation and ( b ) whether such changes are associated with disease incidence or onset .

the colonic mucus barrier is the first line of defence that the underlying mucosa has against the wide range of potentially damaging agents of microbial , endogenous , and dietary origin that occur within the colonic lumen . the functional component of mucus is the secreted , polymeric glycoprotein mucin . the mucus barrier can either act as an energy source or a support medium for growth to the intestinal microflora . the mucus barrier appears to effectively partition the vast number of microbial cells from the underlying epithelium . the normal functionality and biochemistry of this mucus barrier appears to be lost in diseases of the colorectal mucosa . germ - free animal studies have highlighted the necessity of the presence of the colonic microflora to drive the maturation of the colonic mucosa and normal mucus production . a number of by - products of the microflora have been suggested to be key luminal drivers of colonic mucus secretion .

1. Background 2. Colonic Mucus Production and Secretion 3. The Colonic Mucus Barrier as a Microbial Niche 4. Bacterial By-Products and the Colonic Mucus Barrier 5. Summary

the colonic mucosa is constantly exposed to a wide range of luminal agents that have the potential for either mucosal damage , or mucosal protection . therefore , there is a longer exposure time of the colonic mucosa to luminal agents than to the underlying tissues of other areas of the gut . in addition , due to the role of the colon in the salvage of unabsorbed nutrients and absorption of fluid , these luminal agents will be concentrated ( particularly in the distal bowel ) , resulting in further increases of mucosal exposure to these agents . while removal of water from the faecal bulk is likely to reduce the diffusion of agents from the majority of the faecal cross - section , direct contact will still occur between the mucus and the outer surfaces of the colonic luminal contents . as such , changes to the prevalent species within the microfloral population or to microfloral functioning and output within the bowel are likely to be intimately linked with colorectal health and disease . while digestion per se does not tend to occur in the colon , the colonic microflora acts to degrade dietary fibre and/or other dietary factors that escape digestion to produce further agents that could either harm or protect the underlying mucosa . the colonic mucus barrier is the first line of defence the underlying mucosa has against the myriad of damaging agents that occur within the colonic lumen . the colonic mucus barrier also acts to greatly reduce the shear stress caused by the passage of the luminal bolus along the colon . this barrier can also act as an energy source or as a niche for bacteria within the large bowel [ 9 , 10 ] . despite the relatively high potential for luminal exposure , previous studies in healthy humans suggest that bacteria do not routinely associate with the colonic mucosa and only occur at the luminal side of the intestinal mucus layer . the remainder of this paper will focus on current evidence for how the interplay between microflora and mucus may be a key factor in mucosal health and disease and will also highlight what areas of research may be considered in the future to further understanding of this topic . changes in both the muc gene product and glycosylation patterns are believed to be associated with the onset or development of colonic mucosal diseases , such as colorectal cancer and inflammatory bowel disease ( ibd ) . these genes for muc2 , muc5ac , muc5b , and muc6 are all expressed from the same chromosome locus ( 11p15.5 ) . throughout the small and large intestine are highly negatively charged , due to the presence of ester sulphate and terminal sialic acids . reduction of this negative charge in secreted mucins is generally believed to be associated with colorectal disease onset and progression [ 20 , 21 ] . this n - glycosylation is also believed to be important in the subsequent transfer of mucins into the golgi apparatus . within this compartment , mucin granules are subsequently packaged tightly due to the presence of high levels of calcium ions . recent studies in this area have noted that the granules of the secreted gel - forming mucin muc5b ( isolated from saliva ) appeal to each contain somewhere in the region of 50100 sub - units of mucin , organised into 1015 isolated polymers , which are believed to represent the grouping of cysteine - rich c and n - terminal regions . upon their release , mucin molecules become disassociated from the calcium ions and are believed to unfurl in the presence of the aqueous milieu . it has previously been suggested that the rheologically thick mucus secretion seen in cystic fibrosis is a result of incomplete hydration of mucin granules , possibly as a result of defective hco3 transport [ 28 , 29 ] . total mucin output from the colon can be elevated as a result of increased mucin biosynthesis , exocytosis rates , and total goblet cell numbers . increased muc2 mrna was noted by quantitative rt - pcr analysis in human colon cancer cell lines in response to a single stimulation with il-4 ( approximate two - fold increase in comparison to baseline ) , il-13 , and tnf- ( c.2.5-fold increase in muc2 mrna ) via map kinase pathways . n - glycosylation of muc2 monomers appears to be necessary to drive further processing of mucin subunits , and is a required step prior to passing into the golgi apparatus . the expression of mrna of 3 out of 8 tested isoenzymes governing o - glycosylation of mucins ( polypeptide n - acetylgalactosaminyltransferases ) in a colon cancer cell line , was also noted to be upregulated by the th2 cytokine il-4 , resulting in increased incorporation of galnac into mucin o - glycans . within the same study , unglycosylated muc2 precursors were also noted to occur in human ulcerative colitis , even in noninflamed intestinal tissue . in il-10-deficient mice , total muc2 output and synthesis was reduced in germ - free animals . upon application of a commensal microflora , there was a significant reduction in mucin sulphation compared to sulphation in the germ - free animal . within these studies , prostaglandin e2 stimulation did not affect mucin exocytosis from goblet cells but appeared to drive fluid loss from columnar cells , which is likely to wash - out studies on total mucin output from an isolated , vascularly perfused rat colon have noted an increase in the number of cavitated goblet cells following stimulation with cholinergic agonists , prostaglandin e2 , and peptide yy . in further studies using this model , total mucin output ( as assessed by elisa ) was also increased in response to the agents used in the previous study as well as serotonin , vasoactive intestinal peptide ( vip ) , interleukin-1 , and no precursors . while mostly associated with goblet cell proliferation in the lung , recent studies have suggested a role for the ets transcription factor spdef in intestinal goblet cell proliferation [ 37 , 38 ] . in il-9-overexpressing mice , muc2 expression ( as well as other goblet cell - related genes ) knock - out mouse studies suggested a necessity for the presence of il-13 for this hypersecretion and goblet cell hyperplasia to occur . in vivo studies would suggest that the colonic mucus barrier is a functional bilayer [ 8 , 40 ] . upon the application of shear stress , the outer layer of mucus rapidly moves from a gel state to a liquid state . as it is constantly removed , this outer layer may also act to return the material back into the centre of the lumen , thereby aiding in the reduction of mucosal exposure to such material . the inner , adherent mucus can not be removed by suction and is believed to act as a selective physical barrier to the contact of luminal factors with the underlying mucosa , while still allowing absorptive function to occur . due to the large hydration spheres of mucins in the hydrated mucus gel , it is likely that the mucus gel is imbued with a functional pore size anywhere in the region of 10500 nm . diffusion through these pores will be dependent on the charge of the secreted mucins and the properties of the particle crossing the mucus , as well as the thickness of the mucus layer . studies assessing the secretion dynamics of these mucus layers in an anaesthetised rat model would suggest that the outer , lubricative mucus layer equilibrates to maximal thicknesses of over 600 m , and the shear - resistant inner layer is maintained at approximately 100200 m in rats fed a standard diet [ 8 , 40 , 44 ] . it must be noted that within these studies , the mucus layer is measured in the absence of normal colonic contents . the colonic mucus barrier can act as either an energy source , or as a potential support media for growth to the colonic microflora . while a single bacterial species may not possess the necessary enzymes to cleave all the chemical linkages within the mucin structure , it has previously been hypothesised that the ability of each species to thrive as a whole may be dependent on the presence of upstream degradation of mucin by the colonic microbiome . previous faecal culture studies under anaerobic conditions in mucin - agar gels could suggest that enterobacteria and bacteroides species could be the most predominant within the colonic mucus . due to their proximity to the underlying mucosa , it is likely that the bacteria that populate the colonic mucus barrier will have the greatest effect on colonic mucosal responses , including mucus secretion , immunity , and inflammatory responses . however , very little is known about the types of bacteria that inhabit the mucus barrier . perhaps the major finding of this work was that within the normal mouse colonic mucus bilayer , bacteria were only found to occur within the outer , lubricative layer and did not occur within the inner adherent layer ( as evidenced by 16s ribosomal rna in situ hybridisation of histological sections ) . this would therefore suggest that under normal conditions , the adherent layer is impenetrable to colonic bacteria and that the outer layer could be a major habitat for commensal bacteria . while colonic mucus acts as both a barrier and a potential niche for the microflora to exploit , it also appears as if the presence of bacteria in the colon is a major drive of both mucus secretion and normal colonic morphology . this is highlighted by the classic histological observation that germ - free animals have a thinner colonic musculature , with shorter colonic crypts alongside a lack of goblet cells and thin mucus layer [ 5053 ] . in normal human development , the colonic microflora begins to develop from parturition due to indirect maternal inoculation . while germ - free conditions are unlikely in either human physiology or pathophysiology , large - scale changes to bacterial numbers or content could greatly affect mucosal protection . studies in knock - out mice have suggested that deletion of the murine muc2 orthologue muc2 results in the onset of spontaneous ( i.e. within studies on human colorectal adenoma progression , changes to mucin gene product expression and glycosylation patterns have been noted . in the mucinous adenocarcinoma , there is an increased expression of both muc5ac and muc2 in comparison to the nonmucinous form . in the nonmucinous carcinoma , there is a reduction in total mucus output , accompanied by a shortening of the mucin oligosaccharide chains , particularly through the increased presence of two - residue long galnac -sialic acids ( sialyl - tn antigens ) . the change in mucin oligosaccharides is also characterised by a reduction in sulphation levels versus normal mucosa and reduced sialic acid content . the loss of these factors from oligosaccharides results in the reduction of negative charge from the secreted mucin . as with colorectal cancer , there appears to be an increased expression of muc5ac in ulcerative colitis [ 64 , 65 ] , which both animal and patient studies would suggest be linked to pre- or early neoplastic changes [ 66 , 67 ] . such changes are likely to reduce the protective potential of the colonic mucus gel and may lead to an altering of the available microfloral niche within the secreted mucus , thereby potentially changing the bacterial population . recent preliminary metabolic profiling studies have suggested that an increased appearance of cysteine and proline occur in the faecal water extracts of individuals with colorectal cancer compared to controls . both of these amino acids are found in high amounts in mucins ( cysteine is found in globular terminal structures and is necessary for polymerisation whereas proline is found in high amounts in the glycosylated variable number of tandem repeat structures , where it is thought to act as a spacer between glycosylated residues ( serine or threonine ) that imbue the molecule with a greater degree of flexibility ) . the increased presence of these amino acids would be suggestive of elevated mucolysis , yet would more broadly predict an increase in protein degradation in the colorectal lumen . 16s ribosomal rna analysis has previously been used to assess global bacterial make - up of the human colonic microflora . in some cases , this technology has been used to assess the occurrence of bacteria within mucosal biopsies ( referred to as mucosa - associated bacteria but likely to be a mixture of any bacteria adhered to the mucosa and those associated with the outer and inner mucus layers ) . such studies would suggest that the faecal microflora is distinct from that found in mucosal biopsies , with differences occurring in the mucosal biopsy microflora along the length of the large bowel , with marked intra - individual variations being also noted . the mucosal biopsy microflora of ulcerative colitis moving from remission to relapse patients was noted to be considerably less stable over time than healthy control individuals in a small cohort study . a wider diversity of the colonic biopsy tm7-bacteria was also recently noted in crohn 's disease compared to ulcerative colitis patients or controls . it is possible that there is either a direct bacterial degradation of colorectal mucins by colonic bacteria in colorectal mucosal disease or that the presence of certain bacterial species or by - products alters the pathways of mucin biosynthesis and secretion . while the relative ratios of constituent bacterial species within the colonic microflora ( particularly the relative proportion of bifidobacteria and lactobacilli ) have been postulated to be of importance to colonic health and disease [ 77 , 78 ] , it should be noted that the overall enzymatic spectrum of the microflora or the by - products thereof may be of greater relevance to human health . a number of studies have suggested that there is an increase in the numbers of mucosa - associated bacteria , as reviewed by strober et al . there is no consensus on large - scale changes of bacterial populations or obvious mucosal infections that occur within ibd or colorectal cancer . within the case of epithelial damage and/or infiltration , it is likely that bacterial contact with toll - like receptors could trigger inflammatory and immune responses , including increased mucin secretion . although the colonic mucosa is surrounded by a wide variety of potentially damaging agents , the physiological response could be described as a dampened inflammation or general tolerance . as previously discussed , an absence of colonic microflora tends to result in a reduction in the standard maturation of the colonic epithelium , as seen in atypical histology . as very few bacteria cross the colonic epithelia ( and possibly even the inner adherent mucus barrier ) outside of major mucosal trauma , it is unlikely that bacterial presence is having a direct effect on colonic physiology until end - stage mucosal infection . therefore , many of the effects of bacteria on mucus secretion may be elicited indirectly by bacterial by - products . previous evidence from experimental models ( see below ) would support this hypothesis although it must be noted that only a fraction of bacterial by - products have been tested for their potential to affect such processes . upon bacterial cell death , lipopolysaccharides ( lps ) are shed into the colonic lumen . in germ - free rats , the level of interleukin-8 ( il-8 ) and total mucin mrna levels have also been shown to be significantly elevated in a mucus - producing colonic cell culture study in response to lps stimulation . both increased mucin release and increased goblet cell numbers have been reported with direct instillation of low levels of butyrate ( 5 mm ) into a vascularly perfused rat colon model . higher levels of butyrate ( up to 100 mm ) actually lowered the level of mucin secretion whereas the same concentration range of acetate ( 5100 mm ) increased mucin secretion in a dose - dependent manner and propionate had no effect . previous studies on mucus secretion dynamics have suggested that low luminal concentrations of butyrate ( 7 mm ) resulted in an increased rate of mucus barrier secretion following removal , but also resulted in a decrease in the maximal mucus thickness attained ( see figure 2 ) . mechanistic studies have suggested that mucin output and upregulation of muc2 gene expression are dependent on cholinergic pathways and myofibroblast - derived prostaglandins , respectively . reactive oxygen species ( ross ) are a by - product of aerobic respiration that have been shown to occur to millimolar levels in human colonic luminal contents . ros have been shown to increase mucus secretion rates at low luminal concentrations ( 5 mm h2o2 in the presence of fe ) , but lead to mucus degradation and mucosal reddening at higher concentrations ( 25 and 50 mm h2o2 . these data would suggest that the presence of ros could be sensed by the colonic mucosa , with the low levels driving the secretion of a more protective mucus barrier . at higher levels , the degradation of mucus , and potential damage of the underlying mucosa , results in degradation of the mucus barrier that outweighs increased secretion . the colonic mucus bilayer acts to reduce shear stress and protect the underlying mucosa from damaging luminal entities while still allowing colonic salvage to occur . as such there is evidence that the muc gene products secreted are different in the normal state compared to colorectal pathophysiology , such as adenoma formation and ulcerative colitis . however , the inner layer of colonic mucus appears to be generally impermeable to this resident microflora and maintains a physical barrier with an exclusion limit of 100 microns between the overlying bacteria and the underlying epithelium . the outer , lubricative mucus layer appears to act as a niche for bacterial population . 16s ribosomal rna analysis would suggest qualitative differences between the bacterial population that resides within the mucus and that occurring within the lumen ( approximated by faecal sampling ) . it is likely that the bacteria that reside within the mucus will have the greatest impact on the physiology and pathophysiology of the colonic mucosa . development of normal colonic morphology , including production of a functionally relevant mucus barrier , is largely driven by the presence of the resident colonic microflora . as bacteria rarely appear to interact directly with the colonic epithelium under normal physiological conditions , it appears likely that the diffusion of bacterial by - products across the colonic mucus barrier to the underlying mucosa acts as a major drive for mucosal maturation and hence affects the processes that govern mucus secretion ( mucin synthesis , mucin granule exocytosis , and goblet cell proliferation ) previous evidence notes particular roles for lps and scfa in driving mucus secretion . coassessment of faecal mucins and bacterial populations / bacterial by - products could be utilised as a noninvasive screening technique in human participants . this methodology may be an indirect route of testing ( a ) whether changes in the microflora drive changes to mucin secretion / degradation and ( b ) whether such changes are associated with disease incidence or onset .

since the early seventies , a number of european governments have , through the european experimental vehicles committee ( eevc ) , collaborated on the development of test procedures and equipment to assess various aspects of car crash safety . by the middle of 1990s , this research had resulted in completely new full scale crash test procedures for protection of car occupants in frontal and side impacts , and a component test procedure for assessing the protection of pedestrians hit by the fronts of cars . at that time , the only full scale crash test required by european legislation was a full width rigid block impact designed only to control intrusion of the steering column in a frontal crash . in 1979 , the national highway traffic safety administration started the new car assessment programme ( ncap ) , where cars were frontal impact tested at the impact speed of 35 miles per hour . in europe , the german motor club adac and the motoring magazine auto motor und sport started to perform offset rigid wall frontal crash tests and to publish the results as consumer information . at around the same time , a single series of frontal tests , jointly funded by the uk department of transport ( dft ) and international testing and using the eevc offset deformable frontal impact test procedure , were published . these programmes highlighted the beneficial effects that consumer information could offer and got the ball rolling for a permanent programme for the whole of europe . in november 1996 , the swedish national road administration ( snra ) , the federation internationale de l'automobile ( fia ) and international testing were the first organisations to join in the uk dft with the newly formed euro ncap . taking as a starting point , the same eevc procedures that would form the basis of future legislation , dedicated test and rating protocols were developed for front impact and side impact ( including 3-year - old & 18-month - old dummies in manufacturer 's recommended child restraint systems ) and for pedestrian protection . the first results on seven superminis were presented at trl in the uk in february 1997 . the release of these first results caused considerable media interest , fuelled by a strong critical response from some of the car manufacturers . in october 1998 , new directives , based on the eevc 's recommendations , concerning frontal and side vehicle impact ( 96/79/ec and 96/27/ec respectively ) became effective for all new vehicles . in the same year , euro ncap achieved legal status when it became an international association under belgian law . from the formation of euro ncap , the fia took the lead in promoting the programme and in discussions with other potential members . as a consequence , more european governments , automobile clubs and representatives from the insurance industry have joined euro ncap over the years . operational control of euro ncap moved from the uk to a full time secretariat based in brussels in 1999 . from 1997 onwards , new batches of test results were published about twice each year and car manufacturers , setting aside their initial reservations , started to sponsor the testing of their own cars . as new car models replaced those already tested , the improvements in their occupant star ratings could be clearly seen ( fig . 1 ) . in june 2001 , the renault laguna became the first car to be awarded 5 stars for occupant protection , made possible by the introduction of the pole test ( see section below ) . following from this success , manufacturers increasingly saw 5 stars as the goal for all their new models . the first period of euro ncap testing was coincided with the introduction of the first realistic crash tests in european legislation . consequently , the vehicle safety standard in industry was evolved at a fast pace , in particular in occupant protection . from the beginning , it was intended that euro ncap would encourage manufacturers to exceed the legal requirements and this was achieved by applying more stringent and/or additional test conditions and by extending the assessment to new areas of vehicle safety , as illustrated by the examples below . research has shown that pole side impacts are relatively uncommon , but they represent a disproportionately high level of fatalities and ais3 + injuries . in the late nineties , car manufacturers started to introduce countermeasures focussed on preventing head and ( to a lesser extent ) thorax injuries , which together represent the predominant cause of death in such crashes . as head impact did not regularly occur in the barrier test , euro ncap added an optional pole test to demonstrate the benefit of the head protecting airbags for side impact . with no appropriate test being developed in europe , the us side impact pole test was adapted for use with the european side impact dummy eurosid-1 . using this procedure , more recently , euro ncap 's pole test has seen several updates , including the test dummy , performance criteria and scoring . one of the most contentious areas of the adult occupant assessment is related to knee protection . the seating procedure for the adult dummies ensures that the knees always hit the same small areas of the facia in the frontal impact test . with this knowledge however , accident research showed that crash victims can impact their knees on virtually any part of the facia they can reach . before euro ncap , such areas were untested and frequently contained aggressive structures . euro ncap examines the whole facia area and penalties are applied to the euro ncap score where hazards are found . in 2007 , a dedicated knee mapping sled procedure has been adopted to help manufacturers demonstrate the safe design of the facia area for different sizes of front seat occupants and avoid penalties . in contrast to the advances made in occupant protection , improvements in pedestrian protection were initially slow to emerge . in 2002 , the european commission and association des constructeurs europens d'automobiles reached a voluntary agreement on pedestrian protection but failed to implement the state - of - the - art pedestrian protection subsystem tests developed and validated by eevc . this left euro ncap to deal with an industry unwilling to make the necessary investments to improve vehicle front - ends . lack of progress was such that at the beginning of 2002 , euro ncap revised its pedestrian testing and assessment protocols in an attempt to encourage manufacturers to make improvements . at the same time , new developments reported by the eevc were also incorporated . using this revised protocol , encouragingly , in 2005 , the citroen c6 became the first car to achieve 4 stars for pedestrian protection . this car was equipped with sensor technology to detect an impact with a pedestrian and to deploy a however the pop - up bonnet and other innovations in pedestrian protection remained uncommon until euro ncap changed its ratings regime in 2009 and the european commission regulation ( ec ) 78/2009 was adopted in europe . since may 2006 , it has been compulsory to use safety belts and united nations regulation no . it is also mandatory to use child car seats within the eu for children up to the heights of 1.35 m or 1.5 m - depending upon the member state . thanks to these laws and increased consumer awareness and compliance , child deaths in motor vehicle crashes have steadily declined over the last decades . , most car manufacturers relied for many years on the makers of child restraints to provide protection for children in cars . very few offered child restraints through their dealerships or provided any recommendation to their customers , and there were almost no special provisions in the vehicle for the safe transport of children . however , there are many aspects of child protection which can not be influenced by the child restraint manufacturer alone and require action also on the part of the car manufacturer . for this reason , euro ncap introduced an additional star rating in 2003 , specifically addressing the protection of children in the event of a crash . the rating was based on the protection offered in the front and side crash tests to a three year old and 18 month old child seated on the rear seat in a restraint of the type recommended by the car manufacturer . the assessment was complemented with firm incentives with regards to communication ( handbook instructions , information at dealerships , warning labels , etc . ) and availability of isofix attachments and other relevant equipment such as a front passenger airbag deactivation switch . the child occupant protection star rating has motivated all car manufacturers to aim for good child protection and has facilitated a better dialogue between car manufacturers and child restraint suppliers . although euro ncap 's influence was seen to be improving adult occupant protection , there was concern that this improved protection relied on the proper use of seat belts . as protection for belt wearers improved , accident data increasingly showed that a higher proportion of seriously and fatally injured casualties were not wearing their seat belts . to improve this situation , euro ncap developed a protocol to encourage the fitment of intelligent seat belt reminders ( sbr ) . research had shown that most non - wearers could be persuaded to use their seat belt if they were given a suitable reminder . although simple reminders have been available for many years , intelligent systems can be much more effective : almost unnoticed by belt wearers but increasingly aggressive and demanding for those who do not buckle up . for front seats , euro ncap requires a final signal , which has to be audio - visual and must be presented at the latest 60 s after the engine start , after 500 m of vehicle travel or speeds above 25 km / h . the final signal must last for a minimum of 90 s and consist of a loud and clear audible signal and a visual indicator . for rear seats , euro ncap requires a start signal , which may be visual only . for all seats , if a change in belt status occurs at speeds above 25 km / h , i.e. a belt gets unbuckled , an immediate audible signal must be given . initially , most systems covered only the driver 's seat , with the front seat passenger being covered later . by 2004 , intelligent seat belt reminders were becoming more commonplace , the sbr score being an effective way to improve a car 's euro ncap rating . it is well understood that the huge increase in whiplash claims in the last decades were in part the result of the legal system of compensation . nevertheless , whiplash remains the most frequently reported injury on european roads . as whiplash injury to the neck often leads to long term impairment , with 10 percent of people suffering long term discomfort and 1 percent permanent disability , addressing injuries , their causes and prevention has been an important priority for the european commission . whiplash may occur in all impact directions but the injury is most frequently observed , and its risk most effectively addressed , in rear - ends impacts . for this injury type , no biomechanically based vehicle safety regulations exist , mainly as a consequence of the limited ( or inconclusive ) knowledge available on whiplash . however , research has demonstrated that , in the event of a rear - end collision , the vehicle seat and head restraint are the principal means of reducing neck injury . starting from the assumption that lowering loads on the neck lessens the likelihood of whiplash injury , the first stand - alone tests for seats and head restraints were developed by the international insurance whiplash prevention group ( iiwpg ) and the swedish road administration ( sra ) . however , the tests adopted different philosophies with regards to relevant seat performance parameters , one putting heavy emphasis on real world validation ( iiwpg ) , the other using plausible hypotheses regarding the causes of whiplash injury ( sra ) . euro ncap set up a whiplash group in 2002 with the intention of developing a test that could complement the existing whole vehicle consumer crash tests . in 2008 , euro ncap had completed its work and formally launched the first series of results of ( front ) seat testing . by 2007 , the euro ncap ratings for adult protection , child protection and pedestrian protection had been in common use for a decade and had become internationally recognised as a reliable indicator of independent consumer information about car safety . industry 's efforts to deliver increasingly safer cars resulted in many able to achieve 5 stars for adult protection . but while this represented a significant step forwards for consumer protection , worries started to rise over the future of the programme . firstly , it was clear that the high share of cars rated 5 stars for adult protection was diminishing the discriminating factor in the rating with each advancing year leading , amongst other things , to less media interest in the results . at the same time , the successes in adult protection were hiding the less favourable progress in the other areas such as pedestrian protection from the view of the consumer . but it was the rating scheme 's inability to deal with emerging driver - assist and crash avoidance systems that ultimately triggered euro ncap to review and fundamentally overhaul its rating system . in 2009 , euro ncap changed from three individual ratings to a single overall safety rating with a maximum of 5 stars for each vehicle . this overall rating combined the results of assessments in four important areas : adult protection , child protection and pedestrian protection the three areas assessed in the previous scheme and the new area of safety assist technology . the underlying tests included the full - scale frontal offset , side - impact barrier and pole tests carried over from the previous adult and child protection ratings , the new seat tests for whiplash prevention in rear - end crashes , and front - end component tests for pedestrian protection . the assessment of intelligent seat belt reminders was complemented with that of speed limiters and electronic stability control as part of safety assist . in each area of assessment , scores were calculated as a percentage of the maximum points available and a weighted sum of these scores indicated the car 's overall all - round performance . encompass all overall rating was established to provide a more balanced assessment of various vehicle safety aspects and to add more flexibility to the ratings scheme . it allowed future technologies to be added without the need to add stars or introduce new rating categories . a top achiever would not only have to achieve a high overall score , but could also not underachieve in ensure any single area . hence , by moving to the overall rating scheme , euro ncap was able to promote integrated safety solutions , using both active and passive safety technology . with the overall rating scheme in place , euro ncap subsequently worked on a programme of stepwise updates to the rating scheme in following years . during this period , in addition , new functional tests were added , in particular related to crash avoidance and advanced driver assistance technologies . crash avoidance technology is aimed at reducing the likelihood of an accident happening or reducing its severity . research has shown that electronic stability control ( esc ) is effective in helping to prevent certain types of ( loss of control ) crashes , e.g. lie et al 's and thomas 's . already in 2005 , euro ncap gave a strong recommendation to consumers that they should specify esc on any new car purchase . from 2009 onwards , esc became an integral part of the overall rating , first on the basis only of fitment and later , in 2011 , on fitment and functional requirements verified by a series of sine - with - dwell track tests similar to those specified in the global technical regulation 8 . in fact , it has a greater effect on the number of accidents and injury severity than almost all other known risk factors . speed restrictions are intended to promote safe operation of the road network by keeping traffic speeds below the maximum that is appropriate for a given traffic environment . voluntary speed assistance systems ( sas ) are a means to assist drivers to adhere to speed limits , by warning and/or effectively limiting the speed of the vehicle . the only technical requirements for such devices are laid down in united nations regulation no . 89 speed limitation devices , which is not mandatory in europe and does not specifically apply to m1 passenger cars . since 2009 , euro ncap has rewarded manually set speed limitation devices which meet the basic requirements of united nations regulation no . 89 but have additional functionality with regards to the warnings given and the ability to be set - at - speed . by doing so , euro ncap has created a first incentive to manufacturers to promote such speed - limitation devices , to make them available on more models and to fit them as standard equipment . in recent years more advanced speed assistance systems have been introduced onto the market which are able to inform the driver of the speed limit at the vehicle 's current position , based on digital speed maps and/or traffic sign recognition . although there are still limitations to these technologies , intelligent speed assistance systems have much greater potential and will be more readily acceptable to the public . as a result , euro ncap extended the speed limitation protocol in 2013 to include the latest generation of intelligent speed assistance systems . autonomous emergency braking ( aeb ) is without doubt the most important active safety systems that euro ncap has adopted since esc . using technologies such as radar , lasers and optical sensors to identify other vehicles and in many cases pedestrians , aeb automatically applies the brakes if the driver does not respond in time , to avoid or mitigate a collision , saving countless lives , injuries and inconvenience . systems are most effective at lower speeds ( < 40 km / h ) where more than 75% of rear - end crashes occur , but they are also effective in mitigating the devastating effects of higher speed crashes . within europe , four main initiatives have actively contributed to development of test procedures for assessing aeb and forward collision warning ( fcw ) systems for car - to - car crashes . adac , with support from automotive suppliers continental and bosch , developed an inflatable vehicle target in order to perform a comparative test of aeb systems on high - end vehicles . the rcar autonomous emergency braking group , led by thatcham , designed a testing and ( insurance ) rating approach for aeb systems . the group mainly consisted of insurance institutes , but was supported by volvo car corporation and continental . the european commission sponsored research project assess ( assessment of integrated vehicle safety systems for improved vehicle safety ) and the german initiative led by dekra , called vfss ( advanced forward - looking safety systems ) had similar project goals to develop harmonized and standardized assessment procedures and related tools for selected integrated safety systems . the euro ncap aeb test and assessment procedures were born out of the deliverables of these projects . euro ncap adopted both low speed and high speed aeb systems in the rating scheme in 2014 . low speed aeb systems , aeb city , where directly linked to whiplash prevention and therefore added to adult occupant protection . high speed aeb systems , aeb inter - urban , addressing crashes with more variable outcome in terms of injuries were included in safety assist . one of the most overlooked benefits of the consumer rating system in europe is its impact on availability and fitment of equipment across the eu markets . from the start , euro ncap tested the best - selling variant of a model in order to achieve the most relevant rating for the market as a whole . the downside of this approach was that lower specification vehicles were offered in ( often central , east and southeast ) european markets where sales were not expected to be high enough . in 2007 , euro ncap therefore started to demand that all equipment relevant to the safety rating be fitted as standard or , in exceptional cases , meet a minimum fitment percentage over the whole of eu-28 . the latter fitment requirement was ramped up in yearly steps to 100% by 2012 , effectively requiring manufacturers to make safety equipment standard across the board from then on . the fitment policy has been very effective in propagating systems like head curtain airbags ( fig . it has become the norm for new cars tested by euro ncap to be fitted with such systems , despite the fact that these are / were not mandated by law . recently , there has also been a rise in the number of mainstream models available with speed assistance and aeb and/or fcw systems . that is a welcome sign for road safety and helps pave the way for the eventual deployment of highly automated vehicles . during the first years of testing , much of the engineering effort went into achieving higher scores in the frontal and side impact crash tests . the reduction in passenger compartment intrusion in frontal impact is the most visible effect of euro ncap 's influence . by preventing intrusion , the likelihood of the occupant impacting the car 's interior is minimized , providing space for the restraint system to operate effectively . as a result , also , some manufacturers have completely cleared the knee impact zones of potential hazards as a result of euro ncap 's scoring scheme for this area . the provision of side impact and head airbags has helped and it is now normal for the cars to be fitted with side impact airbags and curtains . euro ncap has encouraged improved designs and the fitment of isofix mounts and child restraints . isofix provides a much more secure method of attaching the child restraint to the car , provided that additional provision is made to prevent rotation of the child restraint , which can be caused by seat cushion compression and rebound . as a consequence , euro ncap has seen improved designs , where the child is less likely to strike the car interior whilst at the same time experiencing reduced forces from the restraint system . one of the most remarkable effects of the introduction of the overall rating has been the improved score in pedestrian subsystem tests since 2009 ( fig . 4 ) . supported by an extensive review of protocols that has made the engineering goals more attainable and a soft landing approach in the rating calculation , car manufactures have delivered energy absorbing bumpers , deployable bonnets and external airbags and have repositioned stiff structures in order to boost their performance . although euro ncap 's ratings have been seen to improve over time , the only real proof of euro ncap 's effectiveness lies in real - world accident data . several analyses have been carried out that show the effect of improvements in vehicle safety , many of which have been influenced by euro ncap . in 2000 , cars with three or four adult occupant stars are approximately 30% safer , compared to two star cars or cars without a euro ncap score , in car - to - car collisions . data was also provided which showed that the predicted relative risk of severe or fatal injury was reduced by 12% , for each increase in euro ncap star rating . in 2001 , a monash university report for sarac reported that the euro ncap star rating is able to differentiate with statistical significance both the average crashworthiness and injury severity based on all real crashes of vehicles in star rating categories 1 , 2 and 4 . further evidence of the effectiveness of secondary safety improvements in the early years of euro ncap were reported in the journal accident analysis & prevention . the benefits of intelligent seat belt reminders have been shown in several reports from folksam insurance , the swedish traffic administration and others . the data were collected by field observations in major cities in six european countries and in five cities around sweden . a selection of car models having seat belt reminders ( sbr ) were compared to a fleet of similar car models without such reminders . this study concludes that sbr fulfilling euro ncap 's sbr protocol significantly increase seat belt use in daily traffic . around 80% ( 82.2% 8.6% ) of drivers not wearing a belt in cars with no seat belt reminder do so in cars equipped with a system that has a light signal and an associated loud and clear sound signal . this finding is in line with earlier studies from europe and usa that also found that enhanced sbr with light and sound are most effective . a significant correlation between euro ncap pedestrian scores and injury outcome was reported by pastor using german national accident records from 2009 to 2011 . comparing a vehicle scoring 5 points and a vehicle scoring 22 points , pedestrians ' conditional probability of getting fatally injured was reduced by 35% ( from 0.58% to 0.37% ) for the latter . at the same time the probability of serious injuries could be reduced by 16% ( from 27.4% to 22.9% ) . strandroth et al also showed a significant reduction of injury severity for cars with better pedestrian scoring . in this study , the euro ncap pedestrian scoring was compared with the real - life outcome in pedestrian crashes that occurred in sweden 20032010 . the real - life crash data was obtained from the data acquisition system strada , which combines police records and hospital admission data . the reduction of risk of serious consequences ( rsc ) for medium performing cars in comparison with low performing cars was 17% , 26% and 38% for 1% , 5% and 10% of medical impairment , respectively . these results applied to urban areas with speed limits up to 50 km / h only . kullgren et al carried out an evaluation of the euro ncap , japan ncap and iiwpg whiplash protocols using real - world crash data . three analyses were undertaken comprising an analysis of test outcome data , a logistic regression analysis , a receiver operating characteristic ( roc ) analysis , and a correlation analysis comparing crash and injury outcome . correlations between the test scenarios of each of the three protocols , as well as the outcome associations with crash outcomes , suggested consistent improvements in the risk of permanent medical impairment . finally , euro ncap with support of the australasian ncap studied the effectiveness of low speed aeb systems promoted through the rating scheme since 2014 . real - world evaluations of advanced safety systems are often limited by slow take - up rates , insufficient crash data and lower crash rates of new , safer vehicles . to overcome this , data from five european countries plus australia were pooled using a standard analysis format and a novel prospective meta - analysis approach . the study showed that low speed aeb technology leads to a 38% reduction in real - world rear - end crashes with no significant difference between urban and rural crash benefits being observed . the publication confirmed that autonomous emergency braking is one of the most promising safety technologies in recent years . the increasing globalisation of the car market and the rise of the emerging markets have seen the deployment of new safety rating initiatives in more regions of the world . in 2009 , global ncap initiated the latin ncap for latin america , soon followed by asean ncap in southeast asia . both programmes , taking advantage of the technical know - how and procedures of euro and australasian ncap , have quickly gained momentum . the links between euro ncap and the new ncaps , australasian ncap and to a lesser extend japan ncap , korean ncap and china ncap , have increased euro ncap 's scope and are partly responsible for its evolution from a safety rating for the european market to a new cars today are much safer than they were a decade ago thanks to improved crash test standards , crumple zones , seat belts and airbags , which help to protect occupants in a crash . while most occupant safety measures can be considered mature , more could and should be done to improve their robustness and effectiveness for the general diversity of vehicle occupants and crash scenarios . they should be effectively deployed to address the above key accident scenarios , including those that involve other road users and commercial vehicles . today , the uptake of crash avoidance technology still poses a particular challenge : a large variety of systems is available but only a few are offered as standard . the uptake of optional systems is still low and depends greatly on market incentives . in the coming years , the need for more on - board technologies to support ( partial ) automated driving will probably make crash avoidance systems cheaper and more cost - effective across the european car fleet . besides the price , acceptance and volume of advanced technologies are driven largely by how well consumers understand these features and value them . for this , the vehicle rating must reflect the true contribution of passive and active safety measures to the overall safety performance . the lack of traceability of ( the performance of ) systems in the market , the complex role of driver behaviour and inconsistency in human machine interface ( hmi ) applied across industry , all further complicate the important task of identifying the true potential of avoidance technology . the idea of automated and self - driving cars has been widely aired in technical discussions and in media coverage recently . the rapid development of electronic safety systems has made the concept possible and prototype systems are able to drive in controlled situations . the established vehicle industry is active in this field but new players such as google have also shown prototypes . there is no doubt that greater automation will lead to a revolution in safety , putting it above all other requirements and characteristics of a car . not only will the self - driving car has the technology to sense , avoid and mitigate in potential crash scenarios , it will also drive in a safer manner . besides that , used in a manual way , the vehicle will always carry the safety elements and technologies to intervene when necessary . euro ncap plans to engage in the roll out of vehicle automation as a way to dramatically improve vehicle safety and safe driving . it will continue to promote best safety practice when vehicles start to have elements fitted which support automated driving and to ensure that the vehicle manufacturer remains responsible for safe operation of the system . in 2014 , euro ncap published a roadmap for further developments of the safety rating in the next 5 years . the plan identifies four main domains that focus on the key real life crash scenarios and that can be addressed by new and updated vehicle technology , in particular in the field of crash avoidance . this includes , amongst others , the addition of aeb technology for vulnerable road users ( pedestrians and cyclists ) and the testing of more capable driver support systems for longitudinal and lateral car crashes . most consumers will have no personal experience on which to judge the crash safety of their car . clearly , without objective and transparent safety information , these questions would be impossible to answer . this underlines the importance of public safety ratings and justifies why euro ncap continues to develop its comparative safety tests . moreover , it explains why euro ncap 's online and written publications continue to grow in popularity , not only with consumers but also more and more with public and private fleet managers to help them ensure that the safety of their vehicle fleet provides acceptable levels of protection to their employees . at the time of launch of the first euro ncap results in february 1997 , some critics claimed that the assessment criteria were so severe that no car could ever achieve ( the then maximum ) four stars for occupant protection . in july 1997 results from the second phase of tests were published and included the first 4 star results , for the volvo s40 . this illustrates two important insights that have proven to be true time after time over the last two decades . firstly , given clear , objective targets and sufficient time , the car manufacturers can make great safety improvements . secondly , sufficient time does not always have to mean years , as many manufacturers have responded very quickly to new challenges in the past . a consumer rating system , like euro ncap , that is rooted firmly on real life experiences but which closely follows the technological innovations in the marketplace can therefore deliver the most benefit for society .

established in 1997 , the european new car assessment programme ( euro ncap ) provides consumers with a safety performance assessment for the majority of the most popular cars in europe . thanks to its rigorous crash tests , euro ncap has rapidly become an important driver safety improvement to new cars . after ten years of rating vehicles , euro ncap felt that a change was necessary to stay in tune with rapidly emerging driver assistance and crash avoidance systems and to respond to shifting priorities in road safety . a new overall rating system was introduced that combines the most important aspects of vehicle safety under a single star rating . the overall rating system has allowed euro ncap to continue to push for better fitment and higher performance for vehicles sold on the european market . in the coming years , the safety rating is expected to play an important role in the support of the roll - out of highly automated vehicles .

The origins of consumer testing in Europe The evolution of vehicle safety The advent of crash avoidance The impact of Euro NCAP A view into the future of automotive safety Concluding remarks

since the early seventies , a number of european governments have , through the european experimental vehicles committee ( eevc ) , collaborated on the development of test procedures and equipment to assess various aspects of car crash safety . at that time , the only full scale crash test required by european legislation was a full width rigid block impact designed only to control intrusion of the steering column in a frontal crash . in 1979 , the national highway traffic safety administration started the new car assessment programme ( ncap ) , where cars were frontal impact tested at the impact speed of 35 miles per hour . in europe , the german motor club adac and the motoring magazine auto motor und sport started to perform offset rigid wall frontal crash tests and to publish the results as consumer information . at around the same time , a single series of frontal tests , jointly funded by the uk department of transport ( dft ) and international testing and using the eevc offset deformable frontal impact test procedure , were published . these programmes highlighted the beneficial effects that consumer information could offer and got the ball rolling for a permanent programme for the whole of europe . in november 1996 , the swedish national road administration ( snra ) , the federation internationale de l'automobile ( fia ) and international testing were the first organisations to join in the uk dft with the newly formed euro ncap . in october 1998 , new directives , based on the eevc 's recommendations , concerning frontal and side vehicle impact ( 96/79/ec and 96/27/ec respectively ) became effective for all new vehicles . in the same year , euro ncap achieved legal status when it became an international association under belgian law . from the formation of euro ncap , the fia took the lead in promoting the programme and in discussions with other potential members . as a consequence , more european governments , automobile clubs and representatives from the insurance industry have joined euro ncap over the years . as new car models replaced those already tested , the improvements in their occupant star ratings could be clearly seen ( fig . in june 2001 , the renault laguna became the first car to be awarded 5 stars for occupant protection , made possible by the introduction of the pole test ( see section below ) . the first period of euro ncap testing was coincided with the introduction of the first realistic crash tests in european legislation . consequently , the vehicle safety standard in industry was evolved at a fast pace , in particular in occupant protection . from the beginning , it was intended that euro ncap would encourage manufacturers to exceed the legal requirements and this was achieved by applying more stringent and/or additional test conditions and by extending the assessment to new areas of vehicle safety , as illustrated by the examples below . as head impact did not regularly occur in the barrier test , euro ncap added an optional pole test to demonstrate the benefit of the head protecting airbags for side impact . with no appropriate test being developed in europe , the us side impact pole test was adapted for use with the european side impact dummy eurosid-1 . using this procedure , more recently , euro ncap 's pole test has seen several updates , including the test dummy , performance criteria and scoring . one of the most contentious areas of the adult occupant assessment is related to knee protection . the seating procedure for the adult dummies ensures that the knees always hit the same small areas of the facia in the frontal impact test . before euro ncap , such areas were untested and frequently contained aggressive structures . euro ncap examines the whole facia area and penalties are applied to the euro ncap score where hazards are found . in 2002 , the european commission and association des constructeurs europens d'automobiles reached a voluntary agreement on pedestrian protection but failed to implement the state - of - the - art pedestrian protection subsystem tests developed and validated by eevc . this left euro ncap to deal with an industry unwilling to make the necessary investments to improve vehicle front - ends . lack of progress was such that at the beginning of 2002 , euro ncap revised its pedestrian testing and assessment protocols in an attempt to encourage manufacturers to make improvements . this car was equipped with sensor technology to detect an impact with a pedestrian and to deploy a however the pop - up bonnet and other innovations in pedestrian protection remained uncommon until euro ncap changed its ratings regime in 2009 and the european commission regulation ( ec ) 78/2009 was adopted in europe . , most car manufacturers relied for many years on the makers of child restraints to provide protection for children in cars . very few offered child restraints through their dealerships or provided any recommendation to their customers , and there were almost no special provisions in the vehicle for the safe transport of children . however , there are many aspects of child protection which can not be influenced by the child restraint manufacturer alone and require action also on the part of the car manufacturer . for this reason , euro ncap introduced an additional star rating in 2003 , specifically addressing the protection of children in the event of a crash . the rating was based on the protection offered in the front and side crash tests to a three year old and 18 month old child seated on the rear seat in a restraint of the type recommended by the car manufacturer . although euro ncap 's influence was seen to be improving adult occupant protection , there was concern that this improved protection relied on the proper use of seat belts . to improve this situation , euro ncap developed a protocol to encourage the fitment of intelligent seat belt reminders ( sbr ) . although simple reminders have been available for many years , intelligent systems can be much more effective : almost unnoticed by belt wearers but increasingly aggressive and demanding for those who do not buckle up . for front seats , euro ncap requires a final signal , which has to be audio - visual and must be presented at the latest 60 s after the engine start , after 500 m of vehicle travel or speeds above 25 km / h . for rear seats , euro ncap requires a start signal , which may be visual only . by 2004 , intelligent seat belt reminders were becoming more commonplace , the sbr score being an effective way to improve a car 's euro ncap rating . it is well understood that the huge increase in whiplash claims in the last decades were in part the result of the legal system of compensation . as whiplash injury to the neck often leads to long term impairment , with 10 percent of people suffering long term discomfort and 1 percent permanent disability , addressing injuries , their causes and prevention has been an important priority for the european commission . for this injury type , no biomechanically based vehicle safety regulations exist , mainly as a consequence of the limited ( or inconclusive ) knowledge available on whiplash . however , research has demonstrated that , in the event of a rear - end collision , the vehicle seat and head restraint are the principal means of reducing neck injury . starting from the assumption that lowering loads on the neck lessens the likelihood of whiplash injury , the first stand - alone tests for seats and head restraints were developed by the international insurance whiplash prevention group ( iiwpg ) and the swedish road administration ( sra ) . euro ncap set up a whiplash group in 2002 with the intention of developing a test that could complement the existing whole vehicle consumer crash tests . in 2008 , euro ncap had completed its work and formally launched the first series of results of ( front ) seat testing . by 2007 , the euro ncap ratings for adult protection , child protection and pedestrian protection had been in common use for a decade and had become internationally recognised as a reliable indicator of independent consumer information about car safety . but while this represented a significant step forwards for consumer protection , worries started to rise over the future of the programme . firstly , it was clear that the high share of cars rated 5 stars for adult protection was diminishing the discriminating factor in the rating with each advancing year leading , amongst other things , to less media interest in the results . at the same time , the successes in adult protection were hiding the less favourable progress in the other areas such as pedestrian protection from the view of the consumer . but it was the rating scheme 's inability to deal with emerging driver - assist and crash avoidance systems that ultimately triggered euro ncap to review and fundamentally overhaul its rating system . in 2009 , euro ncap changed from three individual ratings to a single overall safety rating with a maximum of 5 stars for each vehicle . this overall rating combined the results of assessments in four important areas : adult protection , child protection and pedestrian protection the three areas assessed in the previous scheme and the new area of safety assist technology . the underlying tests included the full - scale frontal offset , side - impact barrier and pole tests carried over from the previous adult and child protection ratings , the new seat tests for whiplash prevention in rear - end crashes , and front - end component tests for pedestrian protection . in each area of assessment , scores were calculated as a percentage of the maximum points available and a weighted sum of these scores indicated the car 's overall all - round performance . encompass all overall rating was established to provide a more balanced assessment of various vehicle safety aspects and to add more flexibility to the ratings scheme . hence , by moving to the overall rating scheme , euro ncap was able to promote integrated safety solutions , using both active and passive safety technology . with the overall rating scheme in place , euro ncap subsequently worked on a programme of stepwise updates to the rating scheme in following years . during this period , in addition , new functional tests were added , in particular related to crash avoidance and advanced driver assistance technologies . crash avoidance technology is aimed at reducing the likelihood of an accident happening or reducing its severity . already in 2005 , euro ncap gave a strong recommendation to consumers that they should specify esc on any new car purchase . from 2009 onwards , esc became an integral part of the overall rating , first on the basis only of fitment and later , in 2011 , on fitment and functional requirements verified by a series of sine - with - dwell track tests similar to those specified in the global technical regulation 8 . speed restrictions are intended to promote safe operation of the road network by keeping traffic speeds below the maximum that is appropriate for a given traffic environment . voluntary speed assistance systems ( sas ) are a means to assist drivers to adhere to speed limits , by warning and/or effectively limiting the speed of the vehicle . since 2009 , euro ncap has rewarded manually set speed limitation devices which meet the basic requirements of united nations regulation no . by doing so , euro ncap has created a first incentive to manufacturers to promote such speed - limitation devices , to make them available on more models and to fit them as standard equipment . in recent years more advanced speed assistance systems have been introduced onto the market which are able to inform the driver of the speed limit at the vehicle 's current position , based on digital speed maps and/or traffic sign recognition . as a result , euro ncap extended the speed limitation protocol in 2013 to include the latest generation of intelligent speed assistance systems . autonomous emergency braking ( aeb ) is without doubt the most important active safety systems that euro ncap has adopted since esc . the european commission sponsored research project assess ( assessment of integrated vehicle safety systems for improved vehicle safety ) and the german initiative led by dekra , called vfss ( advanced forward - looking safety systems ) had similar project goals to develop harmonized and standardized assessment procedures and related tools for selected integrated safety systems . the euro ncap aeb test and assessment procedures were born out of the deliverables of these projects . euro ncap adopted both low speed and high speed aeb systems in the rating scheme in 2014 . one of the most overlooked benefits of the consumer rating system in europe is its impact on availability and fitment of equipment across the eu markets . from the start , euro ncap tested the best - selling variant of a model in order to achieve the most relevant rating for the market as a whole . in 2007 , euro ncap therefore started to demand that all equipment relevant to the safety rating be fitted as standard or , in exceptional cases , meet a minimum fitment percentage over the whole of eu-28 . it has become the norm for new cars tested by euro ncap to be fitted with such systems , despite the fact that these are / were not mandated by law . recently , there has also been a rise in the number of mainstream models available with speed assistance and aeb and/or fcw systems . that is a welcome sign for road safety and helps pave the way for the eventual deployment of highly automated vehicles . during the first years of testing , much of the engineering effort went into achieving higher scores in the frontal and side impact crash tests . the reduction in passenger compartment intrusion in frontal impact is the most visible effect of euro ncap 's influence . by preventing intrusion , the likelihood of the occupant impacting the car 's interior is minimized , providing space for the restraint system to operate effectively . the provision of side impact and head airbags has helped and it is now normal for the cars to be fitted with side impact airbags and curtains . euro ncap has encouraged improved designs and the fitment of isofix mounts and child restraints . isofix provides a much more secure method of attaching the child restraint to the car , provided that additional provision is made to prevent rotation of the child restraint , which can be caused by seat cushion compression and rebound . as a consequence , euro ncap has seen improved designs , where the child is less likely to strike the car interior whilst at the same time experiencing reduced forces from the restraint system . one of the most remarkable effects of the introduction of the overall rating has been the improved score in pedestrian subsystem tests since 2009 ( fig . supported by an extensive review of protocols that has made the engineering goals more attainable and a soft landing approach in the rating calculation , car manufactures have delivered energy absorbing bumpers , deployable bonnets and external airbags and have repositioned stiff structures in order to boost their performance . although euro ncap 's ratings have been seen to improve over time , the only real proof of euro ncap 's effectiveness lies in real - world accident data . several analyses have been carried out that show the effect of improvements in vehicle safety , many of which have been influenced by euro ncap . in 2000 , cars with three or four adult occupant stars are approximately 30% safer , compared to two star cars or cars without a euro ncap score , in car - to - car collisions . data was also provided which showed that the predicted relative risk of severe or fatal injury was reduced by 12% , for each increase in euro ncap star rating . in 2001 , a monash university report for sarac reported that the euro ncap star rating is able to differentiate with statistical significance both the average crashworthiness and injury severity based on all real crashes of vehicles in star rating categories 1 , 2 and 4 . further evidence of the effectiveness of secondary safety improvements in the early years of euro ncap were reported in the journal accident analysis & prevention . around 80% ( 82.2% 8.6% ) of drivers not wearing a belt in cars with no seat belt reminder do so in cars equipped with a system that has a light signal and an associated loud and clear sound signal . in this study , the euro ncap pedestrian scoring was compared with the real - life outcome in pedestrian crashes that occurred in sweden 20032010 . the reduction of risk of serious consequences ( rsc ) for medium performing cars in comparison with low performing cars was 17% , 26% and 38% for 1% , 5% and 10% of medical impairment , respectively . kullgren et al carried out an evaluation of the euro ncap , japan ncap and iiwpg whiplash protocols using real - world crash data . correlations between the test scenarios of each of the three protocols , as well as the outcome associations with crash outcomes , suggested consistent improvements in the risk of permanent medical impairment . finally , euro ncap with support of the australasian ncap studied the effectiveness of low speed aeb systems promoted through the rating scheme since 2014 . the publication confirmed that autonomous emergency braking is one of the most promising safety technologies in recent years . the increasing globalisation of the car market and the rise of the emerging markets have seen the deployment of new safety rating initiatives in more regions of the world . both programmes , taking advantage of the technical know - how and procedures of euro and australasian ncap , have quickly gained momentum . the links between euro ncap and the new ncaps , australasian ncap and to a lesser extend japan ncap , korean ncap and china ncap , have increased euro ncap 's scope and are partly responsible for its evolution from a safety rating for the european market to a new cars today are much safer than they were a decade ago thanks to improved crash test standards , crumple zones , seat belts and airbags , which help to protect occupants in a crash . while most occupant safety measures can be considered mature , more could and should be done to improve their robustness and effectiveness for the general diversity of vehicle occupants and crash scenarios . today , the uptake of crash avoidance technology still poses a particular challenge : a large variety of systems is available but only a few are offered as standard . in the coming years , the need for more on - board technologies to support ( partial ) automated driving will probably make crash avoidance systems cheaper and more cost - effective across the european car fleet . for this , the vehicle rating must reflect the true contribution of passive and active safety measures to the overall safety performance . the lack of traceability of ( the performance of ) systems in the market , the complex role of driver behaviour and inconsistency in human machine interface ( hmi ) applied across industry , all further complicate the important task of identifying the true potential of avoidance technology . besides that , used in a manual way , the vehicle will always carry the safety elements and technologies to intervene when necessary . euro ncap plans to engage in the roll out of vehicle automation as a way to dramatically improve vehicle safety and safe driving . it will continue to promote best safety practice when vehicles start to have elements fitted which support automated driving and to ensure that the vehicle manufacturer remains responsible for safe operation of the system . in 2014 , euro ncap published a roadmap for further developments of the safety rating in the next 5 years . the plan identifies four main domains that focus on the key real life crash scenarios and that can be addressed by new and updated vehicle technology , in particular in the field of crash avoidance . this includes , amongst others , the addition of aeb technology for vulnerable road users ( pedestrians and cyclists ) and the testing of more capable driver support systems for longitudinal and lateral car crashes . moreover , it explains why euro ncap 's online and written publications continue to grow in popularity , not only with consumers but also more and more with public and private fleet managers to help them ensure that the safety of their vehicle fleet provides acceptable levels of protection to their employees . at the time of launch of the first euro ncap results in february 1997 , some critics claimed that the assessment criteria were so severe that no car could ever achieve ( the then maximum ) four stars for occupant protection . in july 1997 results from the second phase of tests were published and included the first 4 star results , for the volvo s40 . secondly , sufficient time does not always have to mean years , as many manufacturers have responded very quickly to new challenges in the past . a consumer rating system , like euro ncap , that is rooted firmly on real life experiences but which closely follows the technological innovations in the marketplace can therefore deliver the most benefit for society .

ischemic stroke ( is ) is a frequent pathology whose incidence sharply increases with age , 0.1/1,000 inhabitants per year below the age of 45 and up to 10/1,000 over 75 years of age in france . it is the third cause of death and is responsible for 50% of disabling conditions in survivors in industrialized countries . we suggested that a significant proportion of these unexplained strokes or transient ischemic attacks ( tia ) could be due to undiagnosed atrial fibrillation ( af ) . af is a common cause of embolism , but it can be difficult to identify due to asymptomatic or paroxysmal episodes [ 4 , 5 ] . some authors have advocated studying the atrial vulnerability ( av ) to arrhythmias to explain the mechanism of cryptogenic is [ 6 , 7 ] . in the presence of a low atrial vulnerability index or induction of af , oral anticoagulation and antiarrhythmic therapy were recommended by these same authors , but the predictive value of this electrophysiological method for spontaneous af remains controversial [ 8 , 9 ] . we therefore aimed to document spontaneous af in a population of patients with unexplained is / tia , using invasive continuous cardiac monitoring by means of an implantable loop recorder ( ilr ) . this device , widely used for diagnosis of unexplained syncope , has proven effective in detecting paroxysmal arrhythmias [ 11 , 12 ] . we also wanted to correlate the results of continuous electrocardiogram monitoring with those of av electrophysiological studies in order to test the value of this method for predicting spontaneous af . this is a prospective multicenter study whose aim was to determine the prevalence of af in patients under 75 years of age with a history of is or tia of undetermined cause after complete evaluation . the enrollment took place in the cardiology or neurology units of four hospitals in france . patients were to be between 18 and 75 years old , have a history of cerebral ischemia within 4 months before inclusion , and have recovered neurological status allowing good autonomy ( rankin score between 0 and 2 at inclusion ) . the neurological features of each patient were reviewed by the referent neurologist of the study , who validated the diagnosis of is / tia from anamnesis , clinical presentation , and the results of brain imaging . all patients underwent at least cerebral ct or mri , or both , depending on the equipment of the recruiting centre . undetermined origin was ascertained by negative evaluation requiring ancillary tests performed in all patients to exclude the following conditions : ipsilateral carotid atherosclerotic stenosis > 70% , vertebral or carotid dissection with cervical doppler - echography or angio-mr.documented atrial fibrillation , with a 12-lead electrocardiogram and 24-h holter monitoring.intracardiac thrombus or any other cause of cardiac embolism , with transthoracic ( n = 24/24 ) and transesophageal echocardiography ( n = 22/24).vascular malformation , by means of cerebral ct , mri or angio - mri if necessary.hypercoagulable states , hematological disorders , and inflammatory diseases , with biological tests carried out at least 2 months after stroke : prothrombin ratio , activated partial thromboplastin time , c and s proteins , antithrombin , mutation in factor v leiden gene , circulating anticoagulants , antiphospholipid and anti - 2gp1 antibodies , homocysteinemia , erythrocyte sedimentation rate , and c - reactive protein . ipsilateral carotid atherosclerotic stenosis > 70% , vertebral or carotid dissection with cervical doppler - echography or angio - mr . intracardiac thrombus or any other cause of cardiac embolism , with transthoracic ( n = 24/24 ) and transesophageal echocardiography ( n = 22/24 ) . vascular malformation , by means of cerebral ct , mri or angio - mri if necessary . hypercoagulable states , hematological disorders , and inflammatory diseases , with biological tests carried out at least 2 months after stroke : prothrombin ratio , activated partial thromboplastin time , c and s proteins , antithrombin , mutation in factor v leiden gene , circulating anticoagulants , antiphospholipid and anti - 2gp1 antibodies , homocysteinemia , erythrocyte sedimentation rate , and c - reactive protein . a history of documented cardiac arrhythmias and ineffectively controlled pre - stroke hypertension ( defined by systolic blood pressure > 140 mmhg or diastolic > 90 mmhg ) were also exclusion criteria . the study protocol resulted from a regional 2003 hospital program of clinical research promoted by the university hospital of tours and received approval from the institutional ethics committee ( ccpprb of tours ) on june 29 , 2004 . the electrophysiological study was performed using low - molecular - weight heparin for effective anticoagulation . two quadripolar electrode catheters ( 10-mm interelectrode distance , usci , bard ) were placed in the right cardiac cavities via catheterization of the right femoral vein under local anesthesia . one quadripolar electrode was positioned in the high right atrium ( hra ) in the sinus node area ; the other one was placed in the low right atrium ( lra ) near the atrioventricular node . pacing was performed with a programmable stimulator ( biotronik uhs , germany ) which delivered rectangular pulses of 2-ms duration at twice the diastolic threshold . the study was carried out in three stages : measurement of basic conduction intervals and determination of the wenckebach point of the atrioventricular node.determination of the right atrium refractory periods ( rp ) and the duration of the atrial electrogram . rps were determined by means of an extrastimulus ( s2 ) delivered at three different constant pacing rates 100 , 120 , 150 bpm . effective rp was defined as the longest attainable s1s2 interval that did not produce an atrial electrogram . finally , we measured the duration of the atrial electrogram ( a2 ) following s2 at a pacing rate of 100 bpm . latent vulnerability index ( lvi ) was defined as the ratio between high right atrium effective refractory period ( erp ) and a2 duration at a pacing rate of 100 bpm [ 7 , 14].assessment of the inducibility of atrial arrhythmia was performed by programmed atrial stimulation with up to three extrastimuli ( s2 , s3 , s4 ) at constant pacing rates of 100 , 120 , and 150 bpm at two different stimulation sites : hra and lra . measurement of basic conduction intervals and determination of the wenckebach point of the atrioventricular node . determination of the right atrium refractory periods ( rp ) and the duration of the atrial electrogram . rps were determined by means of an extrastimulus ( s2 ) delivered at three different constant pacing rates 100 , 120 , 150 bpm . effective rp was defined as the longest attainable s1s2 interval that did not produce an atrial electrogram . finally , we measured the duration of the atrial electrogram ( a2 ) following s2 at a pacing rate of 100 bpm . latent vulnerability index ( lvi ) was defined as the ratio between high right atrium effective refractory period ( erp ) and a2 duration at a pacing rate of 100 bpm [ 7 , 14 ] . assessment of the inducibility of atrial arrhythmia was performed by programmed atrial stimulation with up to three extrastimuli ( s2 , s3 , s4 ) at constant pacing rates of 100 , 120 , and 150 bpm at two different stimulation sites : hra and lra . latent atrial vulnerability was defined as lvi < 2.5 , and atrial inducibility as induction of sustained atrial arrhythmia lasting > 60 s [ 7 , 14 ] . minneapolis , usa ) is a small single - use device fitted with two sensing electrodes . it can record the heartbeat loop for 14 months . when interrogated transcutaneously , it can return recorded rhythmic events . the ilr was implanted subcutaneously under local anesthesia and antibiotic prophylaxis in the left pectoral region . the device was programmed similarly for all patients before their discharge : semi - automatic activation mode.recording of 13 automatic events and 1 event by patient activation . patients were provided with an activator at the time of device implantation.the criteria for automatic recording were bradycardia < 30 bpm or pause > 3 s , tachycardia > 165 bpm for more than 32 complexes . the criteria for automatic recording were bradycardia < 30 bpm or pause > 3 s , tachycardia > 165 bpm for more than 32 complexes . patients were instructed to activate the device in case of palpitations , syncope , or recurrence of is / tia . all patients received antiplatelet therapy , aspirin 300 mg / day , or clopidogrel 75 mg / day . patients underwent examination and 12-lead ecg every 4 months , performed by the investigator of the recruiting hospital . interrogation of the ilr was carried out at each visit and stored for review by the principal investigator . af was considered to have occurred when normal sinus rhythm was replaced by irregular tachycardia lasting more than 30 s with no visible p wave or with unorganized f wavelets of af . in this case , antiarrhythmic therapy was prescribed according to the habitual procedure of each investigator , and antiplatelet therapy was replaced by oral anticoagulation treatment with a target international normalized ratio ( inr ) between two and three . in the event of a serious issue ( recurrence of stroke , syncope , death ) , the study ended for a patient when sustained af was documented by the monitor or after the monitoring period of 14 months with the device 's explantation ( end of battery life defined by the manufacturer of the reveal plus ilr 9526 ) . the enrollment took place in the cardiology or neurology units of four hospitals in france . patients were to be between 18 and 75 years old , have a history of cerebral ischemia within 4 months before inclusion , and have recovered neurological status allowing good autonomy ( rankin score between 0 and 2 at inclusion ) . the neurological features of each patient were reviewed by the referent neurologist of the study , who validated the diagnosis of is / tia from anamnesis , clinical presentation , and the results of brain imaging . all patients underwent at least cerebral ct or mri , or both , depending on the equipment of the recruiting centre . undetermined origin was ascertained by negative evaluation requiring ancillary tests performed in all patients to exclude the following conditions : ipsilateral carotid atherosclerotic stenosis > 70% , vertebral or carotid dissection with cervical doppler - echography or angio-mr.documented atrial fibrillation , with a 12-lead electrocardiogram and 24-h holter monitoring.intracardiac thrombus or any other cause of cardiac embolism , with transthoracic ( n = 24/24 ) and transesophageal echocardiography ( n = 22/24).vascular malformation , by means of cerebral ct , mri or angio - mri if necessary.hypercoagulable states , hematological disorders , and inflammatory diseases , with biological tests carried out at least 2 months after stroke : prothrombin ratio , activated partial thromboplastin time , c and s proteins , antithrombin , mutation in factor v leiden gene , circulating anticoagulants , antiphospholipid and anti - 2gp1 antibodies , homocysteinemia , erythrocyte sedimentation rate , and c - reactive protein . ipsilateral carotid atherosclerotic stenosis > 70% , vertebral or carotid dissection with cervical doppler - echography or angio - mr . intracardiac thrombus or any other cause of cardiac embolism , with transthoracic ( n = 24/24 ) and transesophageal echocardiography ( n = 22/24 ) . vascular malformation , by means of cerebral ct , mri or angio - mri if necessary . hypercoagulable states , hematological disorders , and inflammatory diseases , with biological tests carried out at least 2 months after stroke : prothrombin ratio , activated partial thromboplastin time , c and s proteins , antithrombin , mutation in factor v leiden gene , circulating anticoagulants , antiphospholipid and anti - 2gp1 antibodies , homocysteinemia , erythrocyte sedimentation rate , and c - reactive protein . a history of documented cardiac arrhythmias and ineffectively controlled pre - stroke hypertension ( defined by systolic blood pressure > 140 mmhg or diastolic > 90 mmhg ) were also exclusion criteria . the study protocol resulted from a regional 2003 hospital program of clinical research promoted by the university hospital of tours and received approval from the institutional ethics committee ( ccpprb of tours ) on june 29 , 2004 . the electrophysiological study was performed using low - molecular - weight heparin for effective anticoagulation . two quadripolar electrode catheters ( 10-mm interelectrode distance , usci , bard ) were placed in the right cardiac cavities via catheterization of the right femoral vein under local anesthesia . one quadripolar electrode was positioned in the high right atrium ( hra ) in the sinus node area ; the other one was placed in the low right atrium ( lra ) near the atrioventricular node . pacing was performed with a programmable stimulator ( biotronik uhs , germany ) which delivered rectangular pulses of 2-ms duration at twice the diastolic threshold . the study was carried out in three stages : measurement of basic conduction intervals and determination of the wenckebach point of the atrioventricular node.determination of the right atrium refractory periods ( rp ) and the duration of the atrial electrogram . rps were determined by means of an extrastimulus ( s2 ) delivered at three different constant pacing rates 100 , 120 , 150 bpm . effective rp was defined as the longest attainable s1s2 interval that did not produce an atrial electrogram . finally , we measured the duration of the atrial electrogram ( a2 ) following s2 at a pacing rate of 100 bpm . latent vulnerability index ( lvi ) was defined as the ratio between high right atrium effective refractory period ( erp ) and a2 duration at a pacing rate of 100 bpm [ 7 , 14].assessment of the inducibility of atrial arrhythmia was performed by programmed atrial stimulation with up to three extrastimuli ( s2 , s3 , s4 ) at constant pacing rates of 100 , 120 , and 150 bpm at two different stimulation sites : hra and lra . measurement of basic conduction intervals and determination of the wenckebach point of the atrioventricular node . determination of the right atrium refractory periods ( rp ) and the duration of the atrial electrogram . rps were determined by means of an extrastimulus ( s2 ) delivered at three different constant pacing rates 100 , 120 , 150 bpm . effective rp was defined as the longest attainable s1s2 interval that did not produce an atrial electrogram . finally , we measured the duration of the atrial electrogram ( a2 ) following s2 at a pacing rate of 100 bpm . latent vulnerability index ( lvi ) was defined as the ratio between high right atrium effective refractory period ( erp ) and a2 duration at a pacing rate of 100 bpm [ 7 , 14 ] . assessment of the inducibility of atrial arrhythmia was performed by programmed atrial stimulation with up to three extrastimuli ( s2 , s3 , s4 ) at constant pacing rates of 100 , 120 , and 150 bpm at two different stimulation sites : hra and lra . latent atrial vulnerability was defined as lvi < 2.5 , and atrial inducibility as induction of sustained atrial arrhythmia lasting > 60 s [ 7 , 14 ] . minneapolis , usa ) is a small single - use device fitted with two sensing electrodes . it can record the heartbeat loop for 14 months . when interrogated transcutaneously , it can return recorded rhythmic events . the ilr was implanted subcutaneously under local anesthesia and antibiotic prophylaxis in the left pectoral region . the device was programmed similarly for all patients before their discharge : semi - automatic activation mode.recording of 13 automatic events and 1 event by patient activation . patients were provided with an activator at the time of device implantation.the criteria for automatic recording were bradycardia < 30 bpm or pause > 3 s , tachycardia > 165 bpm for more than 32 complexes . the criteria for automatic recording were bradycardia < 30 bpm or pause > 3 s , tachycardia > 165 bpm for more than 32 complexes . patients were instructed to activate the device in case of palpitations , syncope , or recurrence of is / tia . all patients received antiplatelet therapy , aspirin 300 mg / day , or clopidogrel 75 mg / day . patients underwent examination and 12-lead ecg every 4 months , performed by the investigator of the recruiting hospital . interrogation of the ilr was carried out at each visit and stored for review by the principal investigator . af was considered to have occurred when normal sinus rhythm was replaced by irregular tachycardia lasting more than 30 s with no visible p wave or with unorganized f wavelets of af . in this case , antiarrhythmic therapy was prescribed according to the habitual procedure of each investigator , and antiplatelet therapy was replaced by oral anticoagulation treatment with a target international normalized ratio ( inr ) between two and three . in the event of a serious issue ( recurrence of stroke , syncope , death ) , the study ended for a patient when sustained af was documented by the monitor or after the monitoring period of 14 months with the device 's explantation ( end of battery life defined by the manufacturer of the reveal plus ilr 9526 ) . twenty - four patients were included , 15 men and 9 women , from 19 to 74 years old , mean age 48.8 13.6 years . although 50 patients were provided , inclusions were stopped prematurely because of negative results . table 1characteristics of subjects ( n = 24)male n ( % ) 15 ( 62.5)age ( years ) ( mean sd)49 13.6<50 years n ( % ) 14 ( 58.3)diabetes mellitus0hypertension n ( % ) 7 ( 29.2)dyslipidemia n ( % ) 8 ( 33.3)active tobacco use n ( % ) 10 ( 41.7)overweight ( bmi > 27.5 ) n ( % ) 4 ( 16.7)vascular ischemic hereditary n ( % ) 5 ( 20.8 ) characteristics of subjects ( n = 24 ) of the 24 enrolled patients , 3 ( 12.5% ) experienced tia with symptoms lasting less than 1 h ( and normal brain mri ) , and 21 ( 87.5% ) experienced cerebral infarction ; one of them remained with neurological sequelae ( rankin score = 2 ) , symptoms were fully reversible in all the others . the clinical presentation consisted of lateralized motor deficit or aphasia in 75% of the cases , with or without sensory symptoms all patients underwent at least one brain imaging , cerebral ct ( n = 17 ) or mri ( n = 15 ) or both ( n = 8) , and four patients had an angio - mri performed . the most common types of infarcts were superficial middle cerebral artery territory infarcts ( 58.3% ) . one in three patients showed at least two concomitant ischemic locations , lesions were bilateral in 20.8% of cases . table 2neurological features : topography of cerebral infarcts ( n = 24)topography of infarctsnumberpercentagemiddle cerebral artery ( mca ) territory2083.3 superficial mca1458.3 deep mca312.5 global mca312.5vertebrobasilar territory729.2 posterior cerebral artery territory312.5 cerebellum416.7multiple lesions833.3 bilateral520.8 neurological features : topography of cerebral infarcts ( n = 24 ) the results are shown in table 3 . we observed 18 patients with supraventricular arrhythmias , 15 af , 1 common flutter , and 2 other atrial tachycardias . eight of these , termed inducible patients , experienced sustained arrhythmia , lasting more than 60 s. the lvi was calculated whenever possible with the formula described above . it could not be calculated in four cases because of the impossibility of measuring rp or a2 duration due to early onset of sustained arrhythmia . latent av was found in nine patients ( 37.5% ) , among whom , three were inducible . table 3electrophysiological study results ( n = 24 ) mean sdminmaxnumber ( % ) ah interval ( ms)83 22.415125hv interval ( ms)43 9.383065wenckebach point of av node ( bpm)162 35.2110230hra erp ( ms)234 37.1180290a2 duration ( ms)85 26.450140lvi ( n = 20)2.9 1.021.55.6lvi < 2.59 ( 37.5)inducibility of atrial arrhythmia ( including af , flutter , and other atrial tachycardias)18 ( 75.0 ) 030 s8 ( 33.3 ) 3059 s2 ( 8.3 ) 15 min5 ( 20.8 ) > 5 min3 ( 12.5 ) electrophysiological study results ( n = 24 ) the mean time lapse from cerebral accident to initiation of cardiac rhythm monitoring , i.e. , implantation of the ilr , was 3 1.1 months . there was no recurrence of stroke , no episode of syncope or palpitation was reported , and all electrocardiograms were in sinus rhythm . through ilr interrogation , we found short af episodes < 30 s in one patient ( 4.2% ) , detected by automatic record , with a ventricular rate exceeding 200 bpm . this patient was not inducible through programmed atrial stimulation despite a positive latent vulnerability ( lvi = 1.5 ) . no significant af episode or any other sustained arrhythmia was identified , either in inducible patients or in the others . moreover , no significant sinus pause or sinus bradycardia was observed that could have led to a diagnosis of bradycardia tachycardia syndrome . of the 24 enrolled patients , 3 ( 12.5% ) experienced tia with symptoms lasting less than 1 h ( and normal brain mri ) , and 21 ( 87.5% ) experienced cerebral infarction ; one of them remained with neurological sequelae ( rankin score = 2 ) , symptoms were fully reversible in all the others . the clinical presentation consisted of lateralized motor deficit or aphasia in 75% of the cases , with or without sensory symptoms . all patients underwent at least one brain imaging , cerebral ct ( n = 17 ) or mri ( n = 15 ) or both ( n = 8) , and four patients had an angio - mri performed . the most common types of infarcts were superficial middle cerebral artery territory infarcts ( 58.3% ) . one in three patients showed at least two concomitant ischemic locations , lesions were bilateral in 20.8% of cases . table 2neurological features : topography of cerebral infarcts ( n = 24)topography of infarctsnumberpercentagemiddle cerebral artery ( mca ) territory2083.3 superficial mca1458.3 deep mca312.5 global mca312.5vertebrobasilar territory729.2 posterior cerebral artery territory312.5 cerebellum416.7multiple lesions833.3 bilateral520.8 neurological features : topography of cerebral infarcts ( n = 24 ) we observed 18 patients with supraventricular arrhythmias , 15 af , 1 common flutter , and 2 other atrial tachycardias . eight of these , termed inducible patients , experienced sustained arrhythmia , lasting more than 60 s. the lvi was calculated whenever possible with the formula described above . it could not be calculated in four cases because of the impossibility of measuring rp or a2 duration due to early onset of sustained arrhythmia . latent av was found in nine patients ( 37.5% ) , among whom , three were inducible . table 3electrophysiological study results ( n = 24 ) mean sdminmaxnumber ( % ) ah interval ( ms)83 22.415125hv interval ( ms)43 9.383065wenckebach point of av node ( bpm)162 35.2110230hra erp ( ms)234 37.1180290a2 duration ( ms)85 26.450140lvi ( n = 20)2.9 1.021.55.6lvi < 2.59 ( 37.5)inducibility of atrial arrhythmia ( including af , flutter , and other atrial tachycardias)18 ( 75.0 ) 030 s8 ( 33.3 ) 3059 s2 ( 8.3 ) 15 min5 ( 20.8 ) > 5 min3 ( 12.5 ) electrophysiological study results ( n = 24 ) the mean time lapse from cerebral accident to initiation of cardiac rhythm monitoring , i.e. , implantation of the ilr , was 3 1.1 months . there was no recurrence of stroke , no episode of syncope or palpitation was reported , and all electrocardiograms were in sinus rhythm . through ilr interrogation , we found short af episodes < 30 s in one patient ( 4.2% ) , detected by automatic record , with a ventricular rate exceeding 200 bpm . this patient was not inducible through programmed atrial stimulation despite a positive latent vulnerability ( lvi = 1.5 ) . no significant af episode or any other sustained arrhythmia was identified , either in inducible patients or in the others . moreover , no significant sinus pause or sinus bradycardia was observed that could have led to a diagnosis of bradycardia tachycardia syndrome . because of the high recurrence rate of ischemic cerebral accidents , secondary prevention after a first - ever event is essential . despite an extensive evaluation , no determined etiology is found in one third to half of all cases , especially in young subjects . it is currently estimated that 30% of is have a cardioembolic substrate , about half of which are due to af . af is a very common arrhythmia whose prevalence increases with age up to 10% after the age of 75 versus 0.4% in the general population . arterial embolism is a frequent complication of af , with a risk of 5% to 10% per year , especially in subjects with cardiovascular risk factors ( hypertension , diabetes , heart failure ) [ 17 , 18 ] . identifying a potential cardiac source of embolism is of critical importance in the etiological workup of is because of therapeutic and prognostic implications . epidemiological studies have shown a poorer prognosis for is / tia of cardioembolic origin than from other causes , especially those due to af [ 3 , 19 , 20 ] . if af is identified as the cause of a first - ever cerebral infarction , anticoagulation therapy significantly reduces the recurrence rate as compared with antiplatelet therapy and dramatically improves these patients ' prognosis [ 17 , 2123 ] . the yield is low for 12-lead ecgs , as well as for 24- to 72-h holter monitoring ( 2% to 5% ) [ 24 , 25 ] . preliminary studies suggested that extending the duration of heart rate monitoring would increase the probability of detecting paroxysmal af [ 2527 ] . the ilr offers the benefit of continuously monitoring cardiac rhythm over several months . in our study , cerebral ischemic accidents were suspected to be of cardioembolic mechanism , as evidenced by the frequency of multiple , especially bilateral lesions . nevertheless , among the 24 enrolled patients , af was detected as a potential cause of stroke in only one patient . is / tia , the authors found a higher detection rate of af using 21-day monitoring with mobile cardiac outpatient telemetry , 5.3% of af > 30 s and 23% of short episodes < 30 s. we can assume that the difference is due to patient selection and time of initiation of the monitoring . in the tayal study , the patients were at higher risk of af because they were older ( mean age 66 11 versus 49 13 years in our study ) , and the prevalence of diabetes was high . age and diabetes mellitus are well - known risk factors for af [ 16 , 29 ] . it is to be noticed that diabetics were absent from our population . in the tayal study , rhythm monitoring after index event was initiated earlier than in ours ( mean 20 days versus 3 months ) . in their work , the majority of af were diagnosed within the first days of monitoring ( mean 7 days , range 219 ) . however , early transient af has been reported after acute stroke only as a consequence of the stroke , which led us purposely to choose to delay the initiation of monitoring in order to avoid confusion as to the responsibility of the stroke for the onset of paroxysmal arrhythmia . the aim of the av study was to identify subjects with a high risk of atrial arrhythmia . it has been shown that patients with documented af more often have a low lvi and are more easily inducible than patients without af [ 8 , 15 ] . some authors have described similar electrophysiological abnormalities in patients with paroxysmal af and in patients with unexplained ischemic stroke [ 8 , 31 ] . however , the correlation between the presence of an arrhythmogenic substrate and the occurrence of spontaneous af has not been proven . in previous series , the percentage of inducible sustained af in patients with cryptogenic is was 50% to 65% [ 6 , 8 ] . we also found a high rate of inducible sustained atrial arrhythmias ( 33.3% ) and a high rate of latent av ( 37.5% ) , whereas no spontaneous sustained asymptomatic af or af with fast ventricular response was documented over a period of 14 months . moreover , the only patient who experienced short episodes of spontaneous af was not inducible by programmed atrial stimulation . for the first time , continuous monitoring in a prospective study has made it possible to confirm the irrelevance of electrophysiological study in the etiological workup of is / tia , because neither low lvi nor atrial arrhythmia inducibility was predictive of spontaneous af . firstly , the duration of the study was shortened by the device 's limited battery life . rhythm monitoring was thus limited to 14 months , which is brief compared to the period of stroke recurrences . as epidemiological data in the literature reveal that the risk of recurrent stroke is high in the early phase after a first - ever event and remains high for several years thereafter [ 19 , 32 , 33 ] , there is little prospect that the new generation of ilr with a prolonged battery life ( more than 36 months ) would be more informative . the algorithm required 32 consecutive beats at 165 bpm to recognize a rhythm disturbance as an af episode . it was consequently programmed to record af with rapid ventricular response , making it likely that a number of non - rapid episodes may have been missed . regarding self - activation , previous studies have showed the inability of some patients to operate loop recorders properly . this was not a problem in our study because no triggering symptom was experienced resulting from patients ' failure to activate . in the future , the ilr will be designed to record atrial activity more accurately in order to diagnose af with lower ventricular response . the latest model of ilr ( reveal xt , medtronic inc . , minneapolis , usa ) seems to meet this objective , but the device is not yet available in france . finally , it might be objected that a major limitation lies in the limited size of the study , given the low number of 24 subjects included . patients ' inclusion was hampered by difficulties in getting them to accept the protocol , particularly because of the invasiveness of procedures . inclusion was limited to 24 patients for reasons of ethical considerations and cost effectiveness on the grounds that the absence of sustained af in this population made the diagnostic method uneconomical . we suspect that the efficacy of the method could be improved by applying it to a selected population at higher risk of af , specifically including more diabetics and older patients . because of the high recurrence rate of ischemic cerebral accidents , secondary prevention after a first - ever event is essential . despite an extensive evaluation , no determined etiology is found in one third to half of all cases , especially in young subjects . it is currently estimated that 30% of is have a cardioembolic substrate , about half of which are due to af . af is a very common arrhythmia whose prevalence increases with age up to 10% after the age of 75 versus 0.4% in the general population . arterial embolism is a frequent complication of af , with a risk of 5% to 10% per year , especially in subjects with cardiovascular risk factors ( hypertension , diabetes , heart failure ) [ 17 , 18 ] . identifying a potential cardiac source of embolism is of critical importance in the etiological workup of is because of therapeutic and prognostic implications . epidemiological studies have shown a poorer prognosis for is / tia of cardioembolic origin than from other causes , especially those due to af [ 3 , 19 , 20 ] . if af is identified as the cause of a first - ever cerebral infarction , anticoagulation therapy significantly reduces the recurrence rate as compared with antiplatelet therapy and dramatically improves these patients ' prognosis [ 17 , 2123 ] . the yield is low for 12-lead ecgs , as well as for 24- to 72-h holter monitoring ( 2% to 5% ) [ 24 , 25 ] . preliminary studies suggested that extending the duration of heart rate monitoring would increase the probability of detecting paroxysmal af [ 2527 ] . the ilr offers the benefit of continuously monitoring cardiac rhythm over several months . in our study , cerebral ischemic accidents were suspected to be of cardioembolic mechanism , as evidenced by the frequency of multiple , especially bilateral lesions . nevertheless , among the 24 enrolled patients , af was detected as a potential cause of stroke in only one patient . is / tia , the authors found a higher detection rate of af using 21-day monitoring with mobile cardiac outpatient telemetry , 5.3% of af > 30 s and 23% of short episodes < 30 s. we can assume that the difference is due to patient selection and time of initiation of the monitoring . in the tayal study , the patients were at higher risk of af because they were older ( mean age 66 11 versus 49 13 years in our study ) , and the prevalence of diabetes was high . age and diabetes mellitus are well - known risk factors for af [ 16 , 29 ] . it is to be noticed that diabetics were absent from our population . in the tayal study , rhythm monitoring after index event was initiated earlier than in ours ( mean 20 days versus 3 months ) . in their work , the majority of af were diagnosed within the first days of monitoring ( mean 7 days , range 219 ) . however , early transient af has been reported after acute stroke only as a consequence of the stroke , which led us purposely to choose to delay the initiation of monitoring in order to avoid confusion as to the responsibility of the stroke for the onset of paroxysmal arrhythmia . the aim of the av study was to identify subjects with a high risk of atrial arrhythmia . it has been shown that patients with documented af more often have a low lvi and are more easily inducible than patients without af [ 8 , 15 ] . some authors have described similar electrophysiological abnormalities in patients with paroxysmal af and in patients with unexplained ischemic stroke [ 8 , 31 ] . however , the correlation between the presence of an arrhythmogenic substrate and the occurrence of spontaneous af has not been proven . in previous series , the percentage of inducible sustained af in patients with cryptogenic is was 50% to 65% [ 6 , 8 ] . we also found a high rate of inducible sustained atrial arrhythmias ( 33.3% ) and a high rate of latent av ( 37.5% ) , whereas no spontaneous sustained asymptomatic af or af with fast ventricular response was documented over a period of 14 months . moreover , the only patient who experienced short episodes of spontaneous af was not inducible by programmed atrial stimulation . for the first time , continuous monitoring in a prospective study has made it possible to confirm the irrelevance of electrophysiological study in the etiological workup of is / tia , because neither low lvi nor atrial arrhythmia inducibility was predictive of spontaneous af . firstly , the duration of the study was shortened by the device 's limited battery life . rhythm monitoring was thus limited to 14 months , which is brief compared to the period of stroke recurrences . as epidemiological data in the literature reveal that the risk of recurrent stroke is high in the early phase after a first - ever event and remains high for several years thereafter [ 19 , 32 , 33 ] , there is little prospect that the new generation of ilr with a prolonged battery life ( more than 36 months ) would be more informative . the algorithm required 32 consecutive beats at 165 bpm to recognize a rhythm disturbance as an af episode . it was consequently programmed to record af with rapid ventricular response , making it likely that a number of non - rapid episodes may have been missed . regarding self - activation , this was not a problem in our study because no triggering symptom was experienced resulting from patients ' failure to activate . in the future , the ilr will be designed to record atrial activity more accurately in order to diagnose af with lower ventricular response . the latest model of ilr ( reveal xt , medtronic inc . , minneapolis , usa ) seems to meet this objective , but the device is not yet available in france . finally , it might be objected that a major limitation lies in the limited size of the study , given the low number of 24 subjects included . patients ' inclusion was hampered by difficulties in getting them to accept the protocol , particularly because of the invasiveness of procedures . inclusion was limited to 24 patients for reasons of ethical considerations and cost effectiveness on the grounds that the absence of sustained af in this population made the diagnostic method uneconomical . we suspect that the efficacy of the method could be improved by applying it to a selected population at higher risk of af , specifically including more diabetics and older patients . this study suggests that symptomatic atrial fibrillation or af with fast ventricular rate is not a frequent pathology in patients under the age of 75 with unexplained ischemic stroke . these results do not plead for generalizing the use of an implantable loop recorder in the systematic evaluation of these patients . further studies are needed to test the efficacy and cost effectiveness of the method in a selected population with a higher risk of atrial fibrillation , using an updated device that more accurately diagnoses af with low ventricular response . finally , this study asserts that the electrophysiological study of atrial vulnerability is poor at predicting spontaneous atrial fibrillation after cryptogenic cerebral ischemia .

purposeischemic stroke is a frequent pathology with high rate of recurrence and significant morbidity and mortality . there are several causes of stroke , affecting prognosis , outcomes , and management , but in many cases , the etiology remains undetermined . we hypothesized that atrial fibrillation was involved in this pathology but underdiagnosed by standard methods . the aim of the study was to determine the incidence of atrial fibrillation in cryptogenic ischemic stroke by using continuous monitoring of the heart rate over several months . the secondary objective was to test the value of atrial vulnerability assessment in predicting spontaneous atrial fibrillation.methods and resultswe prospectively enrolled 24 patients under 75 years of age , 15 men and 9 women of mean age 49 years , who within the last 4 months had experienced cryptogenic stroke diagnosed by clinical presentation and brain imaging and presumed to be of cardioembolic mechanism . all causes of stroke were excluded by normal 12-lead ecg , 24-h holter monitoring , echocardiography , cervical doppler , hematological , and inflammatory tests . all patients underwent electrophysiological study . of the patients , 37.5% had latent atrial vulnerability , and 33.3% had inducible sustained arrhythmia . patients were secondarily implanted with an implantable loop recorder to look for spontaneous atrial fibrillation over a mean follow - up interval of 14.5 months . no sustained arrhythmia was found . only one patient had non - significant episodes of atrial fibrillation.conclusionin this study , symptomatic atrial fibrillation or af with fast ventricular rate has not been demonstrated by the implantable loop recorder in patients under 75 years with unexplained cerebral ischemia . the use of this device should not be generalized in the systematic evaluation of these patients . in addition , this study attests that the assessment of atrial vulnerability is poor at predicting spontaneous arrhythmia in such patients .

Introduction Patients and methods Patient enrollment Study of atrial vulnerability Implantation of Holter monitor Treatment Patient follow-up Results Neurological features Electrophysiological study Implantable ECG monitor Discussion Atrial fibrillation and stroke Atrial vulnerability Limitations of the study Conclusion

ischemic stroke ( is ) is a frequent pathology whose incidence sharply increases with age , 0.1/1,000 inhabitants per year below the age of 45 and up to 10/1,000 over 75 years of age in france . in the presence of a low atrial vulnerability index or induction of af , oral anticoagulation and antiarrhythmic therapy were recommended by these same authors , but the predictive value of this electrophysiological method for spontaneous af remains controversial [ 8 , 9 ] . we therefore aimed to document spontaneous af in a population of patients with unexplained is / tia , using invasive continuous cardiac monitoring by means of an implantable loop recorder ( ilr ) . we also wanted to correlate the results of continuous electrocardiogram monitoring with those of av electrophysiological studies in order to test the value of this method for predicting spontaneous af . this is a prospective multicenter study whose aim was to determine the prevalence of af in patients under 75 years of age with a history of is or tia of undetermined cause after complete evaluation . patients were to be between 18 and 75 years old , have a history of cerebral ischemia within 4 months before inclusion , and have recovered neurological status allowing good autonomy ( rankin score between 0 and 2 at inclusion ) . the neurological features of each patient were reviewed by the referent neurologist of the study , who validated the diagnosis of is / tia from anamnesis , clinical presentation , and the results of brain imaging . all patients underwent at least cerebral ct or mri , or both , depending on the equipment of the recruiting centre . undetermined origin was ascertained by negative evaluation requiring ancillary tests performed in all patients to exclude the following conditions : ipsilateral carotid atherosclerotic stenosis > 70% , vertebral or carotid dissection with cervical doppler - echography or angio-mr.documented atrial fibrillation , with a 12-lead electrocardiogram and 24-h holter monitoring.intracardiac thrombus or any other cause of cardiac embolism , with transthoracic ( n = 24/24 ) and transesophageal echocardiography ( n = 22/24).vascular malformation , by means of cerebral ct , mri or angio - mri if necessary.hypercoagulable states , hematological disorders , and inflammatory diseases , with biological tests carried out at least 2 months after stroke : prothrombin ratio , activated partial thromboplastin time , c and s proteins , antithrombin , mutation in factor v leiden gene , circulating anticoagulants , antiphospholipid and anti - 2gp1 antibodies , homocysteinemia , erythrocyte sedimentation rate , and c - reactive protein . the study was carried out in three stages : measurement of basic conduction intervals and determination of the wenckebach point of the atrioventricular node.determination of the right atrium refractory periods ( rp ) and the duration of the atrial electrogram . latent vulnerability index ( lvi ) was defined as the ratio between high right atrium effective refractory period ( erp ) and a2 duration at a pacing rate of 100 bpm [ 7 , 14].assessment of the inducibility of atrial arrhythmia was performed by programmed atrial stimulation with up to three extrastimuli ( s2 , s3 , s4 ) at constant pacing rates of 100 , 120 , and 150 bpm at two different stimulation sites : hra and lra . assessment of the inducibility of atrial arrhythmia was performed by programmed atrial stimulation with up to three extrastimuli ( s2 , s3 , s4 ) at constant pacing rates of 100 , 120 , and 150 bpm at two different stimulation sites : hra and lra . latent atrial vulnerability was defined as lvi < 2.5 , and atrial inducibility as induction of sustained atrial arrhythmia lasting > 60 s [ 7 , 14 ] . patients underwent examination and 12-lead ecg every 4 months , performed by the investigator of the recruiting hospital . in the event of a serious issue ( recurrence of stroke , syncope , death ) , the study ended for a patient when sustained af was documented by the monitor or after the monitoring period of 14 months with the device 's explantation ( end of battery life defined by the manufacturer of the reveal plus ilr 9526 ) . patients were to be between 18 and 75 years old , have a history of cerebral ischemia within 4 months before inclusion , and have recovered neurological status allowing good autonomy ( rankin score between 0 and 2 at inclusion ) . the neurological features of each patient were reviewed by the referent neurologist of the study , who validated the diagnosis of is / tia from anamnesis , clinical presentation , and the results of brain imaging . undetermined origin was ascertained by negative evaluation requiring ancillary tests performed in all patients to exclude the following conditions : ipsilateral carotid atherosclerotic stenosis > 70% , vertebral or carotid dissection with cervical doppler - echography or angio-mr.documented atrial fibrillation , with a 12-lead electrocardiogram and 24-h holter monitoring.intracardiac thrombus or any other cause of cardiac embolism , with transthoracic ( n = 24/24 ) and transesophageal echocardiography ( n = 22/24).vascular malformation , by means of cerebral ct , mri or angio - mri if necessary.hypercoagulable states , hematological disorders , and inflammatory diseases , with biological tests carried out at least 2 months after stroke : prothrombin ratio , activated partial thromboplastin time , c and s proteins , antithrombin , mutation in factor v leiden gene , circulating anticoagulants , antiphospholipid and anti - 2gp1 antibodies , homocysteinemia , erythrocyte sedimentation rate , and c - reactive protein . the study was carried out in three stages : measurement of basic conduction intervals and determination of the wenckebach point of the atrioventricular node.determination of the right atrium refractory periods ( rp ) and the duration of the atrial electrogram . latent vulnerability index ( lvi ) was defined as the ratio between high right atrium effective refractory period ( erp ) and a2 duration at a pacing rate of 100 bpm [ 7 , 14].assessment of the inducibility of atrial arrhythmia was performed by programmed atrial stimulation with up to three extrastimuli ( s2 , s3 , s4 ) at constant pacing rates of 100 , 120 , and 150 bpm at two different stimulation sites : hra and lra . assessment of the inducibility of atrial arrhythmia was performed by programmed atrial stimulation with up to three extrastimuli ( s2 , s3 , s4 ) at constant pacing rates of 100 , 120 , and 150 bpm at two different stimulation sites : hra and lra . latent atrial vulnerability was defined as lvi < 2.5 , and atrial inducibility as induction of sustained atrial arrhythmia lasting > 60 s [ 7 , 14 ] . patients underwent examination and 12-lead ecg every 4 months , performed by the investigator of the recruiting hospital . in the event of a serious issue ( recurrence of stroke , syncope , death ) , the study ended for a patient when sustained af was documented by the monitor or after the monitoring period of 14 months with the device 's explantation ( end of battery life defined by the manufacturer of the reveal plus ilr 9526 ) . twenty - four patients were included , 15 men and 9 women , from 19 to 74 years old , mean age 48.8 13.6 years . table 1characteristics of subjects ( n = 24)male n ( % ) 15 ( 62.5)age ( years ) ( mean sd)49 13.6<50 years n ( % ) 14 ( 58.3)diabetes mellitus0hypertension n ( % ) 7 ( 29.2)dyslipidemia n ( % ) 8 ( 33.3)active tobacco use n ( % ) 10 ( 41.7)overweight ( bmi > 27.5 ) n ( % ) 4 ( 16.7)vascular ischemic hereditary n ( % ) 5 ( 20.8 ) characteristics of subjects ( n = 24 ) of the 24 enrolled patients , 3 ( 12.5% ) experienced tia with symptoms lasting less than 1 h ( and normal brain mri ) , and 21 ( 87.5% ) experienced cerebral infarction ; one of them remained with neurological sequelae ( rankin score = 2 ) , symptoms were fully reversible in all the others . the clinical presentation consisted of lateralized motor deficit or aphasia in 75% of the cases , with or without sensory symptoms all patients underwent at least one brain imaging , cerebral ct ( n = 17 ) or mri ( n = 15 ) or both ( n = 8) , and four patients had an angio - mri performed . eight of these , termed inducible patients , experienced sustained arrhythmia , lasting more than 60 s. the lvi was calculated whenever possible with the formula described above . it could not be calculated in four cases because of the impossibility of measuring rp or a2 duration due to early onset of sustained arrhythmia . table 3electrophysiological study results ( n = 24 ) mean sdminmaxnumber ( % ) ah interval ( ms)83 22.415125hv interval ( ms)43 9.383065wenckebach point of av node ( bpm)162 35.2110230hra erp ( ms)234 37.1180290a2 duration ( ms)85 26.450140lvi ( n = 20)2.9 1.021.55.6lvi < 2.59 ( 37.5)inducibility of atrial arrhythmia ( including af , flutter , and other atrial tachycardias)18 ( 75.0 ) 030 s8 ( 33.3 ) 3059 s2 ( 8.3 ) 15 min5 ( 20.8 ) > 5 min3 ( 12.5 ) electrophysiological study results ( n = 24 ) the mean time lapse from cerebral accident to initiation of cardiac rhythm monitoring , i.e. there was no recurrence of stroke , no episode of syncope or palpitation was reported , and all electrocardiograms were in sinus rhythm . no significant af episode or any other sustained arrhythmia was identified , either in inducible patients or in the others . of the 24 enrolled patients , 3 ( 12.5% ) experienced tia with symptoms lasting less than 1 h ( and normal brain mri ) , and 21 ( 87.5% ) experienced cerebral infarction ; one of them remained with neurological sequelae ( rankin score = 2 ) , symptoms were fully reversible in all the others . the clinical presentation consisted of lateralized motor deficit or aphasia in 75% of the cases , with or without sensory symptoms . all patients underwent at least one brain imaging , cerebral ct ( n = 17 ) or mri ( n = 15 ) or both ( n = 8) , and four patients had an angio - mri performed . eight of these , termed inducible patients , experienced sustained arrhythmia , lasting more than 60 s. the lvi was calculated whenever possible with the formula described above . it could not be calculated in four cases because of the impossibility of measuring rp or a2 duration due to early onset of sustained arrhythmia . table 3electrophysiological study results ( n = 24 ) mean sdminmaxnumber ( % ) ah interval ( ms)83 22.415125hv interval ( ms)43 9.383065wenckebach point of av node ( bpm)162 35.2110230hra erp ( ms)234 37.1180290a2 duration ( ms)85 26.450140lvi ( n = 20)2.9 1.021.55.6lvi < 2.59 ( 37.5)inducibility of atrial arrhythmia ( including af , flutter , and other atrial tachycardias)18 ( 75.0 ) 030 s8 ( 33.3 ) 3059 s2 ( 8.3 ) 15 min5 ( 20.8 ) > 5 min3 ( 12.5 ) electrophysiological study results ( n = 24 ) the mean time lapse from cerebral accident to initiation of cardiac rhythm monitoring , i.e. there was no recurrence of stroke , no episode of syncope or palpitation was reported , and all electrocardiograms were in sinus rhythm . in our study , cerebral ischemic accidents were suspected to be of cardioembolic mechanism , as evidenced by the frequency of multiple , especially bilateral lesions . nevertheless , among the 24 enrolled patients , af was detected as a potential cause of stroke in only one patient . is / tia , the authors found a higher detection rate of af using 21-day monitoring with mobile cardiac outpatient telemetry , 5.3% of af > 30 s and 23% of short episodes < 30 s. we can assume that the difference is due to patient selection and time of initiation of the monitoring . in the tayal study , the patients were at higher risk of af because they were older ( mean age 66 11 versus 49 13 years in our study ) , and the prevalence of diabetes was high . the aim of the av study was to identify subjects with a high risk of atrial arrhythmia . however , the correlation between the presence of an arrhythmogenic substrate and the occurrence of spontaneous af has not been proven . we also found a high rate of inducible sustained atrial arrhythmias ( 33.3% ) and a high rate of latent av ( 37.5% ) , whereas no spontaneous sustained asymptomatic af or af with fast ventricular response was documented over a period of 14 months . for the first time , continuous monitoring in a prospective study has made it possible to confirm the irrelevance of electrophysiological study in the etiological workup of is / tia , because neither low lvi nor atrial arrhythmia inducibility was predictive of spontaneous af . firstly , the duration of the study was shortened by the device 's limited battery life . as epidemiological data in the literature reveal that the risk of recurrent stroke is high in the early phase after a first - ever event and remains high for several years thereafter [ 19 , 32 , 33 ] , there is little prospect that the new generation of ilr with a prolonged battery life ( more than 36 months ) would be more informative . in the future , the ilr will be designed to record atrial activity more accurately in order to diagnose af with lower ventricular response . finally , it might be objected that a major limitation lies in the limited size of the study , given the low number of 24 subjects included . inclusion was limited to 24 patients for reasons of ethical considerations and cost effectiveness on the grounds that the absence of sustained af in this population made the diagnostic method uneconomical . in our study , cerebral ischemic accidents were suspected to be of cardioembolic mechanism , as evidenced by the frequency of multiple , especially bilateral lesions . nevertheless , among the 24 enrolled patients , af was detected as a potential cause of stroke in only one patient . is / tia , the authors found a higher detection rate of af using 21-day monitoring with mobile cardiac outpatient telemetry , 5.3% of af > 30 s and 23% of short episodes < 30 s. we can assume that the difference is due to patient selection and time of initiation of the monitoring . in the tayal study , the patients were at higher risk of af because they were older ( mean age 66 11 versus 49 13 years in our study ) , and the prevalence of diabetes was high . the aim of the av study was to identify subjects with a high risk of atrial arrhythmia . some authors have described similar electrophysiological abnormalities in patients with paroxysmal af and in patients with unexplained ischemic stroke [ 8 , 31 ] . however , the correlation between the presence of an arrhythmogenic substrate and the occurrence of spontaneous af has not been proven . in previous series , the percentage of inducible sustained af in patients with cryptogenic is was 50% to 65% [ 6 , 8 ] . we also found a high rate of inducible sustained atrial arrhythmias ( 33.3% ) and a high rate of latent av ( 37.5% ) , whereas no spontaneous sustained asymptomatic af or af with fast ventricular response was documented over a period of 14 months . firstly , the duration of the study was shortened by the device 's limited battery life . as epidemiological data in the literature reveal that the risk of recurrent stroke is high in the early phase after a first - ever event and remains high for several years thereafter [ 19 , 32 , 33 ] , there is little prospect that the new generation of ilr with a prolonged battery life ( more than 36 months ) would be more informative . in the future , the ilr will be designed to record atrial activity more accurately in order to diagnose af with lower ventricular response . finally , it might be objected that a major limitation lies in the limited size of the study , given the low number of 24 subjects included . inclusion was limited to 24 patients for reasons of ethical considerations and cost effectiveness on the grounds that the absence of sustained af in this population made the diagnostic method uneconomical . this study suggests that symptomatic atrial fibrillation or af with fast ventricular rate is not a frequent pathology in patients under the age of 75 with unexplained ischemic stroke . these results do not plead for generalizing the use of an implantable loop recorder in the systematic evaluation of these patients . further studies are needed to test the efficacy and cost effectiveness of the method in a selected population with a higher risk of atrial fibrillation , using an updated device that more accurately diagnoses af with low ventricular response . finally , this study asserts that the electrophysiological study of atrial vulnerability is poor at predicting spontaneous atrial fibrillation after cryptogenic cerebral ischemia .

s. aureus is a causative microorganism for nosocomial infection , and high rates of morbidity and mortality associated with s. aureus infectious diseases have been observed in many areas around the world . moreover , the emergence of multidrug - resistant s. aureus , such as methicillin - resistant s. aureus ( mrsa ) and vancomycin - resistant s. aureus ( vrsa ) , makes the treatment of nosocomial infections more difficult , thereby increasing the mortality of the patients.s . aureus infections occur not only in hospitals but also in various social communities ; furthermore , social community - associated ( ca ) mrsa and vrsa have been frequently observed in recent years . ca - mrsa is an epidemic , particularly observed in the usa , that is characterized by rapid spreading and by the production of panton - valentine leukocidin ( pvl ) , which causes several deadly illnesses and more strongly virulent diseases than hospital - associated mrsa . although new types of resistant s. aureus have been anticipated , the number of new drugs developed against s. aureus has gradually decreased . therefore , the lack of effective antibacterial drugs against the resistant s. aureus strains might become a large threat in the near future . thus , it is important to develop new antibacterial drugs targeting mrsa , vrsa , and multidrug - resistant s. aureus . sadhfr is an enzyme that catalyzes the chemical reaction for the reduction of tetrahydrofolate ( thf ) from dihydrofolate ( dhf ) through nadph . dhfr is essential in the pathways for the intracellular production of purines , such as adenine and guanine . therefore , specific inhibitors of dhfr block dna replication in s. aureus , eventually leading to bacterial death . drugs targeting dhfr include methotrexate ( mtx ) , originally developed as an anticancer drug , and trimethoprim ( tmp ) , developed and still used as an antibacterial drug . tmp is prescribed as cotrimoxazole ( bactrim ) , comprising tmp and sulfamethoxazole , which inhibits the bacterial - specific enzyme dihydropteroate synthase ( dhps).s . aureus that are resistant to tmp with a diaminopyrimidine ( dap ) ring have recently emerged , and the dhfr of the resistant strains contains mutated amino acid residues , including phe 98 to tyr ( f98y ) . the phe to tyr change at position 98 is the most important mutation residue to cause tmp resistance . in addition , it is known that approximately 28% of mrsa show tmp resistance . therefore , the identification of chemical compounds with chemical scaffolds unlike tmp is strongly suggested for the treatment of patients infected with tmp - resistant s. aureus strains . sbds is an effective technique for novel drug discovery . in silico sbds through docking simulations between target proteins and chemical compounds is an efficient screening method to identify candidate compounds from a large chemical database because of the reduced time and cost for hit chemical identification . successful identification of antibacterial chemical compounds through in silico sbds has been reported.in silico sbds has been performed using docking simulation tools , such as gold , dock , glide , fred , and autodock . multistep in silico sbds using combinations of the docking simulation tools have been used to more effectively identify active chemical compounds . in a previous study , we identified potent growth inhibitors targeting mycobacterium through multistep in silico sbds , and the strategy of using multiple chemical conformers could improve the accuracy of docking simulations . in the present study , we performed a three - step in silico sbds to target the crystal structure of sadhfr from 154,118 chemical compounds library . subsequently , we rescreened chemical compounds similar to the active hits obtained from the sbds using 461,397 chemical compounds library . we identified four chemical compounds showing antibacterial effects against a staphylococcal strain and inhibitory effects on the enzymatic activity of the targeted protein . in addition , we confirmed that three of the four identified chemical compounds did not show inhibitory effects on the growth of model enterobacteria or toxic effects on cultured mammalian cells . these results will contribute to the development of novel antibacterial therapies against drug - resistant s. aureus . we performed three - step in silico sbds targeting sadhfr with a virtual chemical compound library ( 154,118 chemical compounds ) . the three - step sbds involved initial screening using dock , followed by screening using gold with a single chemical conformer and a third screening using gold with multiple chemical conformers ( figure 1a ) . the active site of sadhfr , comprises amino acid residues : val 6 , ala 7 , leu 20 , pro 25 , asp 27 , leu 28 , val 31 , ser 49 , ile 50 , arg 57 , phe 92 , and thr 111 ( figure 2 ) . we screened candidate chemical compounds with high potential of binding affinity for the active site of sadhfr . in the first screen , the docking simulations with dock predicted 500 top - ranked chemical compounds ( 0.3% of the primary chemical compound library ) with dock scores of less than 48.5 kcal / mol . the calculation speed of dock - based screening is fast , reflecting grid - based calculations without hydrogen bond ( h - bond ) energy through pc clustering . however , the accuracy of the calculations is relatively low [ the area under the curve ( auc ) values of receiver - operating characteristic ( roc ) = 0.56 ; figure s1 ] . in the second screen , we used the top - ranked 500 chemical compounds with conformations outputted after the first dock screen . gold is a flexible docking simulation tool using genetic algorithm ( the auc values of roc = 0.89 ; figure s1 ) . after the docking simulations with gold , we selected 139 top - ranked chemical compounds ( gold scores > 70 ) from the 500 chemical compounds . in the third screen , we used the multiconformational chemical structures with at most eleven conformations per chemical compound ( the auc values of roc = 0.95 ; figure s1 ) . we removed similar chemical compounds with common structures and selected the candidate chemical compounds that had not previously been evaluated in high throughput screening ( hts ) using as deposited in the pubchem web - based chemical compounds database . finally , we identified five chemical compounds ( kb1-kb5 ) with an average of gold scores > 82 ( table 1 ) . the gold score of kb1 from sadhfr simulation is higher than that of the natural endogenous substrate , dhf ( table 1 ) . flowchart of the in silico sbds and chemical analog search strategy applied in this study . ( a ) flowchart of the identification of candidate chemical compounds using three - step in silico sbds . the secondary structures are indicated using ribbon representations [ -helix ( h1h4 ) : orange , -sheet ( s1s10 ) : green ] . the amino acid residues of the active site are shown as a stick model . the asterisk denotes the following : each value represents the mean sem . we compared the amino acid sequence of human dhfr ( hdhfr ) with that of sadhfr using the basic local alignment search tool ( blast ) and the universal protein resource database ( uniprot ) and observed that the amino acid sequence homology between both proteins was low ( similarity rate = 48% , identity rate = 26% ) . the gold scores of kb1-kb5 when hdhfr is the target are less than those scores arising when targeting sadhfr ( table 1 ) , indicating that kb1-kb5 were predicted to specifically bind to the active site of sadhfr . we examined the inhibitory effects of the five candidate chemical compounds ( kb1-kb5 ) on bacterial growth , as predicted using three - step in silico sbdss . because s. aureus ( biosafety level 2 ) carries infectious risks for humans , we could not perform complex bacterial experiments ( time course growth and ic50 determination assays with 96-well assay plates ) using s. aureus . for the bacterial growth assays , we used wild - type s. epidermidis atcc 12228 , which does not pose a risk of infection to humans ( biosafety level 1 ) , as a model bacterial strain . the blast and the uniprot analyses showed that the amino acid sequence of s. epidermidis dhfr ( sedhfr ) is similar to that of sadhfr ( similarly rate = 94% , identity rate = 82% ) . all of the amino acid residues in the active site of sadhfr are completely conserved in sedhfr ( figure s2a ) . in addition , we generated the three - dimensional structure of sedhfr by homology modeling . the mean values of the rmsd for the structure of the active site pocket ( including substrate recognition sites ) between sadhfr and sedhfr are extremely low ; 0.47 ( figure s2b , c ) . thus , it is expected that the active site structure of sedhfr is extremely similar to that of sadhfr . the five candidate chemical compounds ( 100 m ) showed inhibitory effects on the growth of s. epidermidis . kb1 showed strong inhibitory effects on the growth of s. epidermidis ( figure 3 ) . the other four candidate chemical compounds ( kb2-kb5 ) had no significant or only weak inhibitory effects on bacterial growth compared with that of kb1 . kb1 also showed inhibitory effects on the growth of s. epidermidis similar to those of ampicillin . inhibitory effects of the candidate chemical compounds ( kb1-kb5 ) on the growth of s. epidermidis after 6 h. the concentrations of the chemical compounds were 100 m . dmso ( 0.3% ) and ampicillin ( 100 g / ml ) were used as the negative and positive controls , respectively . bonferroni s all - pair comparison test was performed ( n.s . not significant , * * * p < 0.001 ) . we examined whether kb1 exhibited inhibitory effects on the growth of the model enterobacterium e. coli bl21 and jm109 strains and toxic effects on mammalian cells ( madine - darby canine kidney : mdck and human neuroblastoma : sh - sy5y cells ) . kb1 did not show inhibitory effects on the growth of e. coli bl21 ( figure 4a ) and jm109 ( data not shown ) strains after 4 and 8 h. additionally , kb1 did not show any toxic effects on mdck cells ( figure 4b ) ; however , toxic effects on sh - sy5y cells were observed ( data not shown ; 43.3% of cell death is induced ) . dmso ( 0.3% ) and ampicillin ( 100 m ) were used as the negative and positive controls , respectively . the chemical compounds ( 30 m ; ic99 value for s. epidermidis bacterial growth ) were added to the cultures . dunnett s multiple comparison tests were performed ( n.s . not significant , * * p < 0.01 ) . we screened kb1 analogs expected to have inhibitory effects similar to those of kb1 ( figure 1b ) . we selected 23 kb1 analogs from a web - based database ( hit2lead.com ) . the 23 kb1 analogs had chemical structures with a similarity rate ( tanimoto coefficient ) greater than 85% . subsequently , we performed docking simulations targeting sadhfr with multiconformation of the 23 kb1 analogs using gold . the simulations predicted five top - ranked chemical compounds ( kbs1-kbs5 , table 2 ) , which were not previously identified using hts ( as available in the pubchem database ) . the gold scores of the five kb1 analogs ( kbs1-kbs5 ) were calculated through docking simulations with sadhfr , showing higher score values than those calculated through docking simulations with hdhfr ( table 2 ) . the analogs kbs1-kbs5 have substituted carboxyl and chlorobenzyl groups and similar structures compared with kb1 . kbs1 was the only analog with a gold score higher than that of kb1 ( table 2 ) . we examined whether the five kb1 analogs ( kbs1-kbs5 ) exhibited an inhibitory effect on the growth of s. epidermidis . we did not examine the effects of compound kbs2 , as this compound was not soluble at 100 m in dmso . the three chemical compounds ( kbs1 , kbs3 , and kbs4 ) exhibited significant inhibitory effects on the growth of s. epidermidis ( figure 5 ) . inhibitory effects of the candidate chemical compounds ( kbs1 and kbs3-kbs5 ) on the growth of the s. epidermidis after 6 h. the concentrations of the chemical compounds were 100 m . dmso ( 0.3% ) and ampicillin ( 100 g / ml ) was used as the negative and positive controls , respectively . bonferroni s all - pair comparison test was performed ( n.s . not significant , * * * p < 0.001 ) . furthermore , we examined whether these hit chemical compounds exhibited inhibitory effects on the growth of e. coli ( bl21 and jm109 strains ) and toxic effects on cultured mammalian cells . the results showed that these hit chemical compounds ( kbs1 , kbs3 , and kbs4 ) showed no inhibitory effects on the growth of e. coli bl21 ( figure 6a ) and jm109 ( data not shown ) strains . kbs3 and kbs4 did not exhibit toxic effects on mdck cells , although a weak toxic effect of kbs1 on mdck cells was detected ( figure 6b ) . the hit compounds ( kbs1 , kbs3 , and kbs4 ) also showed toxic effects on sh - sy5y cells ( data not shown ; 35.452.1% of cell death is induced ) . effects of the hit chemical compounds ( kbs1 , kbs3 , and kbs4 ) on e. coli and mdck cells . ( a ) effects of kb1 analogs on e. coli bl21 strains ( 8 h ) . dmso ( 0.3% ) and ampicillin ( 100 m ) were used as the negative and positive controls , respectively . the chemical compounds ( 30 m ; ic99 value for s. epidermidis bacterial growth ) were added to the cultures . multiple comparison tests were performed ( n.s . not significant , * * p < 0.01 ) . we investigated the dose - dependent effects of the four chemical compounds ( kb1 , kbs1 , kbs3 , and kbs4 ) on the growth of s. epidermidis . the experimentally determined ic50 values of kb1 , kbs1 , kbs3 , and kbs4 were 3.84 0.27 , 15.7 5.15 , 12.0 0.60 , and 9.46 0.48 m , respectively ( figure 7a - d , table 3 ) . the dose - dependent effects of compounds kb1 , kbs1 , kbs3 , and kbs4 on the growth of s. epidermidis ( a ) kb1 , ( b ) kbs1 , ( c ) kbs3 , and ( d ) kbs4 . the bacterial growth rate ( % ) during the 6 h cultivation of s. epidermidis . the asterisks denote the following : ( * ) each value represents the mean sem ; ( * * ) not tested . we investigated the enzyme inhibitory activity of the hit compounds ( kb1 , kbs1 , kbs3 , and kbs4 ) . in the enzyme inhibition assay , the reaction carried out with wild - type sadhfr was assessed under saturating conditions of substrate and cofactor , and the level of inhibition was determined relative to an uninhibited negative control reaction . all hit compounds inhibited sadhfr activity in a manner similar to that of the competitive inhibitor tmp ( ic50 value = 0.008 0.003 m ) . the experimentally determined ic50 values of kb1 , kbs1 , kbs3 , and kbs4 were 2.98 0.31 , 0.46 0.17 , 1.77 0.15 , and 1.38 0.10 m , respectively ( figure 8a - d , table 3 ) . the dose - dependent effects ( percent inhibition ) of compounds kb1 , kbs1 , kbs3 , and kbs4 on sadhfr enzymatic activity ( a ) kb1 , ( b ) kbs1 , ( c ) kbs3 , and ( d ) kbs4 . the level of inhibition is expressed as a percentage of the remaining sadhfr activity compared to a reaction with no chemical compound ( i.e. , 0% inhibition ) . the result of each hit compound ( kb1 , kbs1 , kbs3 , and kbs4 ) is shown as a representative of four independent experiments . each ic50 value represents the average of four independent experiments and was determined by performing nonlinear regression analysis . we evaluated the detailed interactions between the target protein ( sadhfr ) and the hit chemical compounds ( kb1 , kbs1 , kbs3 , and kbs4 ) using plif and li analyses . figure 9 shows the predicted binding modes of the hit chemical compounds with the highest gold scores from multiconformation simulations . all of the hit chemical compounds were predicted to be located near the active site of sadhfr . the carboxyl group in the hit compounds forms h - bond with arg 57 ( table 3 ) . in addition , the 4-oxo-2-thioxo-1,3-thiazolidin group in the hit compounds forms van der waals ( vdw ) contacts with leu 28 . the 2-pyrrolidone group in kb1 and kbs4 forms arene - h interactions with ile 50 and the 2-oxo-1,2-dihydro-3h - indol-3-ylidene group in kbs1 , and the 2-pyrrolidone group in kbs1 forms vdw contacts with leu 20 and ile 50 , respectively . moreover , all hit compounds were not predicted to interact with phe 98 ( mutation of the residue in tmp - resistant sadhfr ) . in contrast , the inactive compound kbs5 , without a carboxyl group , did not form an h - bond with arg 57 . moreover , the carboxyl groups of all hit chemical compounds were predicted to be located near one of two carboxyl groups similar to the natural endogenous substrate dhf . the carboxyl group of dhf forms an h - bond with arg 57 ( figure 10 ) . the predicted binding modes of hit chemical compounds with the sadhfr based on plif and li analyses : ( a ) kb1 , ( b ) kbs1 , ( c ) kbs3 , and ( d ) kbs4 . the h - bond , arene - h , and vdw interactions are indicated with orange , light green dashed , and light blue dashed lines , respectively . the color of the amino acid interaction is identical to the colors of the amino acids indicated in table 3 . predicted binding modes of kb1 of the hit chemical compound and the dhf substrate to the sadhfr . the in silico sbds targeting of sadhfr revealed four hit compounds ( kb1 , kbs1 , kbs3 , and kbs4 ) exhibiting inhibitory effects on the growth of s. epidermidis and enzymatic activity of sadhfr . the plif and li analyses predicted that the hit chemical compounds made vdw contacts with leu 20 and leu 28 similar to tmp , an inhibitor of dhfr . particularly , vdw contacts with leu 20 were only predicted in kbs1 , suggesting that this interaction is considerably associated with the highest enzyme inhibitory activity of kbs1 . we performed an sar analysis of kb1 , kbs1 , and kbs3-kbs5 to identify the correlative relationships between the chemical structure scaffolds , biological activities , and interaction residues . the carboxyl groups in r2 were essential for the inhibitory effects on bacterial growth ( table 2 and figure 5 ) . in addition , the carboxyl groups in r2 were predicted to h - bond with arg 57 at an interaction residue similar to that observed in the natural substrate dhf . therefore , the interaction with arg 57 likely plays an important role in the inhibitory effects of this compound . furthermore , the activities of the compounds were altered through differences in the substituent groups in r1 ( tables 2 and 3 ) . although the hit compounds showed inhibitory effects on both sadhfr enzymatic activity and s. epidermidis bacterial growth , different ic50 values were observed . it is possibly suggested that membrane penetration contributes to the inhibitory effects of these compounds on bacterial growth . the results of the bacterial growth assay showed that the activities of those compounds are enhanced by the smaller structure of r2 ( tables 2 and 3 ) . in particular , the r2 of kb1 , with a relatively small carbon chain , exhibits an inhibitory effect on s. epidermidis growth that is five times higher than that of kbs1 . however , the results of enzyme activity assay showed that the longer carbon chain of r2 enhanced the activities of the compounds . drug absorption and membrane permeability are influenced by molecular weight and logp value . although kbs1 and kbs4 with longer r2 carbon chains exhibit higher inhibitory activity against sadhfr , kbs1 and kbs4 have relatively lower inhibitory effects on bacterial growth . an sar analysis of the hit compound will provide important information for development of chemical derivatives targeting s. aureus . we used docking simulations with multiple chemical conformers and plif and li analyses to estimate whether the chemical compounds identified in this study have inhibitory effects on tmp - resistant s. aureus strains . most interaction residues predicted in wild - type sadhfr through plif and li analyses ( table 3 ) were also identified in plif and li analyses of tmp - resistant sadhfr ( data not shown ) . these candidate chemical compounds were predicted not to interact with tyr 98 , which is mutated residues in tmp - resistant sadhfr . therefore , the hit compounds identified in this study are expected to exhibit inhibitory effects on the growth of tmp - resistant s. aureus . in general , hts methods are used for hit identification and in the lead discovery phase of drug development . although hts have the advantage of hit compound identification from chemical libraries containing large numbers of compounds , this technique requires high - cost and long - term experiments , and moreover , the hit rate of hts targeting for s. aureus was approximately 1% . activity relationship ( qsar ) , molecular dynamics ( md ) , and pharmacophore modeling have been developed to identify novel drugs , and these methods can be performed with lower costs and shorter times than hts . the three - step in silico sbds utilized in the present study initially obtained a 20% hit rate , including the identification of a high - efficacy growth inhibitor from five candidate chemical compounds . furthermore , the screening of the active compound analogs considerably improved the hit rate of the drug screening ; three growth inhibitors were identified from four candidate compounds ( 75% hit rate , ic50 < 15.7 m ) . therefore , this in silico drug screening strategy is expected to be effective for the identification of novel active compounds against other disease - causing agents . in the present study , we identified five chemical compounds using three - step in silico sbdss . the in vitro biological assay revealed that one of these compounds , kb1 , exhibited antibiotic effects against s. epidermidis , as a model for the s. aureus strain , and enzyme inhibitory effects against sadhfr . kb1 showed no toxic effects on mdck cells , and no inhibition of e. coli growth was observed . moreover , five kb1 analogs ( kbs1-kbs5 ) were identified using docking simulations . among these , three kb1 analogs ( kbs1 , kbs3 , and kbs4 ) exhibited inhibitory effects on the growth of s. epidermidis and enzyme inhibitory effects against sadhfr . kbs3 and kbs4 did not have any toxic effects on mdck cell growth , and no inhibitory effects on e. coli growth were observed . we performed an sar analysis of kb1 , kbs1 , and kbs3-kbs5 , and the results of sar analysis will likely be useful for the identification and development of more potent chemical derivatives . in addition , the candidate chemical compounds identified in this study are predicted to have potential antibiotic effects against resistant s. aureus stains based on structural and protein ligand interaction . therefore , we expect that these compounds will serve as the lead chemical for the development of novel antimicrobial agents targeting s. aureus . all chemical compounds libraries were obtained from the web - based database , ressource parisienne en bioinformatique structurale ( rpbs ) . for the in silico sbds , we used a single conformation chemical compounds library ( chembridge , 154,118 chemical compounds ) and a multiconformation chemical compounds library including a maximum of eleven conformations per chemical compound . these compound libraries were adme / tox ( absorption , distribution , metabolism , excretion and toxicity ) filtered , using the most lax filtering method . for structure - based screening with hit chemical similarity , we used a multiconformation chemical compounds library ( chembridge , 461,397 chemical compounds ) generated using a lowmode md module in the molecular operating environment ( moe ) version 2010 . 10 . the crystal structure data for the sadhfr ( pdb i d : 2w9 g ) , hdhfr ( pdb i d : 3ntz ) , and tmp - resistant sadhfr ( pdb i d : 3m09 ) were obtained from the research collaboratory for structural bioinformatics protein data bank . before using the sadhfr crystal structure in the in silico sbds , we removed all atoms of the water molecule and tmp atoms of the inhibitor from the original crystal structure data . in addition , hydrogens were added to the sadhfr crystal structure using the mmff94x force field , and the energy was minimized using the protonate 3d and energy minimize modules , with all heavy atoms tethered in moe version 2010 . we performed in silico sbds using ucsf dock version 6.4 and ccdc gold suite version 5.0.1 . in the first screening using dock , we searched and extracted the molecular surface of dhfr using the dms program . in addition , we explored clefts potentially interacting with chemical compounds and determined the potential interaction space using the sphgen program . the protein a scoring function of dock is calculated using vdw and electrostatic interaction energies to estimate the potential binding affinity . we performed the docking simulations using rigid ligand conditions from the virtual chemical compounds library ( 154,118 chemical compounds ) . in the second screening , we estimated the binding affinity between sadhfr and the chemical compounds selected from the first screening using gold , which is a flexible docking simulation tool using genetic algorithm ( ga ) . the docking simulations are performed with default settings in gold . in the final screening using gold , we performed docking simulations with the multiconformation virtual chemical compounds library ( the selected chemical compounds from the second screening ) . we compared the amino acid sequences of sadhfr , sedhfr , and hdhfr . we obtained the sequences of wild - type sadhfr ( entry name : dyr_staau ) , wild - type sedhfr ( entry name : dyr_staes ) , and wild - type hdhfr ( entry name : dyr_human ) from the uniprot web site , and these amino acid sequences were compared using the blastp web tool on the blast web site . the three - dimensional structure of sedhfr is not available on the in pdb web site . therefore , we generated the three - dimensional structure of sedhfr using the homology modeling module in moe version 2010 . based on chemical structure similarities , we searched chemical compounds similar to the initial hit chemical compound kb1 from the data for all chemical compounds using chembridge by 2d and 3d similarity search methods . we performed docking simulations between the target protein and the multiconformation of the 23 virtual chemical compounds using gold . roc analysis was performed to evaluate docking performance using the lactobacillus casei dhfr ( pdb i d : 3dfr(45 ) ) and the directory of useful decoy ( dud ) ligands and decoys set . the chemical structure data for all ligands and decoys were generated using the energy minimize and lowmode md modules in the moe version 2010 . the roc curve and auc values were calculated using the r source of dr . the docking studies were performed using the same methods and parameters as the first step of dock and the second and third steps of the gold screening process . all of the chemical compounds ( kb1-kb5 and kbs1-kbs5 ) were purchased from the chembridge corporation and dissolved in dimethyl sulfoxide ( dmso , sigma ) . the following chemical compounds were obtained from chembridge ( supplier ids given in parentheses ) : kb1 : { 5-[1-(4-chlorobenzyl)-2-oxo-1,2-dihydro-3h - indol-3-ylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}acetic acid ( 7469513 ) ; kb2 : 4-{[7-(4-chlorophenyl)-5-methyl-4,7-dihydrotriazolo[1,5-a]pyrimidin-2-yl]amino}-4-oxobutanoic acid ( 6584644 ) ; kb3 : 4-[(4-{[(4-methoxyphenyl)amino]sulfonyl}phenyl)amino]-4-oxobutanoic acid ( 6952265 ) ; kb4 : [ 4-({[3-(2-chlorophenyl)-5-methyl-4-isoxazolyl]carbonyl}amino)phenyl]acetic acid ( 9009041 ) ; kb5 : 3-[(4,5-diphenyl-1h - imidazol-2-yl)thio]propanoic acid ( 6528030 ) ; kbs1 : 4-{5-[1-(4-chlorobenzyl)-2-oxo-1,2-dihydro-3h - indol-3-ylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}butanoic acid ( 7424742 ) ; kbs2 : { 5-[1-(4-methylbenzyl)-2-oxo-1,2-dihydro-3h - indol-3-ylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}acetic acid ( 6660051 ) ; kbs3 : { 5-[1-(2-chlorobenzyl)-2-oxo-1,2-dihydro-3h - indol-3-ylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}acetic acid ( 7365728 ) ; kbs4 : 3-{5-[1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-3h - indol-3-ylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}propanoic acid ( 6666591 ) ; and kbs5 : 1-(4-chlorobenzyl)-3-[3-(3-ethoxypropyl)-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene]-1,3-dihydro-2h - indol-2-one ( 7397168 ) . the model bacterial strain s. epidermidis atcc 12228 was obtained from the riken bioresource center ( saitama , japan ) . s. epidermidis was incubated overnight in 3 ml of culture medium [ 1% peptone ( bd ) , 1% beef extract ( bd ) and 0.5% nacl ( wako ) , ph 7.1 ] on a rotary shaker ( 37 c , 300 rpm ) . e. coli was incubated overnight in 3 ml of culture medium [ 0.5% yeast extract ( bd ) , 1% tryptone ( bd ) and 0.5% nacl ( wako ) , ph 7.0 ] on a rotary shaker ( 37 c , 300 rpm ) . we used 15 dilutions of the s. epidermidis culture and 10 dilutions of the e. coli culture for the growth assays . the bacterial strains were seeded in 96-well assay plates ( nunc ) with a 100 l total reaction volume and incubated in a culture medium containing each chemical compound , 0.3% dmso as a negative control or ampicillin ( sigma ) as a positive control in the s. epidermidis and e. coli growth assays . after 4 , 6 , and 8 h , we measured the absorbance ( 595 nm ) of the culture media using a micro plate reader ( biorad ) . the effect of the chemical compounds on mammalian cells was assayed using the cell counting kit-8 ( dojin ) , to measure the number of living cells . the mdck and sh - sy5y cells were seeded in 100 mm dishes and maintained in dmem medium ( wako ) supplemented with 10% fbs ( gibco ) , 100 units / ml penicillin , 100 mg / ml streptomycin ( gibco ) , and 2 mm l - glutamine ( gibco ) . the mdck and sh - sy5y cells were seeded in 96-well assay plates ( corning ) with 100 l of total reaction volume at 5.0 10 and 1.5 10 cells / well , respectively , and subsequently incubated for 6 h and overnight , respectively , at 37 c in 5% co2 . for cell starvation , the culture medium of the mdck and sh - sy5y cells was replaced with fresh dmem medium containing 0.25% fbs , and the cells were then incubated overnight and for 2 days , respectively . after cell starvation , the culture medium of the mdck and sh - sy5y cells was replaced with fresh medium ( 0.25% fbs ) containing one of the chemical compounds or ampicillin ( 30 m ) as a negative control . the mdck and sh - sy5y cells subsequently were incubated for 1 and 2 days , respectively . we added 10 l / well of cell counting kit-8 and after 3 h measured the absorbance ( 450 nm ) of wst-8 formazan using a micro plate reader ( biorad ) . purified recombinant wild - type sadhfr protein was used in this enzymatic assay . the assay was carried out in a 96-well assay plate ( bd ) with a 200 l total reaction volume using a biomek 2000 liquid handling robot . the enzyme and dilutions of each chemical compound were preincubated at room temperature for 3 min . subsequently , 120 nm nadph ( sigma ) was added and incubated at 30 c ; the reaction was initiated by the addition of 226 m dhf ( sigma ) and was monitored for 3 min ; the reaction rate remained linear . the enzyme concentration ( 5.0 nm ) yielded an activity of 1.4 nmol dhf reduced per minute for wild - type dhfr . the redox - sensitive tetrazolium dye , 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium ( mts , promega ) , was utilized as a reagent for the detection of dhfr activity . mts is reduced by the product thf to yield an increased absorbance at 450 nm . the absorbance ( 450 nm ) of mts was measured using a dtx880 plate reader ( beckman coulter ) . the change in reaction rate signal was calculated as the percentage of a reaction with no chemical compounds over 2.8 min of reaction . all statistical analyses were performed using r version 2.15.1 ( the r foundation for statistical computing , vienna , austria ) and graphpad prism version 4 ( graphpad prism software , inc . , san diego , ca ) .

the emergence of multidrug - resistant staphylococcus aureus ( s. aureus ) makes the treatment of infectious diseases in hospitals more difficult and increases the mortality of the patients . in this study , we attempted to identify novel potent antibiotic candidate compounds against s. aureus dihydrofolate reductase ( sadhfr ) . we performed three - step in silico structure - based drug screening ( sbds ) based on the crystal structure of sadhfr using a 154,118 chemical compound library . we subsequently evaluated whether candidate chemical compounds exhibited inhibitory effects on the growth of the model bacterium : staphylococcus epidermidis ( s. epidermidis ) . the compound kb1 showed a strong inhibitory effect on the growth of s. epidermidis . moreover , we rescreened chemical structures similar to kb1 from a 461,397 chemical compound library . three of the four kb1 analogs ( kbs1 , kbs3 , and kbs4 ) showed inhibitory effects on the growth of s. epidermidis and enzyme inhibitory effects on sadhfr . we performed structure activity relationship ( sar ) analysis of active chemical compounds and observed a correlative relationship among the ic50 values , interaction residues , and structure scaffolds . in addition , the active chemical compounds ( kb1 , kbs3 , and kbs4 ) had no inhibitory effects on the growth of model enterobacteria ( e. coli bl21 and jm109 strains ) and no toxic effects on cultured mammalian cells ( mdck cells ) . results obtained from protein ligand interaction fingerprint ( plif ) and ligand interaction ( li ) analyses suggested that all of the active compounds exhibited potential inhibitory effects on mutated sadhfr of the drug - resistant strains . the structural and experimental information concerning these novel chemical compounds will likely contribute to the development of new antibiotics for both wild - type and drug - resistant s. aureus .

Introduction Results Discussion Conclusion Materials and Methods

moreover , the emergence of multidrug - resistant s. aureus , such as methicillin - resistant s. aureus ( mrsa ) and vancomycin - resistant s. aureus ( vrsa ) , makes the treatment of nosocomial infections more difficult , thereby increasing the mortality of the patients.s . although new types of resistant s. aureus have been anticipated , the number of new drugs developed against s. aureus has gradually decreased . thus , it is important to develop new antibacterial drugs targeting mrsa , vrsa , and multidrug - resistant s. aureus . aureus that are resistant to tmp with a diaminopyrimidine ( dap ) ring have recently emerged , and the dhfr of the resistant strains contains mutated amino acid residues , including phe 98 to tyr ( f98y ) . therefore , the identification of chemical compounds with chemical scaffolds unlike tmp is strongly suggested for the treatment of patients infected with tmp - resistant s. aureus strains . in silico sbds through docking simulations between target proteins and chemical compounds is an efficient screening method to identify candidate compounds from a large chemical database because of the reduced time and cost for hit chemical identification . multistep in silico sbds using combinations of the docking simulation tools have been used to more effectively identify active chemical compounds . in a previous study , we identified potent growth inhibitors targeting mycobacterium through multistep in silico sbds , and the strategy of using multiple chemical conformers could improve the accuracy of docking simulations . in the present study , we performed a three - step in silico sbds to target the crystal structure of sadhfr from 154,118 chemical compounds library . subsequently , we rescreened chemical compounds similar to the active hits obtained from the sbds using 461,397 chemical compounds library . we identified four chemical compounds showing antibacterial effects against a staphylococcal strain and inhibitory effects on the enzymatic activity of the targeted protein . in addition , we confirmed that three of the four identified chemical compounds did not show inhibitory effects on the growth of model enterobacteria or toxic effects on cultured mammalian cells . these results will contribute to the development of novel antibacterial therapies against drug - resistant s. aureus . we performed three - step in silico sbds targeting sadhfr with a virtual chemical compound library ( 154,118 chemical compounds ) . we screened candidate chemical compounds with high potential of binding affinity for the active site of sadhfr . in the first screen , the docking simulations with dock predicted 500 top - ranked chemical compounds ( 0.3% of the primary chemical compound library ) with dock scores of less than 48.5 kcal / mol . we removed similar chemical compounds with common structures and selected the candidate chemical compounds that had not previously been evaluated in high throughput screening ( hts ) using as deposited in the pubchem web - based chemical compounds database . flowchart of the in silico sbds and chemical analog search strategy applied in this study . ( a ) flowchart of the identification of candidate chemical compounds using three - step in silico sbds . we compared the amino acid sequence of human dhfr ( hdhfr ) with that of sadhfr using the basic local alignment search tool ( blast ) and the universal protein resource database ( uniprot ) and observed that the amino acid sequence homology between both proteins was low ( similarity rate = 48% , identity rate = 26% ) . we examined the inhibitory effects of the five candidate chemical compounds ( kb1-kb5 ) on bacterial growth , as predicted using three - step in silico sbdss . for the bacterial growth assays , we used wild - type s. epidermidis atcc 12228 , which does not pose a risk of infection to humans ( biosafety level 1 ) , as a model bacterial strain . the blast and the uniprot analyses showed that the amino acid sequence of s. epidermidis dhfr ( sedhfr ) is similar to that of sadhfr ( similarly rate = 94% , identity rate = 82% ) . all of the amino acid residues in the active site of sadhfr are completely conserved in sedhfr ( figure s2a ) . in addition , we generated the three - dimensional structure of sedhfr by homology modeling . thus , it is expected that the active site structure of sedhfr is extremely similar to that of sadhfr . the five candidate chemical compounds ( 100 m ) showed inhibitory effects on the growth of s. epidermidis . kb1 showed strong inhibitory effects on the growth of s. epidermidis ( figure 3 ) . the other four candidate chemical compounds ( kb2-kb5 ) had no significant or only weak inhibitory effects on bacterial growth compared with that of kb1 . kb1 also showed inhibitory effects on the growth of s. epidermidis similar to those of ampicillin . inhibitory effects of the candidate chemical compounds ( kb1-kb5 ) on the growth of s. epidermidis after 6 h. the concentrations of the chemical compounds were 100 m . we examined whether kb1 exhibited inhibitory effects on the growth of the model enterobacterium e. coli bl21 and jm109 strains and toxic effects on mammalian cells ( madine - darby canine kidney : mdck and human neuroblastoma : sh - sy5y cells ) . kb1 did not show inhibitory effects on the growth of e. coli bl21 ( figure 4a ) and jm109 ( data not shown ) strains after 4 and 8 h. additionally , kb1 did not show any toxic effects on mdck cells ( figure 4b ) ; however , toxic effects on sh - sy5y cells were observed ( data not shown ; 43.3% of cell death is induced ) . the chemical compounds ( 30 m ; ic99 value for s. epidermidis bacterial growth ) were added to the cultures . subsequently , we performed docking simulations targeting sadhfr with multiconformation of the 23 kb1 analogs using gold . the gold scores of the five kb1 analogs ( kbs1-kbs5 ) were calculated through docking simulations with sadhfr , showing higher score values than those calculated through docking simulations with hdhfr ( table 2 ) . we examined whether the five kb1 analogs ( kbs1-kbs5 ) exhibited an inhibitory effect on the growth of s. epidermidis . the three chemical compounds ( kbs1 , kbs3 , and kbs4 ) exhibited significant inhibitory effects on the growth of s. epidermidis ( figure 5 ) . inhibitory effects of the candidate chemical compounds ( kbs1 and kbs3-kbs5 ) on the growth of the s. epidermidis after 6 h. the concentrations of the chemical compounds were 100 m . furthermore , we examined whether these hit chemical compounds exhibited inhibitory effects on the growth of e. coli ( bl21 and jm109 strains ) and toxic effects on cultured mammalian cells . the results showed that these hit chemical compounds ( kbs1 , kbs3 , and kbs4 ) showed no inhibitory effects on the growth of e. coli bl21 ( figure 6a ) and jm109 ( data not shown ) strains . kbs3 and kbs4 did not exhibit toxic effects on mdck cells , although a weak toxic effect of kbs1 on mdck cells was detected ( figure 6b ) . the hit compounds ( kbs1 , kbs3 , and kbs4 ) also showed toxic effects on sh - sy5y cells ( data not shown ; 35.452.1% of cell death is induced ) . effects of the hit chemical compounds ( kbs1 , kbs3 , and kbs4 ) on e. coli and mdck cells . the chemical compounds ( 30 m ; ic99 value for s. epidermidis bacterial growth ) were added to the cultures . we investigated the dose - dependent effects of the four chemical compounds ( kb1 , kbs1 , kbs3 , and kbs4 ) on the growth of s. epidermidis . the experimentally determined ic50 values of kb1 , kbs1 , kbs3 , and kbs4 were 3.84 0.27 , 15.7 5.15 , 12.0 0.60 , and 9.46 0.48 m , respectively ( figure 7a - d , table 3 ) . the dose - dependent effects of compounds kb1 , kbs1 , kbs3 , and kbs4 on the growth of s. epidermidis ( a ) kb1 , ( b ) kbs1 , ( c ) kbs3 , and ( d ) kbs4 . we investigated the enzyme inhibitory activity of the hit compounds ( kb1 , kbs1 , kbs3 , and kbs4 ) . in the enzyme inhibition assay , the reaction carried out with wild - type sadhfr was assessed under saturating conditions of substrate and cofactor , and the level of inhibition was determined relative to an uninhibited negative control reaction . the experimentally determined ic50 values of kb1 , kbs1 , kbs3 , and kbs4 were 2.98 0.31 , 0.46 0.17 , 1.77 0.15 , and 1.38 0.10 m , respectively ( figure 8a - d , table 3 ) . the dose - dependent effects ( percent inhibition ) of compounds kb1 , kbs1 , kbs3 , and kbs4 on sadhfr enzymatic activity ( a ) kb1 , ( b ) kbs1 , ( c ) kbs3 , and ( d ) kbs4 . the result of each hit compound ( kb1 , kbs1 , kbs3 , and kbs4 ) is shown as a representative of four independent experiments . we evaluated the detailed interactions between the target protein ( sadhfr ) and the hit chemical compounds ( kb1 , kbs1 , kbs3 , and kbs4 ) using plif and li analyses . all of the hit chemical compounds were predicted to be located near the active site of sadhfr . moreover , all hit compounds were not predicted to interact with phe 98 ( mutation of the residue in tmp - resistant sadhfr ) . moreover , the carboxyl groups of all hit chemical compounds were predicted to be located near one of two carboxyl groups similar to the natural endogenous substrate dhf . the predicted binding modes of hit chemical compounds with the sadhfr based on plif and li analyses : ( a ) kb1 , ( b ) kbs1 , ( c ) kbs3 , and ( d ) kbs4 . the in silico sbds targeting of sadhfr revealed four hit compounds ( kb1 , kbs1 , kbs3 , and kbs4 ) exhibiting inhibitory effects on the growth of s. epidermidis and enzymatic activity of sadhfr . we performed an sar analysis of kb1 , kbs1 , and kbs3-kbs5 to identify the correlative relationships between the chemical structure scaffolds , biological activities , and interaction residues . in addition , the carboxyl groups in r2 were predicted to h - bond with arg 57 at an interaction residue similar to that observed in the natural substrate dhf . although the hit compounds showed inhibitory effects on both sadhfr enzymatic activity and s. epidermidis bacterial growth , different ic50 values were observed . in particular , the r2 of kb1 , with a relatively small carbon chain , exhibits an inhibitory effect on s. epidermidis growth that is five times higher than that of kbs1 . an sar analysis of the hit compound will provide important information for development of chemical derivatives targeting s. aureus . we used docking simulations with multiple chemical conformers and plif and li analyses to estimate whether the chemical compounds identified in this study have inhibitory effects on tmp - resistant s. aureus strains . most interaction residues predicted in wild - type sadhfr through plif and li analyses ( table 3 ) were also identified in plif and li analyses of tmp - resistant sadhfr ( data not shown ) . therefore , the hit compounds identified in this study are expected to exhibit inhibitory effects on the growth of tmp - resistant s. aureus . although hts have the advantage of hit compound identification from chemical libraries containing large numbers of compounds , this technique requires high - cost and long - term experiments , and moreover , the hit rate of hts targeting for s. aureus was approximately 1% . activity relationship ( qsar ) , molecular dynamics ( md ) , and pharmacophore modeling have been developed to identify novel drugs , and these methods can be performed with lower costs and shorter times than hts . the three - step in silico sbds utilized in the present study initially obtained a 20% hit rate , including the identification of a high - efficacy growth inhibitor from five candidate chemical compounds . furthermore , the screening of the active compound analogs considerably improved the hit rate of the drug screening ; three growth inhibitors were identified from four candidate compounds ( 75% hit rate , ic50 < 15.7 m ) . therefore , this in silico drug screening strategy is expected to be effective for the identification of novel active compounds against other disease - causing agents . in the present study , we identified five chemical compounds using three - step in silico sbdss . the in vitro biological assay revealed that one of these compounds , kb1 , exhibited antibiotic effects against s. epidermidis , as a model for the s. aureus strain , and enzyme inhibitory effects against sadhfr . kb1 showed no toxic effects on mdck cells , and no inhibition of e. coli growth was observed . among these , three kb1 analogs ( kbs1 , kbs3 , and kbs4 ) exhibited inhibitory effects on the growth of s. epidermidis and enzyme inhibitory effects against sadhfr . kbs3 and kbs4 did not have any toxic effects on mdck cell growth , and no inhibitory effects on e. coli growth were observed . we performed an sar analysis of kb1 , kbs1 , and kbs3-kbs5 , and the results of sar analysis will likely be useful for the identification and development of more potent chemical derivatives . in addition , the candidate chemical compounds identified in this study are predicted to have potential antibiotic effects against resistant s. aureus stains based on structural and protein ligand interaction . therefore , we expect that these compounds will serve as the lead chemical for the development of novel antimicrobial agents targeting s. aureus . for the in silico sbds , we used a single conformation chemical compounds library ( chembridge , 154,118 chemical compounds ) and a multiconformation chemical compounds library including a maximum of eleven conformations per chemical compound . for structure - based screening with hit chemical similarity , we used a multiconformation chemical compounds library ( chembridge , 461,397 chemical compounds ) generated using a lowmode md module in the molecular operating environment ( moe ) version 2010 . the crystal structure data for the sadhfr ( pdb i d : 2w9 g ) , hdhfr ( pdb i d : 3ntz ) , and tmp - resistant sadhfr ( pdb i d : 3m09 ) were obtained from the research collaboratory for structural bioinformatics protein data bank . before using the sadhfr crystal structure in the in silico sbds , we removed all atoms of the water molecule and tmp atoms of the inhibitor from the original crystal structure data . in addition , hydrogens were added to the sadhfr crystal structure using the mmff94x force field , and the energy was minimized using the protonate 3d and energy minimize modules , with all heavy atoms tethered in moe version 2010 . in addition , we explored clefts potentially interacting with chemical compounds and determined the potential interaction space using the sphgen program . we obtained the sequences of wild - type sadhfr ( entry name : dyr_staau ) , wild - type sedhfr ( entry name : dyr_staes ) , and wild - type hdhfr ( entry name : dyr_human ) from the uniprot web site , and these amino acid sequences were compared using the blastp web tool on the blast web site . based on chemical structure similarities , we searched chemical compounds similar to the initial hit chemical compound kb1 from the data for all chemical compounds using chembridge by 2d and 3d similarity search methods . all of the chemical compounds ( kb1-kb5 and kbs1-kbs5 ) were purchased from the chembridge corporation and dissolved in dimethyl sulfoxide ( dmso , sigma ) . we used 15 dilutions of the s. epidermidis culture and 10 dilutions of the e. coli culture for the growth assays . the bacterial strains were seeded in 96-well assay plates ( nunc ) with a 100 l total reaction volume and incubated in a culture medium containing each chemical compound , 0.3% dmso as a negative control or ampicillin ( sigma ) as a positive control in the s. epidermidis and e. coli growth assays . after 4 , 6 , and 8 h , we measured the absorbance ( 595 nm ) of the culture media using a micro plate reader ( biorad ) .

craniopharyngiomas ( cp ) are generally benign , slow - growing tumors that are thought to arise from rests of embryonic cells residing within the remnants of the craniopharyngeal duct or rathke s pouch ( abeloff , 2008 ) . thus , tumors may originate from anywhere along the path of the craniopharyngeal duct , from nasopharynx to tuber cinereum , although most are located in the sellar / parasellar region ( harwood - nash , 1994 ; karavitaki et al . , 2005 ) . although histologically benign , these tumors are often locally aggressive with invasion of adjacent tissues and structures . while mortality is generally low , morbidity as a result of tumor mass effect and/or therapy ( resection radiotherapy ) is substantial and nearly universal ( mortini et al . because of the close proximity to the visual nerve tracts , hypothalamus , pituitary , and ventricular system , cps predispose patients to a number of adverse endocrine , metabolic , psychologic , and neurologic sequelae . as a primary result of the tumor , the majority of patients present with one or more hypothalamic - pituitary deficits including growth hormone ( gh ; 75% ) , gonadotropins ( 60% ) , adrenocorticotropic hormone ( acth ; 30% ) , thyroid - stimulating hormone ( tsh ; 25% ) , and/or antidiuretic hormone ( adh ; 20% ; karavitaki et al . in addition , approximately one - third can develop new or additional endocrinopathies following surgical resection . in addition to pituitary hormone deficiencies , hypothalamic involvement has been shown to result in severe metabolic disturbances and weight gain ( muller et al . , 2001 ; while a minority ( 1520% ) of cp patients complain of weight gain as a presenting symptom , more than 50% develop significant obesity following surgical resection ( muller et al . , 2004 ; ahmet et al . , . this number may be as high as 90% in those with demonstrable hypothalamic damage , whether from direct tumor infiltration or as a result of surgical and/or radiation therapy ( de vile et al . such pathological weight gain , termed hypothalamic obesity ( hyob ) , is often severe , refractory to therapy , and has a significant negative impact on the quality of life for patients with cp ( muller et al . , 2005 ; over the last 75 years , experience with animal and human models has shown the basal medial hypothalamus to be a key center of metabolic regulation by the central nervous system ( cns ; hetherington and ranson , 1940 ; sorva , 1988 ) . the hypothalamus receives and integrates a variety of afferent signals communicating the metabolic state of the organism and adjusts autonomic outputs with the ideal physiologic outcome of maintaining adequate energy stores . the afferent arm of the homeostatic loop consists of hormones , such as leptin , insulin , and peptide yy , that target receptor fields within hypothalamic nuclei to convey information on meal size , nutrient composition , and adipose tissue stores . in addition , neurons within the hypothalamus directly sense and respond to nutrients , such as free fatty acids , glucose , and amino acids . within the hypothalamus , neuropeptides such as neuropeptide y ( npy ) , agouti - related protein ( agrp ) , and alpha - melanocyte stimulating hormone ( -msh ) , integrate and convey this information to other brain centers responsible for controlling appetite / satiety , thermogenic , and motor effectors ( schwartz et al . , 2000 ) . a number of monogenetic obesity syndromes have now been shown to involve mutations in such regulatory hypothalamic pathways ( clement et al . , 1998 ; lubrano - berthelier et al . , 2006 ; savastano et al . , 2009 in addition , several complex genetic syndromes , including prader willi ( pws ) and bardet biedl syndromes ( bbs ) , are associated with obesity , presumably from hypothalamic dysfunction . pws results from the loss of paternally imprinted genes on chromosome 15q11 - 15 and is characterized by an array of multisystemic defects including neonatal hypotonia , short stature , hypogonadism , behavioral and psychiatric phenotypes , aggressive food - seeking behavior , hyperphagia , and obesity ( goldstone et al . , 2008 ) . pws patients have significantly elevated levels of the orexigenic hormone ghrelin , a finding not observed in other causes of hyob ( cummings et al . , 2002a ; delparigi et al . , 2002 interestingly , even young , underweight patients with pws exhibit higher bmi - adjusted body fat and serum leptin levels , suggesting some intrinsic leptin resistance in pws ( eiholzer et al . , 1999 ) . bbs is a multisystemic disorder characterized by retinal degeneration , hypogonadism , polydactyly , renal dysfunction , mental retardation , and obesity resulting from defects in ciliary function . hypothalamic dysfunction in these patients is suggested by the study of mice harboring mutations in bbs genes . bbs mutants have been shown to be hyperleptinemic and have defective hypothalamic leptin signaling ( seo et al . , 2009 ; guo and rahmouni , 2011 ) . thus , damage to , or dysfunction of the hypothalamus results in an inability of the cns to receive proper feedback . patients , thus , inappropriately sense a perpetual state of starvation . to correct the perceived negative energy balance , efferent outputs are adjusted to promote caloric intake and decreased caloric expenditure . decreased sympathetic activation of 2- , 3- , and 2-adrenergic receptors reduce skeletal muscle thermogenesis , reduce adipose tissue lipolysis , and promote pancreatic insulin secretion , respectively . conversely , increased parasympathetic output through the vagus nerve slows the heart rate reducing myocardial oxygen consumption , promotes gastrointestinal peristalsis and substrate absorption , and accentuates post - prandial insulin secretion ( lustig , 2008 ) . the net result of this reduced sympathetic / parasympathetic ratio is significantly decreased energy expenditure with partitioning of calories away from energy consuming tissue such as muscle and toward energy storage depots such as adipose tissue . accumulating evidence supports the hypothesis that cp and its therapies often result in dysfunction of the normal homeostatic mechanisms regulating appetite and metabolism . leptin , a potent anorexigenic peptide secreted by adipocytes , has been found to be elevated in cp patients compared to obese controls suggesting a defect in the normal feedback inhibition of appetite . in addition , some cp patients have a blunted post - meal increase in the anorexigenic hormone peptide yy . hypersecretion of the orexigenic gastric hormone ghrelin is thought to contribute to hyperphagia and obesity in pws , although this does not seem to be the case in cp where pre - meal ghrelin levels are lower than weight - matched controls . found that there may be a reduction in the expected magnitude of post - meal suppression of ghrelin levels in cp patients ( roth et al . , 1998 , 2011 ; holmer et al . , 2010 ) . while there are reports of hyperphagia and obsessive food - seeking behavior after cp ( skorzewska et al . , 1989 ) , a number of studies have now shown that caloric expenditure , and not caloric intake , may be the largest contributor to post - cp hyob ( cp hyob ) . adults and children with cp hyob have been found to have impaired sympathoadrenal activation in response to hypoglycemia , although this defect does not appear to always correlate with development of obesity or hypothalamic involvement ( schofl et al . in a larger study , cp hyob patients were found to have lower levels of urine catecholamines than bmi - matched controls ( roth et al . , 2007 ) as sympathetic tone correlates with spontaneous motor activity , one might also expect lower levels of physical activity in cp patients . indeed , cp patients consistently report reduced physical activity , even when compared to controls with similar bmi ( roth et al . , 2007 ) demonstrated that obese cp patients had decreased spontaneous motor activity compared with age and bmi - matched controls . using food diaries , this same group showed that caloric intake was lower in cp patients with hypothalamic damage , despite a significantly increased bmi ( harz et al . , 2003 ) . these findings were substantiated by a recent study by holmer et al . in 42 adult patients with childhood cp , where cp patients were found to have a significantly reduced basal metabolic rate when adjusted for body weight , than age and sex matched controls . analysis of energy intake showed lower caloric consumption in cp patients which was attributed to cognitive restraint in eating ( holmer et al . , 2010 ) . aside from reduced sympathetic drive , the roadblocks to physical activity are substantial and likely contribute to reduced energy expenditure and risk for obesity . in addition , there is evidence that cp patients have disordered sleep patterns secondary to decreased melatonin and/or secondary narcolepsy causing daytime sleepiness ( muller et al . , 2002 , 2006 ) . obstructive sleep apnea ( osa ) in these patients also likely contributes to poor sleep hygiene . under- or overtreatment of hormonal deficiencies may also contribute to poor metabolic parameters and weight gain in cp patients . glucocorticoid excess is a known cause of significant weight gain , impaired glucose metabolism , sleep disturbance , and defects in bone metabolism and growth ( debono et al . , 2009 ) . most significantly , excess glucocorticoid dosing has been associated with increased mortality from cardiovascular disease . in a large cohort of scandinavian patients with hypopituitarism , filipsson et al . ( 2006 ) showed that those receiving higher doses of glucocorticoid replacement had significantly higher waist circumference , total cholesterol , serum triglycerides , and hba1c . daily cortisol production rates range from 6 to 8 mg / m / day , much less than previous estimates ( linder et al . , 1990 ; thus , the classic adult dosing of 20 mg of hydrocortisone in the a.m. and 10 mg in the p.m. , is likely an excessive dose . unfortunately , no objective test of adequate glucocorticoid replacement has been shown to be sufficiently consistent to aid in dosing management and patients must be followed closely for clinical signs of glucocorticoid deficiency or excess . hypothyroidism also results in poor metabolic health and is associated with fatigue , low basal metabolic rate , increased bmi , and increased cholesterol and triglyceride levels ( kronenberg and williams , 2008 ) and adequate replacement has beneficial effects on these parameters ( slawik et al . , 2007 ) . management of thyroid hormone replacement is made more challenging in cp patients by the absence of the patient s own endogenous the significance of this handicap is highlighted in a recent study comparing free t4 ( ft4 ) levels in tsh deficient patients to those in primary hypothyroid patients where tsh levels are used to guide therapy . nearly 40% of tsh deficient patients were found to have ft4 levels below the 20th centile range compared to 13.4% of primary hypothyroid patients ( koulouri et al . , 2011 ) . growth hormone deficiency is present in a majority of cp patients and likely contributes to the metabolic derangements seen in this patient population . these metabolic effects are particularly important in adults , where gh replacement results in increased lean body mass and decreased body fat ( kronenberg and williams , 2008 ) . gh - deficiency has been shown to contribute to increased cardiovascular risk in hypopituitarism ( abs et al . , 2006 ; verhelst and abs , 2009 ) . the decision to replace gh in patients with a previous history of cp remains controversial although gh replacement does not appear to have any adverse effect on reoccurrence rates of cp in short term follow - up ( muller et al . hypogonadism and inadequately treated diabetes insipidus ( di ) can also potentiate the effects of hypothalamic damage on excessive weight gain . testosterone replacement , in hypogonadal men has been shown to have beneficial effects on body composition , blood pressure , and glucose homeostasis ( katznelson et al . the effects of estrogen replacement on weight gain and body composition are more mixed and unclear , but premenopausal women who are hypogonadal should be physiologically replaced barring other contraindications ( norman et al . , 2000 ) . inadequate control of di can lead to excessive drinking of calories , poor sleep quality , and increased appetite ( beccuti and pannain , 2011 ) . altered carbohydrate and insulin dynamics have been shown to occur with cp and its treatment . while fasting glucose levels are generally no different from controls , cp patients have an exaggerated first and second phase insulin response to carbohydrate challenge ( lustig et al . , 1999 ) . hyperinsulinism in hypothalamic damage is a result of both decreased inhibitory tone by sympathetic pathways and augmented vagal parasympathetic signaling to the pancreatic -cell , and is not primarily a response to insulin resistance . vagally mediated acetylcholine , acting through m3 muscarinic receptors , promotes depolarization of the -cell through increased sodium influx , and mobilization of intracellular calcium stores through activation of the phospholipase c pathway ( miura et al . , 1996 ) . in addition , increased vagal activity stimulates release of the intestinal peptide glucagon - like peptide-1 , itself a potentiator of insulin secretion ( rocca and brubaker , 1999 ; lustig , 2008 ) . increased insulin secretion in turn directs calories toward storage within the adipocyte . thus , the disruption of hypothalamic centers that occurs as a direct result of cp or its treatment creates a situation in which efferent drives to consume and store energy are disengaged from afferent signals that would otherwise dampen such drives . the obesity that results , driven by neural and biochemical stimuli , is often severe and intractable to therapy . while lifestyle changes such as caloric restriction and increased exercise should be encouraged , these interventions alone are often not successful , leaving patients frustrated with cravings , constant hunger , and lack of progress . further , in addition to panhypopituitarism , hyob patients are susceptible to the same metabolic derangements seen in other types of obesity such as diabetes , dyslipidemia , and heart disease ( srinivasan et al . , 2004 ) . for patients already facing significant challenges as a result of their primary disease , the negative impact of such comorbidities on quality of life adds insult to injury . unfortunately , in the last several years pharmacologic agents available for weight loss treatment have significantly declined , as the most effective agents ( i.e. , phen fen , sibutramine ) have been withdrawn from the market for unacceptable side - effect profiles . small studies in cp of stimulants such as modafinil , methylphenidate , and dextroamphetamine have been shown to improve daytime sleepiness , affect , and alertness in hyob ( mason et al . , 2002 ; muller et al . , 2006 ) , but none have proven truly effective at reversing the severe weight gain seen in this condition . targeting the hyperinsulinism seen in hyob , lustig et al . ( 2006 ) demonstrated that octreotide could induce a modest decrease in bmi ( 0.79 kg / m ) . a recent preliminary trial of diazoxide and metformin combined therapy demonstrated a slowing of weight gain with a stabilization of bmi over the 6-months of therapy ( hamilton et al . , 2011 ) . because cp hyob results from damage to the afferent target centers of the hypothalamus , agents targeting the efferent pathways to increase metabolic rate and/or thermogenesis may prove to be more effective ( bays , 2004 ) . given the limited choices and poor results of pharmacotherapy , combined with emerging data suggesting that gastrointestinal surgical procedures effect weight loss by altering the body s weight regulatory signaling pathways , several groups have looked to bariatric surgery as a viable option for producing sustained weight loss for patients with cp hyob . a range of procedures are available and are generally divided into three categories : restrictive , malabsorptive , and hybrid procedures . restrictive procedures , such as gastric banding ( gb ) or vertical banded gastroplasty ( vbg ) , aim to reduce stomach volume resulting in early satiety and smaller meal consumption . pure malabsorptive procedures including jejunoileal and duodenal jejunal bypass , as well as biliopancreatic diversion ( bpd ) involve the diversion of ingested food , bypassing one or more sections of intestine . the former two of these procedures have largely fallen out of favor secondary to associated nutritional deficits ( organ et al . , 1984 ) . a hybrid procedure , the roux - en - y gastric bypass ( rygb ) has become the gold standard and the most commonly performed bariatric surgery for weight loss . it entails dividing the stomach to create a small gastric pouch which is anastomosed to a roux limb of jejunum . an entero - enterostomy is then made between the excluded biliopancreatic limb and the roux alimentary limb ( karra et al . , 2010 ) . bariatric surgery remains the most effective treatment for morbid obesity and is indicated for adult patients with a bmi > 40 kg / m or a bmi of > 35 kg / m and significant obesity - related comorbidities ( pories et al . , 1995 ) . more conservative indications have generally been advocated for weight loss surgery in adolescent patients ( see discussion and future directions ) . an extensive body of research has shown that patients often show measurable improvement in metabolic parameters including glucose homeostasis in the early post - operative period , independent of weight loss . while the exact mechanisms of this phenomenon are still unclear , it appears that gastric bypass alters the secretion of gut hormones that favor improved metabolism and may also counteract certain elements of the orexigenic and energy conserving hypothalamic response to voluntary weight loss . indeed , a number of studies over the last 30 years have shown that glp-1 , an incretin hormone secreted by enteroendocrine cells of the distal ileum , is increased following bariatric surgery . the evidence is especially consistent for bypass procedures , whereas restrictive procedures have been associated with no change or a decrease in glp-1 levels ( sarson et al . , 1981 ; koopmans et al . , 1984 ; laferrere et al . , 2008 ) . glp-1 has been shown to have a number of positive effects on glucose metabolism including potentiating glucose - dependent insulin secretion , improving insulin sensitivity , and suppressing glucagon secretion . in addition , it has been shown to slow gastric emptying and promote early satiety . glp-1 exerts these pleiotropic effect by acting on specific receptors located on pancreatic -cells , peripheral vagal afferent fibers , as well as centrally located neurons ( baggio and drucker , 2007 ) . in addition , bariatric surgery has been shown to increase other anorexigenic hormones such as pyy and decrease orexigenic hormones such as ghrelin ( cummings et al . human and animal studies appear to indicate that exposure of the distal gut to nutrient - rich , partially digested food results in increased secretion of hormones such as glp-1 and pyy . this altered hormonal milieu accounts for the greater than 80% remission in diabetes following rygb , often within days to weeks of surgery , and a combination of malabsorption , gastric restriction , and hormonal changes that accounts for the consistent and durable weight loss seen following such procedures ( buchwald et al . 2010 ) . despite the large body of evidence demonstrating the benefits of bariatric surgery in treating exogenous obesity and its comorbidities , its role in the treatment of hyob has yet to be defined . to date , the existing literature on bariatric surgery in cp hyob is largely limited to case reports and series with short term follow - up . a pubmed search for craniopharyngioma and bariatric surgery results in four citations with a total of eight patients . procedures performed include two rygb , four laparoscopic adjustable band ( lagb ) , one distal gastric bypass , and one biliopancreatic derivation with duodenal switch . in the first report of rygb for cp hyob , we reported on an 18-year - old male who underwent a successful rygb with anterior truncal vagotomy ( due to the presumed importance of vagal efferents in cp - related hyperinsulinemia ) for massive weight gain following the resection of a cp 3 years previous . at the time of cp diagnosis , , he developed hyperphagia and significant weight gain ( 70 kg / year ) despite outpatient and inpatient dietary and physical activity interventions . significant hyperinsulinism was noted and the patient was started on octreotide therapy with significant deceleration in weight gain but no weight loss . at the time of his bariatric surgery consultation , he had developed severe osa , left ventricular hypertrophy , and hypertriglyceridemia . the post - operative course was significant for a 49-kg weight loss over 2.5 years , although weight appeared to stabilize thereafter ( bmi 50 kg / m ) . hyperphagia decreased and food cravings ( as measured by a food craving inventory ) diminished for all food types . ten days following rygb surgery , there was a fivefold decrease in fasting insulin , with normalization of fasting levels by 7 months . as well , measurements of the orexigenic hormone ghrelin revealed a moderate decrease over the same time period . in a different approach , schultes et al . describe their approach to bypass surgery in a 29-year - old male with childhood cp . following cp resection 21 years previous , the patient had experienced continuous weight gain , developed type 2 diabetes , and osa requiring continuous positive airway pressure therapy at night . at the time of hyob surgical consultation , this patient was on hormone replacement for panhypopituitarism as well as metformin and nateglinide for diabetes treatment . to promote preoperative weight loss , he was started on 15 mg sibutramine and enrolled in a rigorous supervised diet and exercise regimen which resulted in 9 kg weight loss over 9 months . because bypass - induced hormonal alterations may exert their influence via hypothalamic mechanisms , it was felt that this patient , with complete hypothalamic atrophy , may not benefit . therefore , a distal gastric bypass , producing a significantly shorter common channel of 80 cm , was performed with the goal of inducing a strong malabsorption component . following surgery , the patient experienced a precipitous weight loss , losing over 50 kg ( 30% of preoperative weight ) over 18 months . in addition , the patient exhibited complete resolution of his diabetes and osa and distinct reduction feelings of hunger and disinhibition on the three factor eating questionnaire ( schultes et al . , 2009 ) . the post - operative course was largely uneventful , although the patient did develop mild nutritional deficiencies despite standardized supplementation . these included anemia , deficiencies in vitamin d , b12 , and zinc as well as hypoproteinemia . in a series of four adolescent patients with childhood cp and obesity , muller et al . found significant weight loss in all patients after lagb . patients participated in the german multicenter surveillance study on childhood cp termed hit - endo - kraniopharyngeom . the age of the patients ranged from 13 to 24 years of age and bmi sds scores improved on average by 0.7 per year of follow - up , with follow - up ranging from 1.5 to 4.5 years . patients also reported significant changes in eating behavior , with reduced food cravings especially for sweets . quality of life and functional capacity , measured by a standardized and validated scale , remained stable following surgery . of the four patients , two experienced dislocation of the lagb resulting in temporary weight gain and requiring laparoscopic revision . no other side - effects were reported ( muller et al . , 2007 ) . ( 2009 ) reported on two adolescents with post - cp hyob who underwent bariatric surgery as treatment for morbid obesity associated with significant comorbidities . the first was a 12-year - old female diagnosed with cp at age of 6 years . after gross total resection she developed panhypopituitarism and exhibited a rapid weight gain of 20 kg in the first 6 months . at the time of rygb her bmi was 65 kg / m and she had developed dyslipidemia , hepatosteatosis , frequent respiratory tract infections , and evidence of hypoventilation . in addition , dyslipidemia normalized , sleep patterns improved , and she had no further respiratory infections . she was subsequently diagnosed with fibromyalgia and symptomatic hyperuricemia , which were treated with regular analgesia and allopurinol . in addition , she developed a dumping - type syndrome ( pallor , diaphoresis , and shakiness following meals ) without associated hypoglycemia . the second patient was a 15-year - old male , diagnosed with cp at age of 4 years , also with panhypopituitarism and rapid weight gain following gross total resection . comorbidities included dyslipidemia , hepatosteatosis , and hyperinsulinism . at the age of 14 years , a trial of octreotide led to no reduction of weight gain . at the time of bariatric surgery , his bmi was 42 kg / m . given that the underlying cause of obesity was biological and would be present life - long , the surgeons involved felt that a diversionary surgical procedure would be a better option than lagb . following a bpd with duodenal switch , his bmi decreased to 32 kg / m over the 2-year follow - up . the post - operative course was complicated by bradycardia requiring pacemaker placement , and stenosis of the distal surgical anastomosis requiring several laparoscopic repairs . overall , both patients exhibited robust , sustained weight loss with resolution of metabolic comorbidities . the efficacy of bariatric surgery for weight loss has also been examined in other hyob patient populations . pws is a complex genetic disorder that results in marked hyperphagia and obesity felt to be secondary to hypothalamic dysfunction . the critical analysis revealed that a number of procedures , including bpd , rygb , vbg , and endoscopic intragastric balloon placement have been used in this population . a review of 60 pws patients highlighted the limited effectiveness and concerning safety profile of operations for weight loss in this unique population ( scheimann et al . in the short term , there appears to be some limited weight loss that is variable across different procedures . for rygb , twelve month and 24 month follow - up revealed 6.5 and 2% weight loss , respectively . this weight loss is far less than the 35% reduction in bmi we see 1 year after rygb for adolescents who do not have pws . five year follow - up , while limited , showed only a 2.4% weight loss in pws patients after rygb ( n = 9 ) . long term weight loss was worse with vbg , with pws patients experiencing a 3.5% weight gain after 5 years ( n = 2 ) . patients undergoing bpd had higher weight loss of 27.6% at 12-months , but nearly half regained weight within 25 years . in addition , the report highlights what appears to be a high post - operative complication rate , with 47% requiring surgical revision after rygb and 27% experiencing acute gastric dilation ( scheimann et al . , 2008 ) . patients with bbs demonstrate hyperphagia and obesity which have been linked to disruption of primary cilia in pomc neurons in the hypothalamus and possibly to altered leptin receptor functioning . in this 16-year - old with a bmi of 53 kg / m , rygb resulted in a 33% reduction in bmi 3 years following surgery ( daskalakis et al . , as melanocortin 4 receptor ( mc4r ) variants represent the most common monogenetic form of hyob in humans , it is relevant to assess the outcome of bariatric surgery in individuals with documented mc4r mutations . surgical experience in this special group of hyob patients a group of investigators at the university of california san francisco ( ucsf ) described surgical outcomes in an adolescent patient with a complete loss of function mutation at mc4r . this 18-year - old male presented with a preoperative weight of 166 kg and bmi of 54 kg / m and underwent bilateral truncal vagotomy and laparoscopic adjustable gb . in this patient , the surgical intervention did not result in successful weight loss . indeed , after an initial modest weight reduction of 12 kg over the first 4 months , the patient regained weight and by 1 year was 6.5 kg over his preoperative weight . first , the patient had moved away to college 4 months prior to the operation , and had in that time experienced a 66-kg weight gain . also , the patient returned for only two band adjustments in the 1-year period of time , which is markedly less than the typically recommended post - operative adjustment schedule . finally , the authors described an insatiable hunger 1 year after the operation , which was almost certainly a factor contributing to weight gain ( aslan et al . , 2011b ) . in a more recent publication from the same group at ucsf , the first report of an experience with gastric bypass in the setting of mutant mc4r contrasts with the results obtained after gb . aslan et al . analyzed 1 year outcomes of four adults with heterozygous mc4r mutations in comparison to matched controls with normal mc4r gene sequences . excess weight loss ( ewl ) in these four subjects was identical to the 6070% ewl seen in matched controls . there was no mention of changes in post - operative appetite in these patients , nor any changes in metabolic features in this cohort ( aslan et al . hypothalamic obesity is observed in a high percentage of patients with cp , resulting in significant comorbidities and negatively impacting quality of life . first line therapy for hyob should include aggressive lifestyle modification including appropriate caloric intake and activity . pituitary hormone deficiencies , if any , should be replaced and doses optimized . as mentioned previously , glucocorticoid replacement should be targeted at replacement doses ( 810 mg / m / day hydrocortisone equivalent ) and patients should be given the lowest dose possible that is adequate to avoid symptoms of adrenal insufficiency . thyroid replacement should generally target the ft4 at the upper third of the normal range and patients should be clinically and biochemically monitored for signs / symptoms of hyper- and hypothyroidism ( rose , 2010 ) . the decision to replace gh should be made in consultation with the patient , family , and other team members such as their oncologist and neurosurgeon . if done , gh doses should be titrated to keep igf-1 levels within the median range . ideally , other medical treatments aimed specifically at ameliorating weight should be pursued through approved protocols at centers capable of collecting outcomes data on a sufficiently large number of patients so that risks and benefits of treatment can be more precisely defined . , we have reviewed the current body of published research on bariatric surgery in cp hyob . we have also included several published reports of bariatric surgery in other populations of hyob , including pws , bbs , and patients with mc4r mutations . while the limited scope of these reports makes comparison difficult , the experience in pws patients demonstrates that all cases of hyob should not be treated equally . likely as a result of the multisystemic nature of their disorder , pws patients have a significantly increased incidence of complications following bariatric surgery without the benefits of sustained weight loss . in addition , it appears that early dietary interventions are able to partially abrogate weight gain this population ( schmidt et al . , 2008 ) . thus , the risk benefit ratio of bariatric surgery in pws does not appear to be favorable . the initial reports of bariatric surgery in cp hyob and patients with mc4rappear more promising , but again , their limited scope makes it difficult to draw any substantial conclusions as to the long term safety and efficacy of these procedures in this population . for instance , how does rygb compare to lagb with regard to long term , sustained weight loss , or with regard to safety and side - effects ? in addition , more work is needed to understand the hormonal alterations that occur in cp hyob patients derive the same benefits given their underlying hypothalamic damage ? if hyperinsulinism is an underlying factor in the development of hyob , will patients benefit or be harmed from increased levels of the insulin secretagogues such as glp-1 . will potentiating insulin secretion place these patients at higher risk of hypoglycemia secondary to the mismatch of insulin secretion and carbohydrate absorption ? these and other questions remain unanswered by the current body of research in this population and should be the subject of future work . in addition , as 50% of patients diagnosed with cp are children , there are serious medical , psychological , and ethical concerns regarding bariatric surgery that must be considered in this population ( inge et al . , 2004 ) . generally , adolescent candidates for bariatric surgery must meet more conservative patient selection criteria than are acceptable in adults . however , exact criteria are still of some debate . in 2008 , the endocrine society , in conjunction with the pediatric endocrine society , published a clinical practice guideline on the prevention and treatment of pediatric obesity , wherein the task force recommended limiting bariatric surgery to adolescents with a bmi 50 kg / m or a bmi 40 kg / m with significant , severe comorbidities . in addition , the task force recommended that candidates have attained tanner 4 or 5 pubertal development and near - final adult height , experienced continued weight gain despite following a formal program of lifestyle modification , and belong to a stable , medically competent family unit . the patient and family should understand that bariatric surgery is not a cure for obesity , but rather an adjunct to a continued commitment to lifestyle modifications in diet and activity . the surgery should be performed by an experienced surgeon in a medical center employing a team capable of long term follow - up and participating in a study of bariatric surgery outcomes . these recommendations largely agree with guidelines previously published and advocate the priority of avoiding unforeseen complications associated with life - long exposure to anatomical and functional alterations above the value of weight loss and amelioration of obesity - related complications provided by bariatric surgery ( august et al . , 2008 ) . 2009 ) suggest that adolescents with a bmi 35 kg / m and serious comorbidities including type 2 diabetes , moderate to severe osa , and/or pseudotumor cerebri or those with bmi 40 kg / m with less serious comorbid conditions such as hypertension , dyslipidemia , gastroesophageal reflux disease , and/or psychosocial stress should be considered as candidates for bariatric surgery . while concurring with the other physical , lifestyle , and psychosocial criteria previously published , they use several lines of evidence to defend the lower bmi criteria . first , they cite data to indicate that patient safety and weight loss outcomes for extremely obese adolescents who undergo bariatric surgery are comparable to , or better than , those seen in adults . second , even with the lower bmi criteria of 35 kg / m , candidates under the age of 18 would still be above the 99th bmi percentile and be at substantially increased risk for short and long term medical comorbidities . third , they stress that selection for bariatric surgery during adolescence should be closely linked to obesity - related comorbidities . the less severe comorbidities and/or fewer risk factors for long term disease , the higher the bmi cut point should be before considering bariatric surgery . finally , some have suggested that because younger patients will generally have fewer advanced comorbidities , early bariatric surgery may decrease the risk of future perioperative mortality , assuming the needed surgery is delayed until adulthood ( garcia and demaria , 2006 ) . importantly , none of the guidelines for bariatric surgery directly addresses the use of bariatric surgery in hyob generally or cp hyob in particular . in general , we believe that most criteria from the published guidelines can be applied to the hyob patients , especially in adults . whether one or more of the published criteria should be modified in the context of children and adolescents given what is known about the natural history of hyob , its resistance to diet and lifestyle modification , and the extreme and progressive weight gain involved , some may consider bariatric surgery appropriate at ages currently considered too young in patients with exogenous obesity . however , in general , the younger the patient the more compelling and serious the comorbidities must be to justify surgical intervention . because of the need for life - long medical support , optimal care of hyob patients will be best met in the setting of tertiary care centers where they can receive treatment in a coordinated , multidisciplinary fashion . team members should include the primary care physician and as needed subspecialists in neurosurgery , neurology , oncology , endocrinology , bariatric surgery , and mental health . ideally , ancillary support by nutrition , physical therapy , nursing , and social work should also be available . patients and families choosing to undergo bariatric surgery should be properly informed regarding specific risks and benefits associated with surgical weight loss and age appropriate consent or assent obtained . to facilitate needed research in this area these early reports of outcome of surgical weight loss treatment of hyob are limited in scope and number , but give us a glimpse of the possible role of surgery in treatment of these conditions . they also make evident the need for more robust , controlled , prospective studies with long term follow - up in order to better define the role of medical as well as surgical therapies in the treatment of hyob . in order to ultimately understand the outcome of surgical therapy for hyob , sufficiently long follow - up of sufficient numbers of similar patients who undergo surgery as well as non - surgical controls non - human surgical models might also in the future permit a better understanding of the mechanisms of obesity , and the importance of tailoring specific operative elements to each unique population . research in this important area is hampered by the difficulty of assembling a sufficiently large cohort of cp hyob patients . given the relative rarity of the condition , it would take a single institution many years to put together a study large enough to reach valid , persuasive conclusions regarding the treatment of hyob surgical or otherwise . in order to increase public awareness of hyob , facilitate future research and ultimately improve the management of hyob , the international registry for hyob disorders ( www.irhod.org ) has been established . the web - based interface will permit both healthcare providers and patient / families to easily enter basic information regarding their hyob case . the systematic accumulation of a large world - wide cohort of patients with hyob will facilitate recognition of individuals eligible for participation in studies of hyob and its treatment . this registry , therefore , has potential to fill many of the numerous knowledge gaps around pathogenesis , coexisting disease , and treatment outcomes for this debilitating form of obesity . 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 .

craniopharyngiomas ( cp ) are epithelial neoplasms generally found in the area of the pituitary and hypothalamus . despite benign histology , these tumors and/or their treatment often result in significant , debilitating disorders of endocrine , neurological , behavioral , and metabolic systems . severe obesity is observed in a high percentage of patients with cp resulting in significant comorbidities and negatively impacting quality of life . obesity occurs as a result of hypothalamic damage and disruption of normal homeostatic mechanisms regulating energy balance . such pathological weight gain , termed hypothalamic obesity ( hyob ) , is often severe and refractory to therapy . unfortunately , neither lifestyle intervention nor pharmacotherapy has proven effective in the treatment of hyob . given the limited choices and poor results of these treatments , several groups have examined bariatric surgery as a treatment alternative for patients with cp hyob . while a large body of evidence exists supporting the use of bariatric surgery in the treatment of exogenous obesity and its comorbidities , its role in the treatment of hyob has yet to be defined . to date , the existing literature on bariatric surgery in cp hyob is largely limited to case reports and series with short term follow - up . here we review the current reports on the use of bariatric surgery in the treatment of cp hyob . we also compare these results to those reported for other populations of hyob , including prader willi syndrome , bardet biedl syndrome , and hypothalamic melanocortin signaling defects . while initial reports of bariatric surgery in cp hyob are promising , their limited scope makes it difficult to draw any substantial conclusions as to the long term safety and efficacy of bariatric surgery in cp hyob . there continues to be a need for more robust , controlled , prospective studies with long term follow - up in order to better define the role of bariatric surgery in the treatment of hyob .

Introduction Hypothalamic Obesity Surgical Treatment of HyOb Discussion and Future Directions Conflict of Interest Statement

craniopharyngiomas ( cp ) are generally benign , slow - growing tumors that are thought to arise from rests of embryonic cells residing within the remnants of the craniopharyngeal duct or rathke s pouch ( abeloff , 2008 ) . thus , tumors may originate from anywhere along the path of the craniopharyngeal duct , from nasopharynx to tuber cinereum , although most are located in the sellar / parasellar region ( harwood - nash , 1994 ; karavitaki et al . while mortality is generally low , morbidity as a result of tumor mass effect and/or therapy ( resection radiotherapy ) is substantial and nearly universal ( mortini et al . because of the close proximity to the visual nerve tracts , hypothalamus , pituitary , and ventricular system , cps predispose patients to a number of adverse endocrine , metabolic , psychologic , and neurologic sequelae . as a primary result of the tumor , the majority of patients present with one or more hypothalamic - pituitary deficits including growth hormone ( gh ; 75% ) , gonadotropins ( 60% ) , adrenocorticotropic hormone ( acth ; 30% ) , thyroid - stimulating hormone ( tsh ; 25% ) , and/or antidiuretic hormone ( adh ; 20% ; karavitaki et al . , 2001 ; while a minority ( 1520% ) of cp patients complain of weight gain as a presenting symptom , more than 50% develop significant obesity following surgical resection ( muller et al . this number may be as high as 90% in those with demonstrable hypothalamic damage , whether from direct tumor infiltration or as a result of surgical and/or radiation therapy ( de vile et al . such pathological weight gain , termed hypothalamic obesity ( hyob ) , is often severe , refractory to therapy , and has a significant negative impact on the quality of life for patients with cp ( muller et al . , 2005 ; over the last 75 years , experience with animal and human models has shown the basal medial hypothalamus to be a key center of metabolic regulation by the central nervous system ( cns ; hetherington and ranson , 1940 ; sorva , 1988 ) . the afferent arm of the homeostatic loop consists of hormones , such as leptin , insulin , and peptide yy , that target receptor fields within hypothalamic nuclei to convey information on meal size , nutrient composition , and adipose tissue stores . , 2009 in addition , several complex genetic syndromes , including prader willi ( pws ) and bardet biedl syndromes ( bbs ) , are associated with obesity , presumably from hypothalamic dysfunction . pws results from the loss of paternally imprinted genes on chromosome 15q11 - 15 and is characterized by an array of multisystemic defects including neonatal hypotonia , short stature , hypogonadism , behavioral and psychiatric phenotypes , aggressive food - seeking behavior , hyperphagia , and obesity ( goldstone et al . pws patients have significantly elevated levels of the orexigenic hormone ghrelin , a finding not observed in other causes of hyob ( cummings et al . accumulating evidence supports the hypothesis that cp and its therapies often result in dysfunction of the normal homeostatic mechanisms regulating appetite and metabolism . leptin , a potent anorexigenic peptide secreted by adipocytes , has been found to be elevated in cp patients compared to obese controls suggesting a defect in the normal feedback inhibition of appetite . hypersecretion of the orexigenic gastric hormone ghrelin is thought to contribute to hyperphagia and obesity in pws , although this does not seem to be the case in cp where pre - meal ghrelin levels are lower than weight - matched controls . found that there may be a reduction in the expected magnitude of post - meal suppression of ghrelin levels in cp patients ( roth et al . , 1989 ) , a number of studies have now shown that caloric expenditure , and not caloric intake , may be the largest contributor to post - cp hyob ( cp hyob ) . adults and children with cp hyob have been found to have impaired sympathoadrenal activation in response to hypoglycemia , although this defect does not appear to always correlate with development of obesity or hypothalamic involvement ( schofl et al . using food diaries , this same group showed that caloric intake was lower in cp patients with hypothalamic damage , despite a significantly increased bmi ( harz et al . under- or overtreatment of hormonal deficiencies may also contribute to poor metabolic parameters and weight gain in cp patients . glucocorticoid excess is a known cause of significant weight gain , impaired glucose metabolism , sleep disturbance , and defects in bone metabolism and growth ( debono et al . in a large cohort of scandinavian patients with hypopituitarism , filipsson et al . , 1990 ; thus , the classic adult dosing of 20 mg of hydrocortisone in the a.m. and 10 mg in the p.m. , is likely an excessive dose . unfortunately , no objective test of adequate glucocorticoid replacement has been shown to be sufficiently consistent to aid in dosing management and patients must be followed closely for clinical signs of glucocorticoid deficiency or excess . management of thyroid hormone replacement is made more challenging in cp patients by the absence of the patient s own endogenous the significance of this handicap is highlighted in a recent study comparing free t4 ( ft4 ) levels in tsh deficient patients to those in primary hypothyroid patients where tsh levels are used to guide therapy . growth hormone deficiency is present in a majority of cp patients and likely contributes to the metabolic derangements seen in this patient population . the decision to replace gh in patients with a previous history of cp remains controversial although gh replacement does not appear to have any adverse effect on reoccurrence rates of cp in short term follow - up ( muller et al . hypogonadism and inadequately treated diabetes insipidus ( di ) can also potentiate the effects of hypothalamic damage on excessive weight gain . altered carbohydrate and insulin dynamics have been shown to occur with cp and its treatment . hyperinsulinism in hypothalamic damage is a result of both decreased inhibitory tone by sympathetic pathways and augmented vagal parasympathetic signaling to the pancreatic -cell , and is not primarily a response to insulin resistance . thus , the disruption of hypothalamic centers that occurs as a direct result of cp or its treatment creates a situation in which efferent drives to consume and store energy are disengaged from afferent signals that would otherwise dampen such drives . the obesity that results , driven by neural and biochemical stimuli , is often severe and intractable to therapy . while lifestyle changes such as caloric restriction and increased exercise should be encouraged , these interventions alone are often not successful , leaving patients frustrated with cravings , constant hunger , and lack of progress . further , in addition to panhypopituitarism , hyob patients are susceptible to the same metabolic derangements seen in other types of obesity such as diabetes , dyslipidemia , and heart disease ( srinivasan et al . for patients already facing significant challenges as a result of their primary disease , the negative impact of such comorbidities on quality of life adds insult to injury . unfortunately , in the last several years pharmacologic agents available for weight loss treatment have significantly declined , as the most effective agents ( i.e. small studies in cp of stimulants such as modafinil , methylphenidate , and dextroamphetamine have been shown to improve daytime sleepiness , affect , and alertness in hyob ( mason et al . , 2006 ) , but none have proven truly effective at reversing the severe weight gain seen in this condition . because cp hyob results from damage to the afferent target centers of the hypothalamus , agents targeting the efferent pathways to increase metabolic rate and/or thermogenesis may prove to be more effective ( bays , 2004 ) . given the limited choices and poor results of pharmacotherapy , combined with emerging data suggesting that gastrointestinal surgical procedures effect weight loss by altering the body s weight regulatory signaling pathways , several groups have looked to bariatric surgery as a viable option for producing sustained weight loss for patients with cp hyob . restrictive procedures , such as gastric banding ( gb ) or vertical banded gastroplasty ( vbg ) , aim to reduce stomach volume resulting in early satiety and smaller meal consumption . a hybrid procedure , the roux - en - y gastric bypass ( rygb ) has become the gold standard and the most commonly performed bariatric surgery for weight loss . bariatric surgery remains the most effective treatment for morbid obesity and is indicated for adult patients with a bmi > 40 kg / m or a bmi of > 35 kg / m and significant obesity - related comorbidities ( pories et al . an extensive body of research has shown that patients often show measurable improvement in metabolic parameters including glucose homeostasis in the early post - operative period , independent of weight loss . indeed , a number of studies over the last 30 years have shown that glp-1 , an incretin hormone secreted by enteroendocrine cells of the distal ileum , is increased following bariatric surgery . despite the large body of evidence demonstrating the benefits of bariatric surgery in treating exogenous obesity and its comorbidities , its role in the treatment of hyob has yet to be defined . to date , the existing literature on bariatric surgery in cp hyob is largely limited to case reports and series with short term follow - up . in the first report of rygb for cp hyob , we reported on an 18-year - old male who underwent a successful rygb with anterior truncal vagotomy ( due to the presumed importance of vagal efferents in cp - related hyperinsulinemia ) for massive weight gain following the resection of a cp 3 years previous . following cp resection 21 years previous , the patient had experienced continuous weight gain , developed type 2 diabetes , and osa requiring continuous positive airway pressure therapy at night . in addition , the patient exhibited complete resolution of his diabetes and osa and distinct reduction feelings of hunger and disinhibition on the three factor eating questionnaire ( schultes et al . in a series of four adolescent patients with childhood cp and obesity , muller et al . the age of the patients ranged from 13 to 24 years of age and bmi sds scores improved on average by 0.7 per year of follow - up , with follow - up ranging from 1.5 to 4.5 years . quality of life and functional capacity , measured by a standardized and validated scale , remained stable following surgery . of the four patients , two experienced dislocation of the lagb resulting in temporary weight gain and requiring laparoscopic revision . ( 2009 ) reported on two adolescents with post - cp hyob who underwent bariatric surgery as treatment for morbid obesity associated with significant comorbidities . the second patient was a 15-year - old male , diagnosed with cp at age of 4 years , also with panhypopituitarism and rapid weight gain following gross total resection . at the time of bariatric surgery , his bmi was 42 kg / m . the post - operative course was complicated by bradycardia requiring pacemaker placement , and stenosis of the distal surgical anastomosis requiring several laparoscopic repairs . the efficacy of bariatric surgery for weight loss has also been examined in other hyob patient populations . the critical analysis revealed that a number of procedures , including bpd , rygb , vbg , and endoscopic intragastric balloon placement have been used in this population . in the short term , there appears to be some limited weight loss that is variable across different procedures . for rygb , twelve month and 24 month follow - up revealed 6.5 and 2% weight loss , respectively . five year follow - up , while limited , showed only a 2.4% weight loss in pws patients after rygb ( n = 9 ) . long term weight loss was worse with vbg , with pws patients experiencing a 3.5% weight gain after 5 years ( n = 2 ) . in addition , the report highlights what appears to be a high post - operative complication rate , with 47% requiring surgical revision after rygb and 27% experiencing acute gastric dilation ( scheimann et al . patients with bbs demonstrate hyperphagia and obesity which have been linked to disruption of primary cilia in pomc neurons in the hypothalamus and possibly to altered leptin receptor functioning . , as melanocortin 4 receptor ( mc4r ) variants represent the most common monogenetic form of hyob in humans , it is relevant to assess the outcome of bariatric surgery in individuals with documented mc4r mutations . first , the patient had moved away to college 4 months prior to the operation , and had in that time experienced a 66-kg weight gain . finally , the authors described an insatiable hunger 1 year after the operation , which was almost certainly a factor contributing to weight gain ( aslan et al . in a more recent publication from the same group at ucsf , the first report of an experience with gastric bypass in the setting of mutant mc4r contrasts with the results obtained after gb . hypothalamic obesity is observed in a high percentage of patients with cp , resulting in significant comorbidities and negatively impacting quality of life . , we have reviewed the current body of published research on bariatric surgery in cp hyob . we have also included several published reports of bariatric surgery in other populations of hyob , including pws , bbs , and patients with mc4r mutations . while the limited scope of these reports makes comparison difficult , the experience in pws patients demonstrates that all cases of hyob should not be treated equally . likely as a result of the multisystemic nature of their disorder , pws patients have a significantly increased incidence of complications following bariatric surgery without the benefits of sustained weight loss . thus , the risk benefit ratio of bariatric surgery in pws does not appear to be favorable . the initial reports of bariatric surgery in cp hyob and patients with mc4rappear more promising , but again , their limited scope makes it difficult to draw any substantial conclusions as to the long term safety and efficacy of these procedures in this population . for instance , how does rygb compare to lagb with regard to long term , sustained weight loss , or with regard to safety and side - effects ? in addition , more work is needed to understand the hormonal alterations that occur in cp hyob patients derive the same benefits given their underlying hypothalamic damage ? if hyperinsulinism is an underlying factor in the development of hyob , will patients benefit or be harmed from increased levels of the insulin secretagogues such as glp-1 . these and other questions remain unanswered by the current body of research in this population and should be the subject of future work . in addition , as 50% of patients diagnosed with cp are children , there are serious medical , psychological , and ethical concerns regarding bariatric surgery that must be considered in this population ( inge et al . in 2008 , the endocrine society , in conjunction with the pediatric endocrine society , published a clinical practice guideline on the prevention and treatment of pediatric obesity , wherein the task force recommended limiting bariatric surgery to adolescents with a bmi 50 kg / m or a bmi 40 kg / m with significant , severe comorbidities . in addition , the task force recommended that candidates have attained tanner 4 or 5 pubertal development and near - final adult height , experienced continued weight gain despite following a formal program of lifestyle modification , and belong to a stable , medically competent family unit . the surgery should be performed by an experienced surgeon in a medical center employing a team capable of long term follow - up and participating in a study of bariatric surgery outcomes . while concurring with the other physical , lifestyle , and psychosocial criteria previously published , they use several lines of evidence to defend the lower bmi criteria . first , they cite data to indicate that patient safety and weight loss outcomes for extremely obese adolescents who undergo bariatric surgery are comparable to , or better than , those seen in adults . the less severe comorbidities and/or fewer risk factors for long term disease , the higher the bmi cut point should be before considering bariatric surgery . finally , some have suggested that because younger patients will generally have fewer advanced comorbidities , early bariatric surgery may decrease the risk of future perioperative mortality , assuming the needed surgery is delayed until adulthood ( garcia and demaria , 2006 ) . importantly , none of the guidelines for bariatric surgery directly addresses the use of bariatric surgery in hyob generally or cp hyob in particular . whether one or more of the published criteria should be modified in the context of children and adolescents given what is known about the natural history of hyob , its resistance to diet and lifestyle modification , and the extreme and progressive weight gain involved , some may consider bariatric surgery appropriate at ages currently considered too young in patients with exogenous obesity . because of the need for life - long medical support , optimal care of hyob patients will be best met in the setting of tertiary care centers where they can receive treatment in a coordinated , multidisciplinary fashion . team members should include the primary care physician and as needed subspecialists in neurosurgery , neurology , oncology , endocrinology , bariatric surgery , and mental health . to facilitate needed research in this area these early reports of outcome of surgical weight loss treatment of hyob are limited in scope and number , but give us a glimpse of the possible role of surgery in treatment of these conditions . they also make evident the need for more robust , controlled , prospective studies with long term follow - up in order to better define the role of medical as well as surgical therapies in the treatment of hyob . in order to ultimately understand the outcome of surgical therapy for hyob , sufficiently long follow - up of sufficient numbers of similar patients who undergo surgery as well as non - surgical controls non - human surgical models might also in the future permit a better understanding of the mechanisms of obesity , and the importance of tailoring specific operative elements to each unique population . research in this important area is hampered by the difficulty of assembling a sufficiently large cohort of cp hyob patients . given the relative rarity of the condition , it would take a single institution many years to put together a study large enough to reach valid , persuasive conclusions regarding the treatment of hyob surgical or otherwise . in order to increase public awareness of hyob , facilitate future research and ultimately improve the management of hyob , the international registry for hyob disorders ( www.irhod.org ) has been established . the systematic accumulation of a large world - wide cohort of patients with hyob will facilitate recognition of individuals eligible for participation in studies of hyob and its treatment . this registry , therefore , has potential to fill many of the numerous knowledge gaps around pathogenesis , coexisting disease , and treatment outcomes for this debilitating form of obesity . 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 .

host systems that can spontaneously self - assemble and stably encapsulate guest molecules under one set of conditions , but disassemble and release the guest molecules when external conditions change , have been of great interest in supramolecular chemistry due to implications in a variety of biomedical applications . while earlier studies have primarily focused on the former ( i.e. , self - assembly and binding ) , there has been a recent surge in interest in the latter features ( disassembly and release in response to an environmental change ) . also , the responsiveness of the assemblies has primarily focused on physical or chemical changes . for example , there has been a significant interest in systems that disassemble in response to chemical stimuli such as ph or redox variation as well as physical stimuli such as light , temperature , or a magnetic field . while systems incorporating features sensitive to light and magnetic field have been designed to respond to external triggers for biological applications , ph- and redox - responsive systems have been designed to respond to the inherent imbalances observed in certain disease tissues . the anomalous ph or redox conditions in disease locations can be considered to be secondary imbalances in biology , as the primary imbalances are often the result of aberrant protein concentrations or enzymatic activity . therefore , there is a growing interest in developing supramolecular systems that respond to these primary factors in biology . covalent modification of the hosts to disable their capacity to hold the guest molecules and noncovalent binding to the host assemblies to produce the same effect . the former is often achieved by an enzyme - driven chemical reaction that modulates the host characteristics of the molecule . in the latter case , this is achieved due to a noncovalent binding interaction . while there have been several systems designed to be degraded or covalently modified by enzymes , supramolecular assemblies that lose their host capacities due to noncovalent binding with proteins have been limited . among the systems that undergo binding - induced disassembly , polyelectrolyte assemblies that can noncovalently bind a complementarily charged surface to cause although nonspecific in its interaction , the simplicity of these systems has proved useful in applications such as separations . it has also been clear that for a binding - induced approach to be useful in applications such as delivery and sensing , where specificity is critical , strategies that use specific ligand protein interactions are needed . since dendritic macromolecules can be produced in high molecular weights , but with a great degree of control , these scaffolds have certain unique advantages for this strategy . for example , the critical aggregate concentrations ( cac ) of the dendrimer - based amphiphilic assemblies are low , an advantage that polymeric systems have over small molecule - based amphiphilic assemblies . similarly , the control over functional group placements in dendritic architectures captures the advantage that small molecules have over their polymeric counterparts . property relationship studies that unravel the factors that underlie the binding - induced supramolecular disassembly process . in this manuscript , we take advantage of this unique feature by incorporating ligands at specific locations within facially amphiphilic dendrons and interrogating the efficiency of the supramolecular disassembly and molecular release in response to a specific protein binding event . we use both experiments and molecular dynamics ( md ) simulations synergistically to gain insights into the ligand facially amphiphilic dendrons , containing a biaryl - based internal repeat unit and an aryl peripheral unit , bear both hydrophilic and hydrophobic functional groups as side chains in each of these repeat units . the amphiphilic functional groups are placed at opposite faces of the biaryl backbone of the dendron such that these molecules are endowed with the capability to form micelle - like assemblies in aqueous solution and form inverse micelle - like assemblies in apolar solvents . in our preliminary findings , we have shown that placing a ligand moiety at the hydrophilic face of the dendron can provide binding - induced disassembly of the micelle - like structure in the aqueous solvent . in that work , the ligand moiety was placed at the focal point of the dendron , as this structure is synthetically most easily accessible . however , the release efficiency of guests , following protein binding , was relatively moderate . in order to fully realize the potential of this process , it is critical that we understand the effect of the functional group placement , thus taking advantage of one of the most prominent features of dendritic architectures in this supramolecular process . therefore , in this manuscript , we investigate the effect of the placement of a protein - specific ligand moiety at specific positions of a dendron upon the accessibility of the complementary protein to the supramolecular assembly and hence the effects on the concomitant guest release response ( figure 1 ) . the relative encapsulation of functional groups , when placed in the periphery vs the focal point of the dendron is well - known . note however that the facially amphiphilic dendrons , utilized here , have two distinct characteristics in this context : ( i ) the ligand moieties placed in the hydrophilic face of the dendron is attached through a rather long ethyleneglycol linker . therefore , the classical encapsulation by the dendritic backbone should not apply here ; ( ii ) it has been previously shown using carboxylic acid based facially amphiphilic dendrimers that these functional groups are available for nonspecific binding to the positively charged surface of chymotrypsin . for these reasons , it is important that we investigate the effect of ligand placement on the binding - induced release . the scheme is an illustration of the self - assembling dendrons with sequestered hydrophobic guest molecules , which are released in response to protein binding . the equilibrium illustrated in the illustration is to indicate the possible unimer - aggregate equilibrium . the cac of even the first generation of these dendrons is substantially lower than the corresponding amphiphilic small molecule ( m compared to mm ) . note that this 3 orders of magnitude difference in cac is not accounted for by the simple difference in molecular weight of the amphiphilic molecules . within each generation of dendrons however , the cac gain has been relatively small , if any , especially when one accounts for the difference in molecular weights . therefore , we focused on the g1 and g2 dendrons , which provide sufficient variations in the functional group placement . we targeted five different dendrons within these two generations , where the ligand placement is the key difference ( see figure 1 for the structures ) . pentaethyleneglycol monomethyl ether ( peg ) was chosen as the hydrophilic moiety not only for its ability to provide the requisite solubility in the aqueous phase but also for its propensity to present a charge neutral surface on the assembly that exhibits minimal nonspecific interactions . biotin was chosen as the ligand , because of its well - established high affinity toward avidin . a ligand can be incorporated on a g1 dendron either at the focal point or at the periphery . on a g2 dendron , a ligand can be incorporated on three different layers : ( i ) the focal point ; ( ii ) the middle layer ; and ( iii ) the periphery . to install a single ligand at a specific place , one of the peg units was replaced by the ligand , attaching it to the dendron backbone via a hydrophilic linker . this placement allows the ligand to be exposed to the aqueous solution on the hydrophilic face of the assembly . note that the focal point is synthetically the easiest place to attach a single functional group , since the focal point is a single and unique position in a dendron . in contrast , to install a single functional group at the periphery , one has to distinguish one of the peripheral monomers from otherwise identical ones within the dendron . the g1 dendron has two such identical positions in the periphery , while the g2 dendron has four such locations . similarly , the middle layer of the g2 dendron contains two similar positions that need to be distinguished for specifically placing a ligand moiety . accordingly , the degree of difficulty in placing a unique functional group in the dendron increases as we move from the focal point to the periphery and as we increase the generation . to achieve these selective functionalization , we and others have developed a variety of synthetic strategies that afford multifunctionalized dendrons and dendrimers . in all our syntheses , we initially targeted dendrons containing an acetylene moiety , which will be used as the handle to click the biotin moiety in the last steps of the syntheses . overall , we made the syntheses of the dendrons modular in order to assemble the targeted dendrons in small number of steps . accordingly , we first synthesized the biaryl , amphiphilic ab2 monomer 1 , the propargyl - functionalized periphery unit 2 , and the nonfunctionalized amphiphilic periphery unit 4 following the previously reported procedures . to achieve the targeted dendron 5 , the biaryl monomer 1 was first monoalkylated with a periphery unit 2 to get the propargyl - functionalized scaffold 3 ( scheme 1 ) in 25% yield . the low isolated yield was because of the statistical distribution of mono- and disubstituted products obtained in this reaction . molecule 3 was then treated with the amphiphilic peripheral monomer 4 under similar alkylation conditions to achieve the g1 dendron 5 containing the reactive propargyl moiety at its periphery . the g1 dendron 5 was then treated with the azide - modified biotin 6 under alkyne azide click chemistry conditions in the presence of cupric sulfate and sodium ascorbate to obtain g1-p in 70% yield . similarly , the molecule g1-f was prepared by clicking the biotin azide to the propargyl moiety present in the biaryl repeat unit ( see supporting information for synthetic details ) . the syntheses of the targeted g2 dendrons were achieved using a similar set of synthetic strategies , as shown in scheme 2 . to achieve the syntheses of these dendrons , the previously reported amphiphilic g1 dendron 7 was treated with the biaryl monomer 1 , where the monoalkylated product 8 was separated from a statistical mixture in 24% yield . this molecule was then treated with the bromomethyl dendron 9 ( obtained from the precursor to g1-f in one step ) or 10 ( obtained from 5 in one step ) to obtain the g2 dendrons 11 or 12 , containing the propargyl moiety at the middle layer or the periphery of the dendron , respectively . copper - catalyzed huisgen reaction of 11 and 12 with the azide - functionalized ligand 6 afforded the g2-m and g2-p dendrons , respectively . as with g1-f , the g2-f dendron was obtained by clicking the biotin azide to the propargyl moiety present in the biaryl repeat unit ( see supporting information for synthetic details ) . first , the self - assembly properties of these dendrons were studied by measuring their cacs using nile red as the hydrophobic , spectroscopic probe ( figure s1 ) . as anticipated , the cacs of final g1 and g2 dendrons were determined to be in the low micromolar range with values of 11.65 and 8.02 m for g1-f and g1-p , respectively ; 7.25 , 7.08 , and 7.01 m for g2-f , g2-m , and g2-p , respectively . with the installment of a single ligand at different layers of a dendron and after knowing the lowest concentration at which they assemble into nanostructures , we investigated the response of the different dendritic aggregates in presence of the complementary protein extravidin and in presence of noncomplementary proteins with diverse pi values and molecular weights , viz . -chymotrypsin ( chy , pi = 8.18.6 ) , pepsin ( pep , pi = 2.9 ) , and myoglobin ( myo , pi = 7.2 ) , as shown in figure 2 . prior to analyzing the interaction between the dendritic assemblies and the proteins , we analyzed the size of the assemblies in aqueous phase using dynamic light scattering ( dls ) , with g1 dendron concentrations of 12.5 m and g2 dendron concentrations of 10 m ( both above their respective cacs ) . the sizes of the assemblies were found to be in a few tens of nanometers , ranging from 30 to 200 nm , 44 nm for g1-f , 220 nm for g1-p , 29 nm for g2-f , 92 nm for g2-m , and 51 nm for g2-p . the reason for the variations in size with the subtle change in the position of the ligand is not clear . however , note that the replacement of a hydrophilic peg chain in a g1 or g2 dendron by a less hydrophilic pendant biotin decreases the peg density on the assembly surface , decreasing hydrophilicity and increasing the chance of nonspecific interactions with noncomplementary proteins . such decrease in peg density could be more relevant in g1 , where substituting one of the three peg chains in a dendron molecule could mean a reduction of up to 33% , while such a reduction in g2 is about 14% . it is possible that these differences confer changes in the way the assemblies pack together in solution , which results in size variations among the biotin - functionalized dendrimeric assemblies . it is interesting that the size of all these assemblies reduced to about 13 nm in presence of the protein , extravidin ( 2 m ) ( figure 2 ) . a particular difference in disassembly among the biotin - functionalized dendrons was observed in the case of the g2-p that disassembled into smaller aggregates ( 78 nm ) in presence of extravidin ( figure 2e ) . g1-p also presented some deviations , showing larger aggregates ( 28 nm ) that could be formed by further aggregation of smaller protein dendron complexes . dls - based measure of size change of dendritic assemblies in aqueous phase upon interaction with different proteins . ( a ) g1-f , ( b ) g1-p , ( c ) g2-f , ( d ) g2-m , ( e ) g2-p , and ( f ) g1 and ( g ) g2 control dendrons . the decrease in the size of the assemblies was observed only in presence of the complementary protein , extravidin . in the presence of the noncomplementary proteins , no size decrease was observed , although a tendency of the biotin - functionalized dendrimeric assemblies to increase in size , forming larger aggregates , was noticed for g1-f and g1-p ( figure 2a , b ) . this aggregate enlargement did not occur when control dendrons with peg replacing the ligand moiety were exposed to the same proteins , even after 15 h ( figure 2f , g ) . this suggested that the higher density of biotin functionalization in the g1 dendrons is the likely reason for such aggregate enlargement . next , we investigated the host capabilities of the dendrons in the presence and absence of complementary and noncomplementary proteins . specifically , we were interested in assessing the effect of incorporating the ligand moieties at different locations within the dendrons upon the disassembly - induced guest release from the dendrimer host . to investigate these differences , nile red was encapsulated in the micelle - like nanoassemblies , and its release was triggered as a consequence of binding - induced disassembly upon exposure to extravidin . nile red is a hydrophobic molecule that exhibits reduced fluorescence in water , unless it is sequestered in a hydrophobic pocket . therefore , the reduction in fluorescence is a good indicator of the binding - induced disassembly event . as shown in figures 3a , b , small differences in placement of the ligand in the dendron produced rather different responses . first , percentage of released dye was assessed after exposing 25 m solutions of the dendrons to increasing concentrations of extravidin . we noticed that g1-p and g2-p responded to increasing concentrations of extravidin more than other dendrons studied . to further evaluate this behavior , we monitored the release profiles over time for all the dendritic assemblies upon exposing these assemblies to 14 m extravidin ( figure 3b ) . in the first hour , the release in the g2-p assembly was as high as 65% , while that of the g1-p assembly was around 22% increasing to 40% after 3 h. interestingly , the release from the g1-p assembly ultimately reached about 77% , which is comparable with the 81% observed for g2-p . in comparison , similar exposures to extravidin resulted in 35% , 25% , and 13% for g1-f , g2-f , and g2-m , respectively . the extent of release observed for g1-f and g2-f is consistent with our prior observation . interestingly , the release from g2-m aggregates is comparable to the release percentages observed due to nonspecific interactions . as a control experiment , the release of the nile red from the dendritic assemblies was also monitored in the absence of any protein ( figure 3c ) . similarly , control dendrons that lack the biotin ligand also did not exhibit appreciable dye release in the presence of extravidin ( figure 3d ) . these results show that the release profiles observed in figure 3a , b are indeed due to the ligand protein binding . moreover , it is clear that among the second generation dendrons , g2-p assembly is the only one that releases the hydrophobic guests efficiently following the extravidin binding . to further test the selectivity in the systems toward the target protein , the biotin - functionalized dendrons were exposed to increasing concentrations of noncomplementary proteins , chy , pep , and myo monitoring the change in nile red fluorescence ( figure 3e g ) . no significant change in the emission intensity was observed for any of the dendrons in the presence of chy and pep . however , while myo did not exhibit any change in the fluorescence intensity in the g1-p , g2-m , and g2-p based assemblies , there was a significant change in fluorescence in the g1-f and g2-f based assemblies ( figure 3 g and s3 ) . interestingly , these latter dendrons also exhibited much smaller release in response to extravidin ( figure 3b ) . it is noteworthy that myo is a metalloprotein , and therefore the cofactors in metalloproteins could be simply quenching the fluorescence of the dye molecule without the need for releasing the contents from the amphiphilic assembly . in fact , such a phenomenon has been previously observed with polymer fluorescence - based percentage of release of guest molecules from the amphiphilic dendritic assemblies : ( a ) with increasing concentration of extravidin , ( b ) time - dependent release in the presence of 14 m of extravidin , ( c ) in buffer solutionin the absence of proteins , ( d ) in assemblies based on control dendrons when exposed to extravidin , ( e ) with increasing concentration of chy , ( f ) with increasing concentration of pep , ( g ) with increasing concentration of myo . ( h ) absorption - based percentage of release upon interaction of dendrons with 14 m of myo . to test this possibility , we investigated the percentage of nile red release in the presence of myo by absorption spectroscopy . if it is simply a quenching phenomenon , there should be no change in the absorption spectrum since all dye molecules are still confined in the amphiphilic assembly without being released . indeed , we noted that there was no change in the absorption spectrum over time , which suggested that the observed increase in release percentage , i.e. , decrease in nile red fluorescence , is likely due to quenching ( figure 3h ) . as a second step , we were interested in gaining insights into the mechanism for the observed fluorescence reduction in the presence of myo , especially for g1-f and g2-f . two limiting mechanisms are possible : ( i ) inherently different encapsulation stabilities among the dendritic assemblies , causing the dye to leak out of g1-f ( or g2-f ) and to move to the hydrophobic pockets in myo , where the proximity between the metalloprotein cofactor and the dye molecule causes fluorescence quenching ; or ( ii ) nonspecific interactions between assemblies formed by g1-f ( or g2-f ) and myo to bring the encapsulated dye molecules in proximity to the metalloprotein cofactor favoring quenching . the mechanism ( i ) implies leakage of the cargo and re - encapsulation in the metalloprotein pockets . to test this possibility , we used a recently reported polymeric nanogel that has been well - established to have cross - link density - dependent encapsulation stabilities . nile red - encapsulated nanogels with 0% , 20% , and 50% cross - link densities were exposed to myo . if mechanism ( i ) was possible , there should be a cross - link density - dependent leakage based quenching . in all cases , we found that the extent of quenching was quite independent of the cross - link density ( figure s4a , b ) . the quenching however was found to increase with increasing concentration of the myo , as observed with g1-f and g2-f assemblies . these results are taken to suggest that , since the 0% cross - linked nanogel assembly is quite leaky , myo itself is not capable of encapsulating nile red molecules in its hydrophobic pockets . since dynamic and static quenching differs in their temperature dependence , we exposed the assemblies g1-f and g2-f encapsulating nile red to increasing concentrations of myo at two different temperatures . the number of collisions between the dendritic assemblies and the protein was expected to increase at a higher temperature as in dynamic quenching , leading to an increase in quenching . on the other hand , in the case of static quenching , the nonspecific and weak dendritic assembly protein complex would dissociate at a higher temperature , leading to a decrease in quenching.figure s4c the fact that quenching decreases with temperature supports the nonspecific and weakly bound complex hypothesis . thus , it is interesting to note that g1-f and g2-f exhibit higher nonspecific interaction and , at the same time , do not exhibit significant release of the guest molecules in response to the target extravidin . on the other hand , the dendrons g1-p and g2-p that exhibited the highest release in response to extravidin did not exhibit any nonspecific quenching with myo . these data and those from the previous sections indicate that if the release of hydrophobic guests is triggered by the specific extravidin biotin interaction , the selective binding with the complementary protein can occur more easily when a biotin ligand is grafted at the periphery than when it is grafted at the middle layer or at the focal point of the dendron and that this makes the release of hydrophobic guests faster for g2-p than for the other constructs , as observed in figure 3a , b . if disassembly is controlled by the same interaction , since the biotin extravidin binding is considered irreversible , when extravidin binds to a dendron in the assembly a reduction in the size of assembly will occur after some time , while the system reorganizes and equilibrates in smaller size assemblies , as observed by dls ( figure 2 ) . thus , although all systems reorganized into smaller size assemblies , the release was higher for the dendron systems with a ligand located at the periphery . based on the release kinetics , which was also faster for the dendritic systems with a ligand at the periphery , we hypothesized that in these cases the assembly reorganization was drastic enough to produce a higher release . on the other hand , in the cases with a ligand at the middle layer and focal point the assemblies rearranged slower into smaller size structures , allowing for the encapsulated hydrophobic small molecules to still be accommodated in hydrophobic pockets . in fact , figure 3b shows that even after 6 h of g2-m ( 25 m ) exposure to a constant concentration of extravidin ( 14 m ) , the release was as low as 13% . at this point , some interesting questions , including why is the release from g2-p so high compared to the release from g2-m and g2-f remain still open . for this reason , we have employed md simulations to gain additional insights into this process . we have used md simulations focusing on the second generation dendrons in order to understand why the different positioning of a biotin ligand within the dendron scaffold has such a strong effect on the final properties . in particular , we were interested in gaining insight on ligand accessibility from the external solution if it is tethered at the dendron periphery , at the focal point , or at the middle layer . in fact , it is known that similar dendritic structures can undergo strong folding in solution , so that if the biotin ligand is backfolded and surrounded by peg in the experimental conditions the specific binding with extravidin will be unlikely . the entire simulation work was carried out with the amber 12 suite of programs . molecular models were created with three different functionalization points for biotin to understand how the individual dendrons arrange in solution . g2-p , g2-f , and g2-m dendron models were created and parametrized according to our similar studies on dendrons interacting with proteins . starting configurations of the dendrons were then immerged in a simulation box ( figure 4a ) containing explicit water molecules ( figure s6 and details in the supporting information ( si ) ) . all systems underwent 200 ns of md in periodic boundary conditions at 25 c ( 298 k ) of temperature and 1 atm of pressure . during this time the root - mean - square displacement ( rmsd ) and the radius of gyration ( rg ) data extracted from the md simulations were used to assess the system s equilibration . computational details for the simulation procedure and data analysis are available in the si . initially , all dendrons were constructed with all peg chains on one side and all decyl chains on the other side . molecular models for the g2 dendrons were also constructed with alternated chains to avoid configuration - dependent results . however , md simulations of those systems suggested that the initial configuration does not have any impact on the shape and equilibrium configuration assumed by such small and flexible molecules in water , in terms of density distribution , radius of gyration , etc . these findings are consistent with the idea of treating these dendrons as facially amphiphilic structures . g2-p scaffold is colored in black , hydrophobic decyl chains in red , hydrophilic peg in blue , and biotin ligand in green . ( b ) final snapshot taken from the md simulation of g2-p . during the md run all dendrons undergo strong folding in solution assuming a globular shape ( rg = 9.410 ) . extravidin interaction is composed of two steps : first , unfolding of the biotin ligand ( green ) that becomes available for the protein at the dendron surface , and second , the specific binding between biotin and extravidin . the size of g2 in water , predicted by md simulation , does not change substantially depending on the tethering position of biotin ; the rg in the three cases is 9.410 ( figure 4b ) . in general , the dendrons tend to compact the hydrophobic decyl chains at the core and to surround them with hydrophilic peg . on the other hand , in terms of distribution of the biotin ligand the situation is different . the plots in figure 4c report the radial distribution function g(r ) of the biotin ligand calculated with respect to the dendrons center and expressed as a function of the dendron radius ( rg ) for the cases where biotin is grafted at the periphery ( g2-p : red ) , the middle layer ( g2-m : blue ) , or at the focal point ( g2-f : black ) . in general , the g(r ) values give indication on the relative probability to find the biotin ligand at a certain distance from the dendron center , being the position of the g(r ) maximum peak the most probable one . the biotin density going from the center to the surface is calculated at each simulation step , and the reported g(r ) data are averaged in time over the equilibrated phase md trajectories ( the last 100 ns ) . thus , high and sharp peaks in g(r ) identify high biotin density regions , but they also indicate high localization , confinement and backfolding ( namely , atoms that can not move are counted at each step in the same region of space ) . on the contrary , flexible and fluctuating figure 4c shows that at the equilibrium biotin distribution is very different for g2-p , g2-m , and g2-f . in particular , the biotin g(r ) maximum peak for g2-p ( red curve ) corresponds well with its rg indicating that , on average , the ligand availability on the surface is very good . on the contrary , the maximum peaks of black and blue g(r ) curves at a distance r 0.5 rg suggest that biotin is considerably more backfolded in the case of g2-f and g2-m . these data give indication on how much the biotin ligand is available at the surface of the dendron and thus also on the probability to have a specific binding with extravidin . in fact , we built a simplified model describing the dendron extravidin specific interaction as composed of two phases , namely , the unfolding of the biotin ligand to make it available for the protein and then the specific biotin avidin interaction ( figure 4d ) . according to this scheme , we can obtain information on the overall affinity of g2-p , g2-m , and g2-f for extravidin by evaluating the free energy of the dendron - protein binding process as : abind = aspecific + aunfold ; where the specific biotin avidin affinity is known experimentally ( absolute free energy of binding aspecific = 20.4 kcal mol ) and can be considered as a constant for all cases ( all dendrons bear the same number of biotin ligands ) , aunfold is the free energy necessary to drag out the biotin ligand from its backfolded state to make it available at the dendron surface . aunfold values for the different cases can be extracted directly from the g(r ) and depend on how much the ligand is backfolded within the dendron structure ( details in the si ) . in particular , if g2 dendrons are thought of as spheres with radius rg , aunfold = 0 for g2-p as in this case the biotin ligand is most probably available at the dendron surface ( g(r ) peak position coincides with rg ) . on the other hand , for g2-f and g2-m aunfold is an unfavorable term ( > 0 ) that depends on the level of backfolding , namely , the lower the biotin ligand availability at the surface ( high level of backfolding ) , the higher the necessary free energy to make it available at the dendrons surface for avidin binding . table 1 reports the free energy of binding values calculated for the different dendrons . we also calculated the statistical weight for the different dendrons from the dendron avidin affinity energies as exp(abindkbt ) ( kbt = 0.593 kcal mol at room temperature ) , which provides qualitative indication on the relative probability for a specific binding with extravidin in the case of g2-p , g2-m , and g2-f , depending on how much the biotin ligand is available at the surface . in particular , these data suggest that if we set the probability of having extravidin specifically bound to the biotin moiety in g2-p to 1 , then the probability of having extravidin bound to the biotin moiety in g2-f and g2-m is reduced to 0.1 due to reduced ligand availability ; a difference of 1 order of magnitude indicating that the extravidin specific binding to biotin ligands from the dendrons at the surface of g2-p aggregates is 10 times higher than in the case of g2-f and g2-m . it can be calculated for all cases directly from the g(r ) ; see si . experimental biotin avidin affinity aspecific = 20.4 kcal mol . global dendron then we aimed at studying the specific binding between one dendron and extravidin . since we did not have any information regarding the conformation assumed by the dendron during the binding with extravidin , we started from the unfolded configuration of one dendron ( figure 5d ) . initially , the biotin ligand of one unfolded g2-p was superimposed to the native biotin present in chain a of the crystallographic structure for the extravidin tetramer ( pdb : 1avd ) replacing it and thus obtaining the first g2-p+avd molecular complex . ( a ) when biotin is tethered at the periphery , it is more available for extravidin binding . on the contrary , when it is grafted at the middle layer ( b ) or at the focal point ( c ) , it is surrounded by an environment rich in peg , so that the deep penetration of the ligand in one of the binding pockets of extravidin is hindered . g2-p backbone is colored in black , hydrophobic decyl chains in red , hydrophilic peg in blue , and biotin in green . oxygen atoms of water molecules and cl counterions are represented as transparent cyan and purple spheres , respectively . ( e ) last equilibrated snapshot taken from the md simulation of the g2-p+avd system . during the md extravidin binding pocket is colored in yellow , and water molecules and counterions are not shown for clarity . when biotin is grafted at the middle layer ( figure 5b ) or the focal point ( figure 5c ) it is surrounded by an environment rich in peg . this makes the specific binding with extravidin , which implies the deep penetration of the ligand inside one of the four binding pockets of tetrameric extravidin , very difficult compared to g2-p ( figure 5a ) . in fact , it was not possible to create the initial complex with extravidin for g2-f and g2-m without incurring intractable distortions . for this reason and in light of the results on biotin availability discussed in the previous section , for what pertains to the modeling study of the interactions with the complementary protein , we focused only on g2-p . the starting configuration of the g2-p+avd complex ( figure 5d ) was immerged in a periodic box containing explicit water molecules and the minimum number of counterions necessary to guarantee the system neutrality ( details in the si ) . g2-p+avd system was equilibrated for 200 ns of npt md simulation at 25 c ( 298 k ) and 1 atm of pressure . the binding between biotin and extravidin remained very strong and stable ( see si for details ) . the interaction energy ( ebind ) between g2-p and extravidin was extracted directly from the md trajectories according to the mm - pbsa approach . in particular , ebind is the sum of the gas - phase in vacuo interaction energy ( egas ) and the solvation term ( esol ) . in general , the more negative the ebind value , the stronger the binding . in the case of a 1:1 binding between g2-p and extravidin , ebind was calculated to be as strong as 93.9 8.7 kcal mol at the equilibrium . in our preliminary communication , we had suggested that the change in hydrophilic lipophilic balance upon protein binding likely drives the binding - induced disassembly . to further test this hypothesis , we first modeled the amphiphilic assembly of g2-p dendrons in water . then , we aimed at understanding how the dendron assembly changes upon extravidin addition to the system . md simulation allows for the study of hydrophobic aggregation in water , as it was recently reported in the case of the self - assembly of hydrophobic drug molecules and amphiphilic diblock copolymer micelles . according to the same protocol , nine copies of g2-p dendron arranged on a plane were immerged in a simulation box filled with explicit water molecules . in particular , as a starting configuration for the g2-p dendron ( figure 6a ) , we chose the final equilibrated configuration produced by the simulation of g2-p+avd binding ( figure 5e ) , with the biotin ligand initially extended in extravidin - bound conformation , so that it was also possible to add a bound extravidin to the central dendron without difficulties . we thus generated two molecular systems both containing nine g2-p dendrons solvated in water , where the central dendron is respectively unbound ( figure 6a : 9g2-p ) or bound to one extravidin protein ( figure 6c ; 9g2-p+avd ) , in such a case the central dendron was simply replaced by the g2-p+avd complex from figure 5e . both systems were equilibrated for 200 ns of md simulation at 25 c and 1 atm pressure . during the simulation of the 9g2-p system , we found that the nine dendrons by themselves showed a strong propensity to self - assemble . the aggregate also reorganized in order to decrease the exposure of the hydrophobic regions ( red ) to the external solution . in general , figure 6b shows that the red decyl chains converge at the core of the aggregate and are surrounded by blue peg chains . structural reorganization during self - assembly is also demonstrated by the rg plots in figure 6e obtained from the md simulation of the 9g2-p system . red decyl chains converge to a lower rg value than peg ( blue ) and biotin ( green ) , indicating structural reorganization within the aggregate due to hydrophobic effects . ( a ) initially , nine copies of unbound g2-p dendrons were immerged in a simulation box containing water ( 9g2-p system ) . ( b ) during 200 ns of md simulation the nine dendrons aggregate and rearrange : hydrophilic peg ( blue ) and biotins ( green ) surround the hydrophobic parts ( red decyl chains ) . ( c ) the starting configuration for the g2-p+avd system is the same of g2-p , but a tetrameric extravidin protein ( avd : black ribbons ) is bound to the central g2-p dendron . ( d ) the 9g2-p+avd system was equilibrated for 200 ns of md simulation , during which the specific binding between biotin ( yellow ) and avd remains stable and the nine g2-p dendrons in the system self - assemble . rg plots are obtained also for the biotin ligands ( green ) , peg ( blue ) , and hydrophobic decyl chains ( red ) to understand molecular reorganization in solution . ( f ) the central dendron ( b , d : red ) was used as a reference , and the self - assembly energy ( eass ) was extracted for the md simulations . eass values were obtained for the 9g2-p ( black ) and 9g2-p+avd ( blue ) systems and plotted as a function of simulation time . ( g ) the energetic difference eass ( red ) identifies the g2-p self - assembly destabilization due to avd binding . similar aggregation occurred also in the 9g2-p+avd system containing eight g2-p unbound dendrons surrounding a single g2-p+avd complex ( figure 6c , d ) . however , interestingly in this case the aggregate was clearly less tight and less ordered at the equilibrium than in the 9g2-p case . we were interested in evaluating the native propensity of g2-p dendrons to self - assemble and in understanding how much the latter is affected when one specific binding with a complementary protein occurs at the surface of the aggregate . thus , we obtained from the md simulations the dendrons self - assembly energy ( eass ) for the two systems . the latter was evaluated as the binding energy between the central dendron with the other eight surrounding ones in the systems ( schemes in figure 6b , d : red and blue dendrons , respectively ) . eass was calculated as described in the previous section , taking into account for solute solute and solute solvent interactions ( details on energetic analysis in the si ) . eass calculated values were further normalized per dendron ( i.e. , divided per 8) , so that they become general indicators of how stable is the aggregation within the g2-p aggregates . data reported in figure 6f show that , at the equilibrium , the self - assembly energy triggering aggregation is as high as eass = 32.5 1.4 kcal mol in absence of extravidin binding ( 9g2-p : black curve ) . on the other hand , in the 9g2-p+avd system eass the destabilization induced by the specific binding with extravidin is represented by the energetic difference eass = eass ( 9g2-p+avd ) eass ( 9g2-p ) . positive values for eass indicate that , in general , when an avd protein binds specifically to one biotin ligand at the surface of a g2-p aggregate , the self - assembly is locally destabilized , which could be the trigger for disassembly . in our case , the energetic destabilization due to extravidin binding converged to the value of eass = + 15.7 1.7 kcal mol ( figure 6 g , red curve ) , indicating that when one avd binding occurs , the stability of g2-p dendron self - assembly ( eass ) is reduced to the half . ( a , b ) during the md simulation the dendrons self - assemble and blue dendrons surround the red one , which is specifically bound to avd ( biotin in green ) . ( c ) the dendrons can interact with extravidin specifically ( red ) and also nonspecifically ( blue ) . ( d ) specific ( red ) and nonspecific ( blue ) interaction energies normalized per dendron expressed as a function of simulation time . ( e ) nonspecific interactions with avd ( blue ) are sensibly lower than the dendrons native self - assembly energy ( green , the same 9g2-p values reported in black in figure 6f ) , demonstrating why nonspecific interactions alone are energetically not strong enough to trigger disassembly . we also extracted useful information from the md simulations regarding specific and nonspecific interactions ( figure 7 ) . in fact , during the 200 ns of md simulation of the 9g2-p+avd system , we saw that all nine g2-p dendrons interact not only with each other but also with extravidin ( figure 7a c ) . importantly , while the central dendron ( red ) is specifically bound to the protein through the biotin ligand , the other eight interact with avd nonspecifically . to obtain insights into the differences between specific and nonspecific interactions with avd , we extracted the binding energy ( ebind ) between the dendron aggregate and extravidin from the md simulation of 9g2-p+avd through the same approach adopted previously . ebind measures the global interaction of the aggregate ( composed of nine dendrons ) for avd , and it was calculated as ebind = 162.5 14.3 kcal mol , equivalent to ebind = 18.1 1.6 kcal mol per dendron , at the equilibrium . thus , this result shows that in general extravidin is attracted by g2-p aggregates , which suggests that as soon as extravidin is added to a solution containing dendron aggregates there will be a long - range trigger for molecular recognition . how much of this attraction is due to the specific interaction ( red dendron ) and how much to nonspecific interactions ? we obtained this information via a simple decomposition of the global ebind energy on a per dendron basis . this analysis shows that most of the interaction is due to the specific binding between the red dendron in figure 7 and avd , that is as strong as ebind = 91.6 5.4 kcal mol . such a high ebind value is very close to that found for the 1:1 g2-p+avd binding in figure 5 , which demonstrates that the presence of the other blue dendrons in the aggregate does not affect the strength of the specific binding . on the other hand , nonspecific interactions with extravidin are on average sensibly lower ( ebind = 8.8 1.9 kcal mol ) . this result shows that in this case the strength of nonspecific interactions is 1 order of magnitude weaker than specific interactions and also less persistent and more discontinuous , as demonstrated by the standard deviation which is 20% of the ebind average value ( for specific interactions the latter is 6% ) . interestingly , nonspecific interactions , even if present , are weaker than the intrinsic self - assembly energy of g2-p dendrons ( figure 7e : 9 vs. 33 kcal mol ) . for this reason , they are probably not strong enough to perturb the stability of the aggregates , which is consistent with our dls evidence ( figure 2 ) showing no disassembly in the case of noncomplementary proteins . all these results suggest that as soon as extravidin gets in contact with the surface of a g2-p aggregate , the formation of a specific binding will be an energetically favored event . in addition , it is worth noting that extravidin is a protein tetramer possessing four binding sites for biotin . thus , after a first specific binding occurs between avd with one biotin at the g2-p aggregate surface , the protein can find also other biotin ligands available in the neighborhood to establish many more specific bindings in a cooperative way . according to the so - called multivalent effect , this will be an energetically favored thus highly probable event . to test this hypothesis , we performed the md simulation , similar to that in figure 5 , but with four g2-p dendrimers bound to all of the biotin binding sites of tetrameric avd ( figure 8a , b ) . the result of the energetic analysis for this case is in agreement with the multivalency principle . in fact , the interaction energy ( ebind ) between avd and all four g2-p dendrons extracted from this simulation and reported in figure 8b is sensibly higher than that related to the binding of a single dendron reported in figure 5 ( multiplied per four , which equals to : ebind = 375.6 34.9 kcal mol ) . this large difference of 137 kcal mol is a clear signal of multivalency , and it demonstrates that avd is energetically favored to bind more biotins at the same time . ( a ) an additional system was simulated with four g2-p dendrons ( yellow , green , magenta , and red ) specifically bound to the four biotin binding sites of extravidin ( pdb : 1avd ) . ( b ) binding energy extracted from this md simulation indicates that the specific binding of avd to multiple g2-p dendrons is energetically favored . in fact , this tendency of avd to bind more biotin ligands at the same time , when available , can in principle speed up the disassembly process . conceptually , since avd will tend to preserve its structure much more than the dendron aggregate , in the case of cooperative binding to multiple ligands , it is reasonable to think that the dendrons from the aggregate will adapt over extravidin , rather than the contrary ( unlikely protein collapsing over the g2-p surface ) . one key factor allowing for multivalent binding is biotin availability , i.e. , biotin ligands must be accessible at the aggregate surface and free to complete specific interactions with avd . in fact , as we already discussed with figure 4c , d , the chance of having multivalent binding between the dendrons with avd will be extremely sensitive to biotin availability at the surface of the aggregates . our md simulations suggested that biotin availability is high for g2-p and low for g2-m and g2-f ( figure 4c , d ) . thus , at the g2-p aggregates surface avd will find many accessible binding spots for completing specific interactions . moreover , after a first specific binding is established , the same avd protein will be then energetically favored to bind more biotins from other g2-p dendrons . on the other hand , since the probability to have specific binding for g2-m and g2-f is reduced , even if a first specific interaction occurs between avd and one biotin ligand , it is reasonable to hypothesize that the chance of having multivalent avd binding at the surface of g2-m and g2-f aggregates will be even lower . ( a ) due to high biotin availability at the surface of g2-p ( red ) aggregates , the formation of multivalent avd - biotin bindings is an energetically favored phenomenon . while completing multiple bindings , avd would trigger the fast disassembly of the aggregates into small aggregates ( avd micelles ) via exfoliation , leading to the release of hydrophobic fluorescent guest molecules in solution ( green ) . ( b ) on the other hand , even if a less favored specific binding event occurs at the surface of g2-m and g2-f aggregates ( blue ) , multivalent binding of the same avd to multiple biotins is unlikely due to low ligand availability . the dendron aggregates would disassemble in larger and more ordered assemblies that would be able to retain the guests in their interior . ( c ) starting configuration of an additional system containing one avd ( black ribbons ) with four specific bindings with four g2-p dendrons ( red ) and surrounded by other 36 g2-p dendrons ( transparent blue ) . the number of g2-p dendrons that can surround one avd being in contact with the surface was calculated as 40 according to the mansfield ( d ) final snapshot of the md simulation of the large aggregate in solution ( oxygen atoms of water molecules in transparent cyan and counterions in purple ) . during the simulation all g2-p dendrons ( transparent black ) collapse and ( e ) gyration radius of the large aggregate over simulation time ( black ) . during the md run the dendrimers first aggregate around avd and then undergo rearrangement , surrounding the hydrophobic decyl chains ( red ) with the hydrophilic peg chains ( blue ) . rg plots for biotin ligands are represented in green . in light of these results , we propose two possible mechanisms for the observed supramolecular disassembly ( schematized in figure 9 ) . a first one for the g2-p case based on multivalent binding of avd , leading to the rapid disassembly of the dendron aggregates in solution due to exfoliation ( figure 9a ) . a second one for g2-m and g2-f , where the aggregates would disassemble more slowly due to the destabilization ( figure 6 ) induced by the specific binding with avd ( figure 9b ) . in principle , the first mechanism would result in the rapid production of smaller aggregates ( since they are limited by avd size ) and in a higher level of hydrophobic guests release . in fact , the velocity of the process and the small size of the aggregates would not allow for the structural rearrangement necessary to retain the guest molecules . the second proposed mechanism would most likely produce larger size aggregates and lower levels of release . in fact , the high level of biotin availability of g2-p ( figure 4c , d ) is compatible with the fast disassembly and hydrophobic guests release by g2-p aggregates in presence of extravidin , as shown by our dls and fluorescence experiments . at the same time , our data demonstrate that g2-m and g2-f aggregates also disassemble in presence of extravidin ( figure 2 ) , but more slowly . in addition , the final size of the disassembled aggregates is larger than that of g2-p ( 14 vs 7 nm ) , and no appreciable guest release is present during the disassembly of the g2-m and g2-f aggregates ( figure 3 ) . finally , we performed another md simulation aiming at representing the final step of the disassembly mechanism based on multivalent binding and exfoliation represented in figure 9a . first , we calculated how many g2-p dendrons , thought of as small spheres , would be necessary to surround completely an avd molecule , thought of as a larger sphere , while being in direct contact with the protein surface . using the gyration radii of a single g2-p dendron and avd obtained from our md simulations ( respectively , rg = 0.97 and 2.2 nm on average ) , we found that in our case this ratio equals to 40 g2-p dendrons for a single avd according to the mansfield then we started from the final configuration obtained from the simulation of avd specifically bound to four g2-p dendrons and have added other 36 equilibrated g2-p dendrons surrounding the complex ( figure 9c ) . the system was then immerged in a simulation box containing explicit solvent molecules and simulated for 150 ns in npt conditions , at 25 c of temperature and 1 atm of pressure . during this time , all 40 g2-p dendrons collapsed over the surface of avd surrounding the protein ( figure 9d ) and forming a real protein micelle . furthermore , during the md simulation the entire aggregate underwent structural reorganization attempting to surround the surface with hydrophilic peg , limiting as much as possible the exposure of the hydrophobic parts to water . this structural rearrangement is testified by the rg plots of figure 9e , demonstrating how during the md simulation the peg chains surround the surface of the aggregate . we also calculated the equilibrated size of the whole aggregate of figure 9d , which resulted to be rg = 3.3 nm . considering the compact and globular nature of this large aggregate , the hydrodynamic size can be calculated as rh 1.29 rg . for this case the obtained size is thus dh 8.5 nm , which is in good agreement with the size obtained for the disassembled aggregates in the case of g2-p ( figure 2 ) . our research reveals that ligand placement on a supramolecular scaffold for binding - induced disassembly greatly impacts disassembly and release of encapsulated guest molecules , as we have shown from the high release difference observed , for example , between g2-p and g2-m . the best place to attach a ligand , looking for a protein triggered release from a dendritic micelle - like nanostructure , is the periphery . md simulations show backfolding of the ligand , when attached to middle layer and focal point ; and a better availability for protein binding , when the ligand is attached at the periphery . incorporation of the biotin ligand in the dendrons gives the dendritic assemblies selectivity toward the target protein extravidin , regardless of the ligand position . nonetheless , ligand positioning in the dendrons gives the assemblies sensitivity toward release upon binding of a target protein . md simulations show that after avd binding the stability of g2-p assembly is strongly affected , possible signal of avd binding - induced disassembly . in addition , our computational efforts show that once a first specific binding between avd and one g2-p dendron occurs , the multivalent binding of the same protein to other g2-p dendrons via specific biotin this evidence allowed us to hypothesize two different mechanisms of disassembly induced by avd binding . the divergence of the mechanism also allows us to explain the observed experimental differences in guest release that depend on the ligand location in the scaffold . a fast one for g2-p based on high biotin availability at the aggregate surface , multivalent interactions , and aggregates exfoliation . this mechanism leads to fast formation of small disassembled aggregates and to a high release of hydrophobic guests . a second mechanism for g2-m and g2-p , slower , based on avd binding - induced aggregate destabilization and producing larger and more ordered aggregates in solution that are still capable of retaining the guest molecules in their interior . substitution of a peg unit in the dendrimer with a pendant biotin increased nonspecific interactions of the assemblies with proteins , which was seen as the formation of larger aggregates in solution . this became more evident when the density of peg chains on the hydrophilic face was low , as it is in a g1 dendron compared to a g2 . this , in turn , facilitates the formation of a weak complex with proteins , which was evidenced when a metalloprotein acted as a quencher , generating static quenching of the encapsulated fluorophore molecules . the combination of these results shows that g2 dendrons are better for selectivity and that the periphery is the best location for achieving binding - induced disassembly . thus , g2-p exhibits most guest release and least nonspecific interactions with other proteins . our md simulations of g2-p show that , despite the general affinity between the aggregates and avd , nonspecific interactions alone are too weak , and a specific binding ( more than 1 order of magnitude stronger ) is needed to trigger the release of the hydrophobic guest molecules . the research reported here gives a picture of how supramolecular disassembly and release might be largely affected by choosing a specific location for a trigger , rather than a random placement based on molecular architecture . also , we have shown how controlled variations in peg density could affect interactions of nanoparticles with proteins . developing zwitterionic moieties , which have the potential to circumvent the steric hindrance exhibited by lengthy peg chains in amphiphilic systems , for use in protein - binding - induced disassembly is part of the ongoing focus in our laboratories .

we use monodisperse dendrons that allow control over functional group presentation to investigate the influence of the location of a ligand on protein - induced disassembly and release of encapsulated small molecules . based on both experiments and molecular dynamics simulations , we demonstrate that ligand location greatly influences release of guest molecules from the dendron - based supramolecular assembly . we show that a ligand moiety grafted to the dendron periphery is more accessible for the target protein in aqueous solution . on the other hand , the ligand moiety placed at the focal point or at the intermediate layer within the dendritic scaffold is less accessible , since it is surrounded by an environment rich in peg chains , which hinders binding and even influences nonspecific interactions . we also demonstrate that the specific binding between one ligand and the target protein can destabilize the dendritic assembly . furthermore , if more ligands are available , multivalent interactions are also possible with extravidin , which speed up disassembly and trigger the release of hydrophobic guests .

Introduction Results and Discussion Conclusions

similarly , the control over functional group placements in dendritic architectures captures the advantage that small molecules have over their polymeric counterparts . in this manuscript , we take advantage of this unique feature by incorporating ligands at specific locations within facially amphiphilic dendrons and interrogating the efficiency of the supramolecular disassembly and molecular release in response to a specific protein binding event . we use both experiments and molecular dynamics ( md ) simulations synergistically to gain insights into the ligand facially amphiphilic dendrons , containing a biaryl - based internal repeat unit and an aryl peripheral unit , bear both hydrophilic and hydrophobic functional groups as side chains in each of these repeat units . the amphiphilic functional groups are placed at opposite faces of the biaryl backbone of the dendron such that these molecules are endowed with the capability to form micelle - like assemblies in aqueous solution and form inverse micelle - like assemblies in apolar solvents . in our preliminary findings , we have shown that placing a ligand moiety at the hydrophilic face of the dendron can provide binding - induced disassembly of the micelle - like structure in the aqueous solvent . in that work , the ligand moiety was placed at the focal point of the dendron , as this structure is synthetically most easily accessible . therefore , in this manuscript , we investigate the effect of the placement of a protein - specific ligand moiety at specific positions of a dendron upon the accessibility of the complementary protein to the supramolecular assembly and hence the effects on the concomitant guest release response ( figure 1 ) . the relative encapsulation of functional groups , when placed in the periphery vs the focal point of the dendron is well - known . for these reasons , it is important that we investigate the effect of ligand placement on the binding - induced release . therefore , we focused on the g1 and g2 dendrons , which provide sufficient variations in the functional group placement . a ligand can be incorporated on a g1 dendron either at the focal point or at the periphery . to install a single ligand at a specific place , one of the peg units was replaced by the ligand , attaching it to the dendron backbone via a hydrophilic linker . this placement allows the ligand to be exposed to the aqueous solution on the hydrophilic face of the assembly . note that the focal point is synthetically the easiest place to attach a single functional group , since the focal point is a single and unique position in a dendron . in contrast , to install a single functional group at the periphery , one has to distinguish one of the peripheral monomers from otherwise identical ones within the dendron . accordingly , the degree of difficulty in placing a unique functional group in the dendron increases as we move from the focal point to the periphery and as we increase the generation . prior to analyzing the interaction between the dendritic assemblies and the proteins , we analyzed the size of the assemblies in aqueous phase using dynamic light scattering ( dls ) , with g1 dendron concentrations of 12.5 m and g2 dendron concentrations of 10 m ( both above their respective cacs ) . however , note that the replacement of a hydrophilic peg chain in a g1 or g2 dendron by a less hydrophilic pendant biotin decreases the peg density on the assembly surface , decreasing hydrophilicity and increasing the chance of nonspecific interactions with noncomplementary proteins . specifically , we were interested in assessing the effect of incorporating the ligand moieties at different locations within the dendrons upon the disassembly - induced guest release from the dendrimer host . therefore , the reduction in fluorescence is a good indicator of the binding - induced disassembly event . in the first hour , the release in the g2-p assembly was as high as 65% , while that of the g1-p assembly was around 22% increasing to 40% after 3 h. interestingly , the release from the g1-p assembly ultimately reached about 77% , which is comparable with the 81% observed for g2-p . interestingly , the release from g2-m aggregates is comparable to the release percentages observed due to nonspecific interactions . as a control experiment , the release of the nile red from the dendritic assemblies was also monitored in the absence of any protein ( figure 3c ) . these results show that the release profiles observed in figure 3a , b are indeed due to the ligand protein binding . in fact , such a phenomenon has been previously observed with polymer fluorescence - based percentage of release of guest molecules from the amphiphilic dendritic assemblies : ( a ) with increasing concentration of extravidin , ( b ) time - dependent release in the presence of 14 m of extravidin , ( c ) in buffer solutionin the absence of proteins , ( d ) in assemblies based on control dendrons when exposed to extravidin , ( e ) with increasing concentration of chy , ( f ) with increasing concentration of pep , ( g ) with increasing concentration of myo . two limiting mechanisms are possible : ( i ) inherently different encapsulation stabilities among the dendritic assemblies , causing the dye to leak out of g1-f ( or g2-f ) and to move to the hydrophobic pockets in myo , where the proximity between the metalloprotein cofactor and the dye molecule causes fluorescence quenching ; or ( ii ) nonspecific interactions between assemblies formed by g1-f ( or g2-f ) and myo to bring the encapsulated dye molecules in proximity to the metalloprotein cofactor favoring quenching . on the other hand , in the case of static quenching , the nonspecific and weak dendritic assembly protein complex would dissociate at a higher temperature , leading to a decrease in quenching.figure s4c the fact that quenching decreases with temperature supports the nonspecific and weakly bound complex hypothesis . thus , it is interesting to note that g1-f and g2-f exhibit higher nonspecific interaction and , at the same time , do not exhibit significant release of the guest molecules in response to the target extravidin . on the other hand , the dendrons g1-p and g2-p that exhibited the highest release in response to extravidin did not exhibit any nonspecific quenching with myo . these data and those from the previous sections indicate that if the release of hydrophobic guests is triggered by the specific extravidin biotin interaction , the selective binding with the complementary protein can occur more easily when a biotin ligand is grafted at the periphery than when it is grafted at the middle layer or at the focal point of the dendron and that this makes the release of hydrophobic guests faster for g2-p than for the other constructs , as observed in figure 3a , b . thus , although all systems reorganized into smaller size assemblies , the release was higher for the dendron systems with a ligand located at the periphery . based on the release kinetics , which was also faster for the dendritic systems with a ligand at the periphery , we hypothesized that in these cases the assembly reorganization was drastic enough to produce a higher release . on the other hand , in the cases with a ligand at the middle layer and focal point the assemblies rearranged slower into smaller size structures , allowing for the encapsulated hydrophobic small molecules to still be accommodated in hydrophobic pockets . in particular , we were interested in gaining insight on ligand accessibility from the external solution if it is tethered at the dendron periphery , at the focal point , or at the middle layer . in fact , it is known that similar dendritic structures can undergo strong folding in solution , so that if the biotin ligand is backfolded and surrounded by peg in the experimental conditions the specific binding with extravidin will be unlikely . extravidin interaction is composed of two steps : first , unfolding of the biotin ligand ( green ) that becomes available for the protein at the dendron surface , and second , the specific binding between biotin and extravidin . on the other hand , in terms of distribution of the biotin ligand the situation is different . the plots in figure 4c report the radial distribution function g(r ) of the biotin ligand calculated with respect to the dendrons center and expressed as a function of the dendron radius ( rg ) for the cases where biotin is grafted at the periphery ( g2-p : red ) , the middle layer ( g2-m : blue ) , or at the focal point ( g2-f : black ) . in general , the g(r ) values give indication on the relative probability to find the biotin ligand at a certain distance from the dendron center , being the position of the g(r ) maximum peak the most probable one . these data give indication on how much the biotin ligand is available at the surface of the dendron and thus also on the probability to have a specific binding with extravidin . in fact , we built a simplified model describing the dendron extravidin specific interaction as composed of two phases , namely , the unfolding of the biotin ligand to make it available for the protein and then the specific biotin avidin interaction ( figure 4d ) . according to this scheme , we can obtain information on the overall affinity of g2-p , g2-m , and g2-f for extravidin by evaluating the free energy of the dendron - protein binding process as : abind = aspecific + aunfold ; where the specific biotin avidin affinity is known experimentally ( absolute free energy of binding aspecific = 20.4 kcal mol ) and can be considered as a constant for all cases ( all dendrons bear the same number of biotin ligands ) , aunfold is the free energy necessary to drag out the biotin ligand from its backfolded state to make it available at the dendron surface . aunfold values for the different cases can be extracted directly from the g(r ) and depend on how much the ligand is backfolded within the dendron structure ( details in the si ) . on the other hand , for g2-f and g2-m aunfold is an unfavorable term ( > 0 ) that depends on the level of backfolding , namely , the lower the biotin ligand availability at the surface ( high level of backfolding ) , the higher the necessary free energy to make it available at the dendrons surface for avidin binding . we also calculated the statistical weight for the different dendrons from the dendron avidin affinity energies as exp(abindkbt ) ( kbt = 0.593 kcal mol at room temperature ) , which provides qualitative indication on the relative probability for a specific binding with extravidin in the case of g2-p , g2-m , and g2-f , depending on how much the biotin ligand is available at the surface . in particular , these data suggest that if we set the probability of having extravidin specifically bound to the biotin moiety in g2-p to 1 , then the probability of having extravidin bound to the biotin moiety in g2-f and g2-m is reduced to 0.1 due to reduced ligand availability ; a difference of 1 order of magnitude indicating that the extravidin specific binding to biotin ligands from the dendrons at the surface of g2-p aggregates is 10 times higher than in the case of g2-f and g2-m . since we did not have any information regarding the conformation assumed by the dendron during the binding with extravidin , we started from the unfolded configuration of one dendron ( figure 5d ) . on the contrary , when it is grafted at the middle layer ( b ) or at the focal point ( c ) , it is surrounded by an environment rich in peg , so that the deep penetration of the ligand in one of the binding pockets of extravidin is hindered . when biotin is grafted at the middle layer ( figure 5b ) or the focal point ( figure 5c ) it is surrounded by an environment rich in peg . this makes the specific binding with extravidin , which implies the deep penetration of the ligand inside one of the four binding pockets of tetrameric extravidin , very difficult compared to g2-p ( figure 5a ) . in general , figure 6b shows that the red decyl chains converge at the core of the aggregate and are surrounded by blue peg chains . ( d ) the 9g2-p+avd system was equilibrated for 200 ns of md simulation , during which the specific binding between biotin ( yellow ) and avd remains stable and the nine g2-p dendrons in the system self - assemble . on the other hand , in the 9g2-p+avd system eass the destabilization induced by the specific binding with extravidin is represented by the energetic difference eass = eass ( 9g2-p+avd ) eass ( 9g2-p ) . this analysis shows that most of the interaction is due to the specific binding between the red dendron in figure 7 and avd , that is as strong as ebind = 91.6 5.4 kcal mol . such a high ebind value is very close to that found for the 1:1 g2-p+avd binding in figure 5 , which demonstrates that the presence of the other blue dendrons in the aggregate does not affect the strength of the specific binding . on the other hand , nonspecific interactions with extravidin are on average sensibly lower ( ebind = 8.8 1.9 kcal mol ) . conceptually , since avd will tend to preserve its structure much more than the dendron aggregate , in the case of cooperative binding to multiple ligands , it is reasonable to think that the dendrons from the aggregate will adapt over extravidin , rather than the contrary ( unlikely protein collapsing over the g2-p surface ) . in fact , as we already discussed with figure 4c , d , the chance of having multivalent binding between the dendrons with avd will be extremely sensitive to biotin availability at the surface of the aggregates . on the other hand , since the probability to have specific binding for g2-m and g2-f is reduced , even if a first specific interaction occurs between avd and one biotin ligand , it is reasonable to hypothesize that the chance of having multivalent avd binding at the surface of g2-m and g2-f aggregates will be even lower . while completing multiple bindings , avd would trigger the fast disassembly of the aggregates into small aggregates ( avd micelles ) via exfoliation , leading to the release of hydrophobic fluorescent guest molecules in solution ( green ) . ( b ) on the other hand , even if a less favored specific binding event occurs at the surface of g2-m and g2-f aggregates ( blue ) , multivalent binding of the same avd to multiple biotins is unlikely due to low ligand availability . a first one for the g2-p case based on multivalent binding of avd , leading to the rapid disassembly of the dendron aggregates in solution due to exfoliation ( figure 9a ) . in fact , the velocity of the process and the small size of the aggregates would not allow for the structural rearrangement necessary to retain the guest molecules . our research reveals that ligand placement on a supramolecular scaffold for binding - induced disassembly greatly impacts disassembly and release of encapsulated guest molecules , as we have shown from the high release difference observed , for example , between g2-p and g2-m . md simulations show backfolding of the ligand , when attached to middle layer and focal point ; and a better availability for protein binding , when the ligand is attached at the periphery . incorporation of the biotin ligand in the dendrons gives the dendritic assemblies selectivity toward the target protein extravidin , regardless of the ligand position . in addition , our computational efforts show that once a first specific binding between avd and one g2-p dendron occurs , the multivalent binding of the same protein to other g2-p dendrons via specific biotin this evidence allowed us to hypothesize two different mechanisms of disassembly induced by avd binding . the combination of these results shows that g2 dendrons are better for selectivity and that the periphery is the best location for achieving binding - induced disassembly . our md simulations of g2-p show that , despite the general affinity between the aggregates and avd , nonspecific interactions alone are too weak , and a specific binding ( more than 1 order of magnitude stronger ) is needed to trigger the release of the hydrophobic guest molecules . developing zwitterionic moieties , which have the potential to circumvent the steric hindrance exhibited by lengthy peg chains in amphiphilic systems , for use in protein - binding - induced disassembly is part of the ongoing focus in our laboratories .

nondopaminergic and nonmotor symptoms of parkinson 's disease ( pd ) , which include psychiatric , autonomic , and gastrointestinal symptoms , are sometimes present before a diagnosis of pd and almost inevitably emerge with the disease progression . in contrast to motor symptoms of pd , nonmotor symptoms have been poorly recognized and inadequately treated . however , methods for recognizing and quantifying the nonmotor symptoms of pd have become a new focus of research that will form the basis for improved treatments . among the nonmotor symptoms , cognitive impairment adversely influences their activities of daily living and quality of life and worsens caregiver burden . a community - based study found that within a group of patients with a diagnosis of pd of at least 20 years , 83% had experienced dementia , and a similar number of patients had experienced frequent falls ( 87% ) , moderate dysarthria ( 81% ) , visual hallucinations ( 74% ) , or urinary incontinence ( 71% ) . in pd dementia ( pd - d ) patients , -synuclein pathology can be widespread in the cerebral cortex . in addition , there is less acetylcholinesterase activity in the cerebral cortex of pd - d patients than of alzheimer 's disease patients . moreover , clinical studies have reported on the effectiveness of cholinesterase inhibitors for treating cognitive impairment in pd - d patients . the prevalence of cognitive dysfunction among pd patients has led to the recognition that a diagnostic criterion is needed for pd - d . as a result , the movement disorder society task force published diagnostic criteria for pd - d in 2007 and simultaneously proposed a practical diagnostic procedure for pd - d with the aim of its widespread international application . level i testing includes a simple and short algorithm based on current cognitive tools that can be used in an office or at the bedside . it is considered a screening tool for diagnosing pd - d , whereas level ii testing provides a more detailed assessment that characterizes the components of pd - d and monitors the elements that may be responsive to intervention . level i testing was designed as a screening tool for use in the clinic and in clinical trials . although it has been used in practice , no study has yet verified its validity or utility in a large sample of pd patients . in this study , we used level i testing on 304 pd patients with the objective of determining which patients would be diagnosed with pd - d and what impairments would be diagnosed among the 4 cognitive domains tested . the subjects included in our study were japanese patients with idiopathic pd who were clinically diagnosed and treated by board - certified neurologists at 1 of the 13 participating institutions between july and september 2011 . a diagnosis of pd was made according to the uk parkinson 's disease society brain bank criteria as well as a differential diagnosis . the study design and protocol were approved by the ethics committees for human research of the keio university school of medicine and all the participating institutions . this was a cross - sectional study which examined the clinical and neurological features of these pd patients , including sex , age at the assessment , education , disease duration , and the hoehn and yahr ( h&y ) stage . their cognitive function was evaluated with the japanese versions of the mini - mental state examination ( mmse ) and montreal cognitive assessment ( moca ) . the mmse consists of 10 subtests scored to a total of 30 points : pentagon copying ( 1 point ) , serial 7 subtraction ( 5 points ) , immediate ( 3 points ) and delayed 3-word recall ( 3 points ) , naming ( 2 points ) , repeating a sentence ( 1 point ) , 3-stage commands ( 3 point ) , reading a sentence ( 1 point ) , writing a sentence ( 1 point ) , and orientation to time and place ( 10 points ) . the moca consists of 12 subtests also scored to a total of 30 points : cube copying ( 1 point ) , clock drawing ( 3 points ) , trail making ( 1 point ) , phonemic fluency ( 1 point ) , verbal abstraction ( 2 points ) , digit span ( 2 points ) , serial 7 subtraction ( 3 points ) , target tapping ( 1 point ) , delayed 5-word recall ( 5 points ) , naming ( 3 points ) , repeating 2 sentences ( 2 points ) , and orientation to time and place ( 6 points ) . we used the algorithm for level i testing to examine the present patient population ( table 1 ) . in this algorithm , the diagnosis of pd - d is based on the following 5 criteria : ( 1 ) diagnosis of pd , ( 2 ) pd developed prior to the onset of dementia , ( 3 ) a decreased global cognitive efficiency , ( 4 ) a cognitive deficiency that impairs daily life , and ( 5 ) impairment in > 1 cognitive domain . in this study , however , we did not investigate whether cognitive deficits were severe enough to affect daily living ( item 4 , typically assessed with a caregiver interview or the pill questionnaire ) . for item 5 , the results of the following subtests of the moca and mmse were used : serial 7 subtraction ( seven backward in table 1 ) , phonemic fluency ( lexical fluency ) , and clock drawing from the moca , and pentagon copying ( mmse pentagons ) and delayed 3-word recall from the mmse . memory for delayed 3-word recall ( table 1 ) . in the months reversed test , the patient must name the months in reverse order . however , japanese months are named by number , which is different from their naming in english ; thus , the months reversed test was excluded from this study . according to the level i testing procedures , test results that indicated impairment were : 2 incorrect responses on the seven backward test , a score of 9 words in the lexical fluency test , an inability to correctly draw the clock face or clock hands pointing to the correct time in the clock drawing test , an inability to correctly draw 2 pentagons that overlap in the pentagon copying test , and 1 word missing in the 3-word recall test . the jmp software version 8.0 ( sas institute , tokyo , japan ) was used for all statistical analyses . the level of statistical significance was defined as p < 0.05 . a one - way anova and the tukey - kramer hsd test the subjects included in our study were japanese patients with idiopathic pd who were clinically diagnosed and treated by board - certified neurologists at 1 of the 13 participating institutions between july and september 2011 . a diagnosis of pd was made according to the uk parkinson 's disease society brain bank criteria as well as a differential diagnosis . the study design and protocol were approved by the ethics committees for human research of the keio university school of medicine and all the participating institutions . this was a cross - sectional study which examined the clinical and neurological features of these pd patients , including sex , age at the assessment , education , disease duration , and the hoehn and yahr ( h&y ) stage . their cognitive function was evaluated with the japanese versions of the mini - mental state examination ( mmse ) and montreal cognitive assessment ( moca ) . the mmse consists of 10 subtests scored to a total of 30 points : pentagon copying ( 1 point ) , serial 7 subtraction ( 5 points ) , immediate ( 3 points ) and delayed 3-word recall ( 3 points ) , naming ( 2 points ) , repeating a sentence ( 1 point ) , 3-stage commands ( 3 point ) , reading a sentence ( 1 point ) , writing a sentence ( 1 point ) , and orientation to time and place ( 10 points ) . the moca consists of 12 subtests also scored to a total of 30 points : cube copying ( 1 point ) , clock drawing ( 3 points ) , trail making ( 1 point ) , phonemic fluency ( 1 point ) , verbal abstraction ( 2 points ) , digit span ( 2 points ) , serial 7 subtraction ( 3 points ) , target tapping ( 1 point ) , delayed 5-word recall ( 5 points ) , naming ( 3 points ) , repeating 2 sentences ( 2 points ) , and orientation to time and place ( 6 points ) . we used the algorithm for level i testing to examine the present patient population ( table 1 ) . in this algorithm , the diagnosis of pd - d is based on the following 5 criteria : ( 1 ) diagnosis of pd , ( 2 ) pd developed prior to the onset of dementia , ( 3 ) a decreased global cognitive efficiency , ( 4 ) a cognitive deficiency that impairs daily life , and ( 5 ) impairment in > 1 cognitive domain . in this study , however , we did not investigate whether cognitive deficits were severe enough to affect daily living ( item 4 , typically assessed with a caregiver interview or the pill questionnaire ) . for item 5 , the results of the following subtests of the moca and mmse were used : serial 7 subtraction ( seven backward in table 1 ) , phonemic fluency ( lexical fluency ) , and clock drawing from the moca , and pentagon copying ( mmse pentagons ) and delayed 3-word recall from the mmse . however , japanese months are named by number , which is different from their naming in english ; thus , the months reversed test was excluded from this study . according to the level i testing procedures , test results that indicated impairment were : 2 incorrect responses on the seven backward test , a score of 9 words in the lexical fluency test , an inability to correctly draw the clock face or clock hands pointing to the correct time in the clock drawing test , an inability to correctly draw 2 pentagons that overlap in the pentagon copying test , and 1 word missing in the 3-word recall test . the jmp software version 8.0 ( sas institute , tokyo , japan ) was used for all statistical analyses . the level of statistical significance was defined as p < 0.05 . a one - way anova and the tukey - kramer hsd test a detailed description of the demographics of the 304 pd patients included in our study has been reported elsewhere . briefly , the subjects were 166 men and 138 women with a mean age sd of 70.6 8.3 ( range : 42 - 90 ) years ; their education lasted 12.5 2.6 ( range : 1 - 20 ) years , the disease duration was 6.6 5.1 ( range : 0.2 - 30.1 ) years , and their h&y stage was 2.7 0.7 ( range : 1 - 5 ) . the subjects were grouped into tertiles according to their total moca score ( table 2 ) . similarly , the subjects were grouped into tertiles according to their total mmse score ( table 3 ) . the single subtest scores decreased significantly as the total moca scores decreased from the high - moca - score group to the middle- and low - moca - score groups ( p < 0.05 ) . exceptionally , the decreases in scores for the naming , target tapping , and orientation tests from the high- to the middle - moca - score group were not significantly different . therefore , the decline in the total moca score mirrors a decline in the scores for almost all the subtests . naming and orientation were preserved ( > 80% ) even in the low - moca - score group . in contrast , sentence repeating , phonemic fluency , and delayed recall scores decreased remarkably ( < 70% ) even in the high - moca - score group . trail making was preserved in the high - moca - score group but decreased steeply in the middle- and low - moca - score groups . the scores for the 10 mmse subtests are presented by group in figure 1b . for most of the subtests ( 7 of 10 ) , the scores failed to show a significant decrease from the high- to the middle - mmse - score group , and the scores for the naming subtest failed to decrease even in the low - mmse - score group . all domains were preserved ( > 90% ) in the high - mmse - score group , indicating that no mmse subtest was significative for these pd patients ( fig . a significant decrease was observed only for the serial 7 subtraction and delayed recall subtests in the low - mmse - score group ( < 50% , p < 0.05 ) . for example , the moca includes a 5-word delayed recall test , whereas the mmse provides a 3-word delayed recall test ; 5-word recall according to the moca was < 70% even in the high - moca - score group , whereas 3-word recall according to the mmse was relatively preserved ( fig . 1 , 2 ) . in addition , the moca includes a subtest in which 2 sentences are repeated , whereas the mmse includes a subtest in which 1 sentence is repeated . the repeating 2 sentences subtest of the moca showed a score < 70% in the high - moca - score group , but the score for the repeating a sentence subtest of the mmse was preserved even in the low - mmse - score group . items 1 ( diagnosis of pd ) and 2 ( dementia preceding pd ) in level i testing ( table 1 ) were applicable to the subjects participating in this study . for item 3 ( decreased global cognitive efficiency ) , 105 subjects ( 34.5% ) showed an mmse score < 26 . with respect to item 5 ( dysfunction in > 1 cognitive domain ) , we observed that 53 subjects ( 17.4% ) showed no impairment in any cognitive domain , whereas the remaining subjects showed impairments in 1 ( n = 74 ; 24.3% ) , 2 ( n = 87 ; 28.6% ) , 3 ( n = 74 ; 24.3% ) , or 4 ( n = 16 ; 5.3% ) cognitive domains . therefore , 177 subjects ( 58.2% ) showed impairments in 2 cognitive domains . among these 177 subjects , 99 also had mmse scores < 26 ( 32.6% of all subjects ; 94.3% of the total group with mmse scores < 26 ) and therefore met the diagnostic criteria for pd - d . only 6 subjects ( 5.7% of 105 subjects ) with mmse scores < 26 did not meet the diagnostic criteria for pd - d , because they had only 1 impaired cognitive domain . of the 105 subjects with mmse scores < 26 , most showed impairments in 3 cognitive domains ( n = 62 ; 59.0% ) . figure 3 shows the number of subjects with mmse scores < 26 or 26 , along with the number of impaired domains . no subject with an mmse score < 26 was without impairment in any domain . among the subjects with mmse scores 26 ( n = 199 ) , we observed 1 ( n = 68 ; 34.2% ) , 2 ( n = 65 ; 32.7% ) , 3 ( n = 12 ; 6.0% ) , and 4 ( n = 1 ; 0.5% ) impaired domains ; 53 ( 26.6% ) had no impairment in any cognitive domain . figure 4 shows the proportions of subjects with mmse scores < 26 or 26 , along with the number of impaired domains . subjects with mmse scores 26 accounted for the majority of subjects with 1 impaired domain , and they accounted for 74.7% of subjects with 2 impaired domains . in contrast , subjects with mmse scores < 26 accounted for the majority of subjects with 3 or 4 impaired domains . this finding was as expected because patients with lower mmse scores should have an increased likelihood of being affected in more cognitive domains . figure 5a shows the numbers of subjects with impairments for each of the 4 cognitive domains . the most common impairment was executive dysfunction ( 224 subjects ; 73.7% ) , followed by memory ( n = 161 ; 53.0% ) and attention ( n = 119 ; 39.1% ) impairments . visuospatial functional impairments were the least common and were apparent in only 30 subjects ( 9.9% ) . of the 224 subjects who showed executive dysfunction , most exhibited impairment in lexical fluency ( 180 subjects ; 80.4% ) or clock drawing ( n = 133 ; 59.4% ) , whereas 89 subjects ( 39.7% ) displayed impairments in both ( fig . the most common combinations in the group with mmse scores 26 were executive dysfunction alone ( 49 subjects ; 24.6% ) and executive dysfunction with memory impairment ( n = 47 ; 23.6% ) , followed by executive dysfunction with attention impairment ( n = 16 ; 9.5% ) and memory impairment alone ( n = 15 ; 7.5% ) . the most common combination of impaired cognitive domains in the group with mmse scores < 26 was executive dysfunction with both memory and attention impairments ( n = 54 ; 51.4% ) , followed by impairments in all 4 domains ( n = 15 ; 14.3% ) . interestingly , visuospatial impairment was always accompanied by executive dysfunction ; however , visuospatial impairment alone , or in combination with attention , memory , or both attention and memory impairments , did not occur in any of the subjects . for the purpose of screening pd patients for dementia , it is necessary to use a test in which achievement does not decline in pd patients who have normal global cognitive efficiency ( as measured by the total mmse or moca score ) but declines in those patients with impaired global cognitive efficiency indicative of dementia . with regard to the hypothesis that an appropriate test is one in which the high- and middle - total - score groups by the mmse or the moca maintain a score 70% while the low - total - score group scores < 70% , the copy cube , draw clock , verbal abstraction , digit span , target tapping , and serial 7 subtraction tests of the moca ( marked # in fig . 2a ) and the serial 7 subtraction and 3-word delayed recall tests of the mmse ( marked # in fig . 2b ) are considered appropriate . in level i testing , this includes the clock drawing , serial 7 subtraction , and delayed 3-word recall subtests , but not the phonemic fluency or pentagon copying subtests . as regards evaluating executive function , scores for the phonemic fluency test declined markedly to approximately 50% in the high - moca - score group , whose scores ( 24 - 30 ) were consistent with pd without dementia . the other subtest that evaluates executive function in level i testing is the clock drawing test . scores for this test declined to < 70% only in the low - moca - score group . this suggests the clock drawing test may be more appropriate than the phonemic fluency test for screening for a decline in executive function in pd - d . on the other hand , when evaluating visuospatial function , scores for the pentagon copying test stayed at 70% even in the low - mmse - score group , whose scores ( 12 - 25 ) were below the 26-point cutoff for pd - d . the authors of the original moca test indicated that the cube copying and clock drawing tests are concerned with visuospatial function , but in level i testing , the clock drawing test is used for executive function . for the cube copying test , the authors stated that if any of the following criteria are not met , the abnormal result was assigned : ( 1 ) drawing must be 3 dimensional , ( 2 ) all lines are drawn , ( 3 ) no line is added , ( 4 ) lines are relatively parallel and their length is similar . applied to the 304 subjects in the present study , 101 were classified as abnormal according to the cube copying test , including 43 subjects in the group with mmse scores 26 and 58 subjects in the group with mmse scores < 26 . these are far more than the 30 subjects with abnormal results for the pentagon copying test , and almost as many as the 119 subjects who were categorized as abnormal in attention by the serial 7 subtraction test . a detailed description of the demographics of the 304 pd patients included in our study has been reported elsewhere . briefly , the subjects were 166 men and 138 women with a mean age sd of 70.6 8.3 ( range : 42 - 90 ) years ; their education lasted 12.5 2.6 ( range : 1 - 20 ) years , the disease duration was 6.6 5.1 ( range : 0.2 - 30.1 ) years , and their h&y stage was 2.7 0.7 ( range : 1 - 5 ) . the subjects were grouped into tertiles according to their total moca score ( table 2 ) . similarly , the subjects were grouped into tertiles according to their total mmse score ( table 3 ) . the single subtest scores decreased significantly as the total moca scores decreased from the high - moca - score group to the middle- and low - moca - score groups ( p < 0.05 ) . exceptionally , the decreases in scores for the naming , target tapping , and orientation tests from the high- to the middle - moca - score group were not significantly different . therefore , the decline in the total moca score mirrors a decline in the scores for almost all the subtests . the differences between the 12 moca subtests are presented in figure 2a . naming and orientation were preserved ( > 80% ) even in the low - moca - score group . in contrast , sentence repeating , phonemic fluency , and delayed recall scores decreased remarkably ( < 70% ) even in the high - moca - score group . trail making was preserved in the high - moca - score group but decreased steeply in the middle- and low - moca - score groups . the scores for the 10 mmse subtests are presented by group in figure 1b . for most of the subtests ( 7 of 10 ) , the scores failed to show a significant decrease from the high- to the middle - mmse - score group , and the scores for the naming subtest failed to decrease even in the low - mmse - score group . all domains were preserved ( > 90% ) in the high - mmse - score group , indicating that no mmse subtest was significative for these pd patients ( fig . a significant decrease was observed only for the serial 7 subtraction and delayed recall subtests in the low - mmse - score group ( < 50% , p < 0.05 ) . for example , the moca includes a 5-word delayed recall test , whereas the mmse provides a 3-word delayed recall test ; 5-word recall according to the moca was < 70% even in the high - moca - score group , whereas 3-word recall according to the mmse was relatively preserved ( fig . 1 , 2 ) . in addition , the moca includes a subtest in which 2 sentences are repeated , whereas the mmse includes a subtest in which 1 sentence is repeated . the repeating 2 sentences subtest of the moca showed a score < 70% in the high - moca - score group , but the score for the repeating a sentence subtest of the mmse was preserved even in the low - mmse - score group . items 1 ( diagnosis of pd ) and 2 ( dementia preceding pd ) in level i testing ( table 1 ) were applicable to the subjects participating in this study . for item 3 ( decreased global cognitive efficiency ) , 105 subjects ( 34.5% ) showed an mmse score < 26 . with respect to item 5 ( dysfunction in > 1 cognitive domain ) , we observed that 53 subjects ( 17.4% ) showed no impairment in any cognitive domain , whereas the remaining subjects showed impairments in 1 ( n = 74 ; 24.3% ) , 2 ( n = 87 ; 28.6% ) , 3 ( n = 74 ; 24.3% ) , or 4 ( n = 16 ; 5.3% ) cognitive domains . therefore , 177 subjects ( 58.2% ) showed impairments in 2 cognitive domains . among these 177 subjects , 99 also had mmse scores < 26 ( 32.6% of all subjects ; 94.3% of the total group with mmse scores < 26 ) and therefore met the diagnostic criteria for pd - d . only 6 subjects ( 5.7% of 105 subjects ) with mmse scores < 26 did not meet the diagnostic criteria for pd - d , because they had only 1 impaired cognitive domain . of the 105 subjects with mmse scores < 26 , most showed impairments in 3 cognitive domains ( n = 62 ; 59.0% ) . figure 3 shows the number of subjects with mmse scores < 26 or 26 , along with the number of impaired domains . no subject with an mmse score < 26 was without impairment in any domain . among the subjects with mmse scores 26 ( n = 199 ) , we observed 1 ( n = 68 ; 34.2% ) , 2 ( n = 65 ; 32.7% ) , 3 ( n = 12 ; 6.0% ) , and 4 ( n = 1 ; 0.5% ) impaired domains ; 53 ( 26.6% ) had no impairment in any cognitive domain . figure 4 shows the proportions of subjects with mmse scores < 26 or 26 , along with the number of impaired domains . subjects with mmse scores 26 accounted for the majority of subjects with 1 impaired domain , and they accounted for 74.7% of subjects with 2 impaired domains . in contrast , subjects with mmse scores < 26 accounted for the majority of subjects with 3 or 4 impaired domains . this finding was as expected because patients with lower mmse scores should have an increased likelihood of being affected in more cognitive domains . figure 5a shows the numbers of subjects with impairments for each of the 4 cognitive domains . the most common impairment was executive dysfunction ( 224 subjects ; 73.7% ) , followed by memory ( n = 161 ; 53.0% ) and attention ( n = 119 ; 39.1% ) impairments . visuospatial functional impairments were the least common and were apparent in only 30 subjects ( 9.9% ) . of the 224 subjects who showed executive dysfunction , most exhibited impairment in lexical fluency ( 180 subjects ; 80.4% ) or clock drawing ( n = 133 ; 59.4% ) , whereas 89 subjects ( 39.7% ) displayed impairments in both ( fig . the most common combinations in the group with mmse scores 26 were executive dysfunction alone ( 49 subjects ; 24.6% ) and executive dysfunction with memory impairment ( n = 47 ; 23.6% ) , followed by executive dysfunction with attention impairment ( n = 16 ; 9.5% ) and memory impairment alone ( n = 15 ; 7.5% ) . the most common combination of impaired cognitive domains in the group with mmse scores < 26 was executive dysfunction with both memory and attention impairments ( n = 54 ; 51.4% ) , followed by impairments in all 4 domains ( n = 15 ; 14.3% ) . interestingly , visuospatial impairment was always accompanied by executive dysfunction ; however , visuospatial impairment alone , or in combination with attention , memory , or both attention and memory impairments , did not occur in any of the subjects . for the purpose of screening pd patients for dementia , it is necessary to use a test in which achievement does not decline in pd patients who have normal global cognitive efficiency ( as measured by the total mmse or moca score ) but declines in those patients with impaired global cognitive efficiency indicative of dementia . with regard to the hypothesis that an appropriate test is one in which the high- and middle - total - score groups by the mmse or the moca maintain a score 70% while the low - total - score group scores < 70% , the copy cube , draw clock , verbal abstraction , digit span , target tapping , and serial 7 subtraction tests of the moca ( marked # in fig . 2a ) and the serial 7 subtraction and 3-word delayed recall tests of the mmse ( marked # in fig . in level i testing , this includes the clock drawing , serial 7 subtraction , and delayed 3-word recall subtests , but not the phonemic fluency or pentagon copying subtests . as regards evaluating executive function , scores for the phonemic fluency test declined markedly to approximately 50% in the high - moca - score group , whose scores ( 24 - 30 ) were consistent with pd without dementia . the other subtest that evaluates executive function in level i testing is the clock drawing test . scores for this test declined to < 70% only in the low - moca - score group . this suggests the clock drawing test may be more appropriate than the phonemic fluency test for screening for a decline in executive function in pd - d . on the other hand , when evaluating visuospatial function , scores for the pentagon copying test stayed at 70% even in the low - mmse - score group , whose scores ( 12 - 25 ) were below the 26-point cutoff for pd - d . the authors of the original moca test indicated that the cube copying and clock drawing tests are concerned with visuospatial function , but in level i testing , the clock drawing test is used for executive function . for the cube copying test , the authors stated that if any of the following criteria are not met , the abnormal result was assigned : ( 1 ) drawing must be 3 dimensional , ( 2 ) all lines are drawn , ( 3 ) no line is added , ( 4 ) lines are relatively parallel and their length is similar . applied to the 304 subjects in the present study , 101 were classified as abnormal according to the cube copying test , including 43 subjects in the group with mmse scores 26 and 58 subjects in the group with mmse scores < 26 . these are far more than the 30 subjects with abnormal results for the pentagon copying test , and almost as many as the 119 subjects who were categorized as abnormal in attention by the serial 7 subtraction test . some previous studies have examined the screening properties of level i testing for pd - d . have demonstrated that level i testing is more sensitive for the detection of pd - d , detecting up to 22% more cases than the dsm - iv . di battista et al . validated level i testing in comparison with the dsm - iv criteria for pd - d and revealed its relatively lower sensitivity ( 78% ) but high specificity ( 95.5% ) with a 12% prevalence of pd - d in 76 pd patients . the authors raised their doubts about the sensitivity of an mmse cutoff value of 26 , stating this to be the main cause of false negatives , and also pointed out that more sensitive cognitive domain - related psychometric tests would maximize the screening property of level i testing . compared level i testing with full neuropsychological testing for detecting pd - d in 91 pd patients and found that 7.7% of the subjects met the level i testing criteria , while 16.5% of the subjects were classified as pd - d by full neuropsychological testing . they revealed that level i testing showed 100% specificity but only 46.7% sensitivity for diagnosing pd - d compared with the full neuropsychological assessment . they showed that the low sensitivity is largely due to the total mmse cutoff values for decreased global cognitive efficiency and the 15-item geriatric depression scale score for excluding depression . we found that 34.5% of all pd patients examined in the present study had mmse scores < 26 . most of these patients ( 94.3% ) were impaired in 2 cognitive domains and therefore were cases of probable pd - d . many of these patients ( 59% ) showed impairment in 3 cognitive domains , and patients rarely showed impairment in a single cognitive domain . at least 1 of these 4 cognitive domains was always impaired in pd patients with mmse scores < 26 . even among pd patients with mmse scores 26 , many had impairments in 1 or 2 cognitive domains , and the proportion of those with no impairment in any cognitive domain was 26.6% . subjects with mmse scores 26 accounted for 75% of pd patients who had impairments in 2 cognitive domains , which was far more than in the group with mmse scores < 26 . among the cognitive domains , executive function , which was tested by the phonemic fluency and clock drawing subtests , was most frequently impaired ; visuospatial function , which was tested by the pentagon copying test , was least frequently impaired . no patient showed visuospatial impairment alone , and an impaired visuospatial function was always accompanied by executive dysfunction . among the subjects in the group with mmse scores 26 , the most common combinations were executive dysfunction alone or executive dysfunction with memory impairment , each of which accounted for approximately 25% of these subjects . among the subjects in the group with mmse scores < 26 , the combination of executive dysfunction with both attention and memory impairment was most frequent , accounting for approximately 50% of these subjects . the inclusion of impairments in 2 cognitive domains as an item in level i testing stems from the conventional thinking behind the diagnostic criteria for dementia resulting from research on alzheimer 's disease . level i testing includes 4 cognitive domains that may often be impaired in pd patients . we showed that for the mmse and moca subtests , some test results were abnormal even among pd patients with mmse scores 26 . this suggests that for dementia screening tools such as level i testing , it may be appropriate to use tests in which abnormalities are rare in patients with mmse scores 26 but tend to be common in those scoring < 26 . thus , once tests that tend to yield abnormal scores even for pd patients with mmse scores 26 are included in a screening tool , its sensitivity for pd - d diagnosis will increase , even if its specificity will decrease . a total of 32.6% of the pd patients had mmse scores < 26 and impairments in 2 cognitive domains , and may therefore have met the diagnostic criteria for probable pd - d by level i testing . this point prevalence of pd - d in pd is similar to the mean value of 31.1% ( 95% ci : 20.1 - 42.1 ) reported in a systematic and critical review of previous studies , thus indicating the validity of level i testing . the fact that 1 of the 5 tests concerning the 4 cognitive domains included in these diagnostic tests showed abnormal scores for the group of patients with mmse scores < 26 indicates the validity of these tests as a whole for pd - d screening . however , the results of this study show that the moca clock drawing subtest might be more appropriate than the phonemic fluency test for revealing executive dysfunction when screening for pd - d . the frequency of abnormal results for the mmse pentagon copying subtest was < 10% , and thus was markedly lower than for the other tests . this is inconsistent with previous reports which found that visuospatial function tends to be impaired in pd - d , and it may be necessary to replace this test with another that also examines visuospatial function . item 4 ( activities of daily living ) in level i testing was not evaluated and no extensive neuropsychological assessment was done , because of which dementia was not accurately diagnosed for each subject . therefore , this study could not address the validity , sensitivity , or specificity of level i testing regarding clinically diagnosed pd - d . the months reversed test was not used as there was no japanese equivalent to this english subtest in level i testing ; therefore , we could not examine its validity . by using the mmse and moca , level i testing for pd - d can evaluate both decreased global cognitive efficiency and impairments in cognitive domains that are vulnerable in pd - d . level i testing is therefore regarded as a practical and useful tool for screening dementia in pd patients . a total of 32.6% of the pd patients met the diagnostic criteria for probable pd - d . among the subjects with mmse scores < 26 , the combination of impaired attention , executive dysfunction , and memory impairment was the most common ( 51.4% ) . the months reversed test can not be used in japanese as a test for attention owing to linguistic nonequivalence . the moca clock drawing test may be more appropriate than the phonemic fluency test as a measure of executive function . perhaps consideration should be given to replacing the mmse pentagon copying test with a different test of visuospatial function , such as the moca cube copying test , to improve sensitivity when screening for pd - d .

backgrounddementia is a new focus of research on improved treatment for parkinson 's disease ( pd ) . in 2007 , a screening tool for pd dementia ( pd - d ) was developed by the movement disorder society ( level i testing ) , which still requires verification by a large population study.methodswe conducted a cross - sectional and multicenter study including 13 institutions administering the mini - mental state examination ( mmse ) and montreal cognitive assessment ( moca ) to 304 pd patients ( mean age : 70.6 8.3 years ; mean hoehn and yahr stage : 2.7 0.7).resultsin all , 34.5% of the patients had mmse scores < 26 ; 94.3% of these patients had impairments in 2 cognitive domains and met the criteria for probable pd - d by level i testing . executive dysfunction combined with attention and memory impairment was most common ( 51.4% ) . in the level i subtests of executive function , the score for phonemic fluency declined by < 50% in patients with high moca scores ( 24 - 30 points ) and lacked specificity for pd - d . no patient had visuospatial impairment ( measured by the pentagon copying subtest ) alone , and the score for pentagon copying stayed at 70% even in patients with low mmse scores ( 12 - 25 points ) , therefore lacking sensitivity for pd-d.conclusionslevel i testing with administration of the mmse and moca is a practical and efficient screening tool for pd - d . however , the phonemic fluency and pentagon copying tests should be replaced by more specific / sensitive ones when screening for pd - d .

Introduction Subjects and Methods Subjects Assessments of Clinical and Neurological Features Algorithm for Level I Testing Statistical Analysis Results Subject Demographics Differences among the MoCA and MMSE Tests Application of Diagnostic Criteria for PD-D Screening (Level I Testing) Possible Alternatives to Level I Testing Discussion Conclusions Disclosure Statement

nondopaminergic and nonmotor symptoms of parkinson 's disease ( pd ) , which include psychiatric , autonomic , and gastrointestinal symptoms , are sometimes present before a diagnosis of pd and almost inevitably emerge with the disease progression . however , methods for recognizing and quantifying the nonmotor symptoms of pd have become a new focus of research that will form the basis for improved treatments . in pd dementia ( pd - d ) patients , -synuclein pathology can be widespread in the cerebral cortex . as a result , the movement disorder society task force published diagnostic criteria for pd - d in 2007 and simultaneously proposed a practical diagnostic procedure for pd - d with the aim of its widespread international application . it is considered a screening tool for diagnosing pd - d , whereas level ii testing provides a more detailed assessment that characterizes the components of pd - d and monitors the elements that may be responsive to intervention . level i testing was designed as a screening tool for use in the clinic and in clinical trials . in this study , we used level i testing on 304 pd patients with the objective of determining which patients would be diagnosed with pd - d and what impairments would be diagnosed among the 4 cognitive domains tested . this was a cross - sectional study which examined the clinical and neurological features of these pd patients , including sex , age at the assessment , education , disease duration , and the hoehn and yahr ( h&y ) stage . their cognitive function was evaluated with the japanese versions of the mini - mental state examination ( mmse ) and montreal cognitive assessment ( moca ) . the mmse consists of 10 subtests scored to a total of 30 points : pentagon copying ( 1 point ) , serial 7 subtraction ( 5 points ) , immediate ( 3 points ) and delayed 3-word recall ( 3 points ) , naming ( 2 points ) , repeating a sentence ( 1 point ) , 3-stage commands ( 3 point ) , reading a sentence ( 1 point ) , writing a sentence ( 1 point ) , and orientation to time and place ( 10 points ) . the moca consists of 12 subtests also scored to a total of 30 points : cube copying ( 1 point ) , clock drawing ( 3 points ) , trail making ( 1 point ) , phonemic fluency ( 1 point ) , verbal abstraction ( 2 points ) , digit span ( 2 points ) , serial 7 subtraction ( 3 points ) , target tapping ( 1 point ) , delayed 5-word recall ( 5 points ) , naming ( 3 points ) , repeating 2 sentences ( 2 points ) , and orientation to time and place ( 6 points ) . for item 5 , the results of the following subtests of the moca and mmse were used : serial 7 subtraction ( seven backward in table 1 ) , phonemic fluency ( lexical fluency ) , and clock drawing from the moca , and pentagon copying ( mmse pentagons ) and delayed 3-word recall from the mmse . according to the level i testing procedures , test results that indicated impairment were : 2 incorrect responses on the seven backward test , a score of 9 words in the lexical fluency test , an inability to correctly draw the clock face or clock hands pointing to the correct time in the clock drawing test , an inability to correctly draw 2 pentagons that overlap in the pentagon copying test , and 1 word missing in the 3-word recall test . this was a cross - sectional study which examined the clinical and neurological features of these pd patients , including sex , age at the assessment , education , disease duration , and the hoehn and yahr ( h&y ) stage . their cognitive function was evaluated with the japanese versions of the mini - mental state examination ( mmse ) and montreal cognitive assessment ( moca ) . the mmse consists of 10 subtests scored to a total of 30 points : pentagon copying ( 1 point ) , serial 7 subtraction ( 5 points ) , immediate ( 3 points ) and delayed 3-word recall ( 3 points ) , naming ( 2 points ) , repeating a sentence ( 1 point ) , 3-stage commands ( 3 point ) , reading a sentence ( 1 point ) , writing a sentence ( 1 point ) , and orientation to time and place ( 10 points ) . the moca consists of 12 subtests also scored to a total of 30 points : cube copying ( 1 point ) , clock drawing ( 3 points ) , trail making ( 1 point ) , phonemic fluency ( 1 point ) , verbal abstraction ( 2 points ) , digit span ( 2 points ) , serial 7 subtraction ( 3 points ) , target tapping ( 1 point ) , delayed 5-word recall ( 5 points ) , naming ( 3 points ) , repeating 2 sentences ( 2 points ) , and orientation to time and place ( 6 points ) . for item 5 , the results of the following subtests of the moca and mmse were used : serial 7 subtraction ( seven backward in table 1 ) , phonemic fluency ( lexical fluency ) , and clock drawing from the moca , and pentagon copying ( mmse pentagons ) and delayed 3-word recall from the mmse . according to the level i testing procedures , test results that indicated impairment were : 2 incorrect responses on the seven backward test , a score of 9 words in the lexical fluency test , an inability to correctly draw the clock face or clock hands pointing to the correct time in the clock drawing test , an inability to correctly draw 2 pentagons that overlap in the pentagon copying test , and 1 word missing in the 3-word recall test . briefly , the subjects were 166 men and 138 women with a mean age sd of 70.6 8.3 ( range : 42 - 90 ) years ; their education lasted 12.5 2.6 ( range : 1 - 20 ) years , the disease duration was 6.6 5.1 ( range : 0.2 - 30.1 ) years , and their h&y stage was 2.7 0.7 ( range : 1 - 5 ) . for most of the subtests ( 7 of 10 ) , the scores failed to show a significant decrease from the high- to the middle - mmse - score group , and the scores for the naming subtest failed to decrease even in the low - mmse - score group . for example , the moca includes a 5-word delayed recall test , whereas the mmse provides a 3-word delayed recall test ; 5-word recall according to the moca was < 70% even in the high - moca - score group , whereas 3-word recall according to the mmse was relatively preserved ( fig . the repeating 2 sentences subtest of the moca showed a score < 70% in the high - moca - score group , but the score for the repeating a sentence subtest of the mmse was preserved even in the low - mmse - score group . items 1 ( diagnosis of pd ) and 2 ( dementia preceding pd ) in level i testing ( table 1 ) were applicable to the subjects participating in this study . among these 177 subjects , 99 also had mmse scores < 26 ( 32.6% of all subjects ; 94.3% of the total group with mmse scores < 26 ) and therefore met the diagnostic criteria for pd - d . only 6 subjects ( 5.7% of 105 subjects ) with mmse scores < 26 did not meet the diagnostic criteria for pd - d , because they had only 1 impaired cognitive domain . of the 105 subjects with mmse scores < 26 , most showed impairments in 3 cognitive domains ( n = 62 ; 59.0% ) . the most common impairment was executive dysfunction ( 224 subjects ; 73.7% ) , followed by memory ( n = 161 ; 53.0% ) and attention ( n = 119 ; 39.1% ) impairments . of the 224 subjects who showed executive dysfunction , most exhibited impairment in lexical fluency ( 180 subjects ; 80.4% ) or clock drawing ( n = 133 ; 59.4% ) , whereas 89 subjects ( 39.7% ) displayed impairments in both ( fig . the most common combinations in the group with mmse scores 26 were executive dysfunction alone ( 49 subjects ; 24.6% ) and executive dysfunction with memory impairment ( n = 47 ; 23.6% ) , followed by executive dysfunction with attention impairment ( n = 16 ; 9.5% ) and memory impairment alone ( n = 15 ; 7.5% ) . the most common combination of impaired cognitive domains in the group with mmse scores < 26 was executive dysfunction with both memory and attention impairments ( n = 54 ; 51.4% ) , followed by impairments in all 4 domains ( n = 15 ; 14.3% ) . interestingly , visuospatial impairment was always accompanied by executive dysfunction ; however , visuospatial impairment alone , or in combination with attention , memory , or both attention and memory impairments , did not occur in any of the subjects . with regard to the hypothesis that an appropriate test is one in which the high- and middle - total - score groups by the mmse or the moca maintain a score 70% while the low - total - score group scores < 70% , the copy cube , draw clock , verbal abstraction , digit span , target tapping , and serial 7 subtraction tests of the moca ( marked # in fig . in level i testing , this includes the clock drawing , serial 7 subtraction , and delayed 3-word recall subtests , but not the phonemic fluency or pentagon copying subtests . as regards evaluating executive function , scores for the phonemic fluency test declined markedly to approximately 50% in the high - moca - score group , whose scores ( 24 - 30 ) were consistent with pd without dementia . this suggests the clock drawing test may be more appropriate than the phonemic fluency test for screening for a decline in executive function in pd - d . on the other hand , when evaluating visuospatial function , scores for the pentagon copying test stayed at 70% even in the low - mmse - score group , whose scores ( 12 - 25 ) were below the 26-point cutoff for pd - d . the authors of the original moca test indicated that the cube copying and clock drawing tests are concerned with visuospatial function , but in level i testing , the clock drawing test is used for executive function . briefly , the subjects were 166 men and 138 women with a mean age sd of 70.6 8.3 ( range : 42 - 90 ) years ; their education lasted 12.5 2.6 ( range : 1 - 20 ) years , the disease duration was 6.6 5.1 ( range : 0.2 - 30.1 ) years , and their h&y stage was 2.7 0.7 ( range : 1 - 5 ) . for most of the subtests ( 7 of 10 ) , the scores failed to show a significant decrease from the high- to the middle - mmse - score group , and the scores for the naming subtest failed to decrease even in the low - mmse - score group . for example , the moca includes a 5-word delayed recall test , whereas the mmse provides a 3-word delayed recall test ; 5-word recall according to the moca was < 70% even in the high - moca - score group , whereas 3-word recall according to the mmse was relatively preserved ( fig . the repeating 2 sentences subtest of the moca showed a score < 70% in the high - moca - score group , but the score for the repeating a sentence subtest of the mmse was preserved even in the low - mmse - score group . items 1 ( diagnosis of pd ) and 2 ( dementia preceding pd ) in level i testing ( table 1 ) were applicable to the subjects participating in this study . among these 177 subjects , 99 also had mmse scores < 26 ( 32.6% of all subjects ; 94.3% of the total group with mmse scores < 26 ) and therefore met the diagnostic criteria for pd - d . only 6 subjects ( 5.7% of 105 subjects ) with mmse scores < 26 did not meet the diagnostic criteria for pd - d , because they had only 1 impaired cognitive domain . of the 105 subjects with mmse scores < 26 , most showed impairments in 3 cognitive domains ( n = 62 ; 59.0% ) . the most common impairment was executive dysfunction ( 224 subjects ; 73.7% ) , followed by memory ( n = 161 ; 53.0% ) and attention ( n = 119 ; 39.1% ) impairments . of the 224 subjects who showed executive dysfunction , most exhibited impairment in lexical fluency ( 180 subjects ; 80.4% ) or clock drawing ( n = 133 ; 59.4% ) , whereas 89 subjects ( 39.7% ) displayed impairments in both ( fig . the most common combinations in the group with mmse scores 26 were executive dysfunction alone ( 49 subjects ; 24.6% ) and executive dysfunction with memory impairment ( n = 47 ; 23.6% ) , followed by executive dysfunction with attention impairment ( n = 16 ; 9.5% ) and memory impairment alone ( n = 15 ; 7.5% ) . the most common combination of impaired cognitive domains in the group with mmse scores < 26 was executive dysfunction with both memory and attention impairments ( n = 54 ; 51.4% ) , followed by impairments in all 4 domains ( n = 15 ; 14.3% ) . interestingly , visuospatial impairment was always accompanied by executive dysfunction ; however , visuospatial impairment alone , or in combination with attention , memory , or both attention and memory impairments , did not occur in any of the subjects . with regard to the hypothesis that an appropriate test is one in which the high- and middle - total - score groups by the mmse or the moca maintain a score 70% while the low - total - score group scores < 70% , the copy cube , draw clock , verbal abstraction , digit span , target tapping , and serial 7 subtraction tests of the moca ( marked # in fig . in level i testing , this includes the clock drawing , serial 7 subtraction , and delayed 3-word recall subtests , but not the phonemic fluency or pentagon copying subtests . as regards evaluating executive function , scores for the phonemic fluency test declined markedly to approximately 50% in the high - moca - score group , whose scores ( 24 - 30 ) were consistent with pd without dementia . this suggests the clock drawing test may be more appropriate than the phonemic fluency test for screening for a decline in executive function in pd - d . on the other hand , when evaluating visuospatial function , scores for the pentagon copying test stayed at 70% even in the low - mmse - score group , whose scores ( 12 - 25 ) were below the 26-point cutoff for pd - d . the authors of the original moca test indicated that the cube copying and clock drawing tests are concerned with visuospatial function , but in level i testing , the clock drawing test is used for executive function . some previous studies have examined the screening properties of level i testing for pd - d . validated level i testing in comparison with the dsm - iv criteria for pd - d and revealed its relatively lower sensitivity ( 78% ) but high specificity ( 95.5% ) with a 12% prevalence of pd - d in 76 pd patients . compared level i testing with full neuropsychological testing for detecting pd - d in 91 pd patients and found that 7.7% of the subjects met the level i testing criteria , while 16.5% of the subjects were classified as pd - d by full neuropsychological testing . they revealed that level i testing showed 100% specificity but only 46.7% sensitivity for diagnosing pd - d compared with the full neuropsychological assessment . we found that 34.5% of all pd patients examined in the present study had mmse scores < 26 . most of these patients ( 94.3% ) were impaired in 2 cognitive domains and therefore were cases of probable pd - d . many of these patients ( 59% ) showed impairment in 3 cognitive domains , and patients rarely showed impairment in a single cognitive domain . at least 1 of these 4 cognitive domains was always impaired in pd patients with mmse scores < 26 . even among pd patients with mmse scores 26 , many had impairments in 1 or 2 cognitive domains , and the proportion of those with no impairment in any cognitive domain was 26.6% . subjects with mmse scores 26 accounted for 75% of pd patients who had impairments in 2 cognitive domains , which was far more than in the group with mmse scores < 26 . among the cognitive domains , executive function , which was tested by the phonemic fluency and clock drawing subtests , was most frequently impaired ; visuospatial function , which was tested by the pentagon copying test , was least frequently impaired . no patient showed visuospatial impairment alone , and an impaired visuospatial function was always accompanied by executive dysfunction . among the subjects in the group with mmse scores 26 , the most common combinations were executive dysfunction alone or executive dysfunction with memory impairment , each of which accounted for approximately 25% of these subjects . among the subjects in the group with mmse scores < 26 , the combination of executive dysfunction with both attention and memory impairment was most frequent , accounting for approximately 50% of these subjects . the inclusion of impairments in 2 cognitive domains as an item in level i testing stems from the conventional thinking behind the diagnostic criteria for dementia resulting from research on alzheimer 's disease . we showed that for the mmse and moca subtests , some test results were abnormal even among pd patients with mmse scores 26 . this suggests that for dementia screening tools such as level i testing , it may be appropriate to use tests in which abnormalities are rare in patients with mmse scores 26 but tend to be common in those scoring < 26 . thus , once tests that tend to yield abnormal scores even for pd patients with mmse scores 26 are included in a screening tool , its sensitivity for pd - d diagnosis will increase , even if its specificity will decrease . a total of 32.6% of the pd patients had mmse scores < 26 and impairments in 2 cognitive domains , and may therefore have met the diagnostic criteria for probable pd - d by level i testing . the fact that 1 of the 5 tests concerning the 4 cognitive domains included in these diagnostic tests showed abnormal scores for the group of patients with mmse scores < 26 indicates the validity of these tests as a whole for pd - d screening . however , the results of this study show that the moca clock drawing subtest might be more appropriate than the phonemic fluency test for revealing executive dysfunction when screening for pd - d . by using the mmse and moca , level i testing for pd - d can evaluate both decreased global cognitive efficiency and impairments in cognitive domains that are vulnerable in pd - d . level i testing is therefore regarded as a practical and useful tool for screening dementia in pd patients . a total of 32.6% of the pd patients met the diagnostic criteria for probable pd - d . among the subjects with mmse scores < 26 , the combination of impaired attention , executive dysfunction , and memory impairment was the most common ( 51.4% ) . perhaps consideration should be given to replacing the mmse pentagon copying test with a different test of visuospatial function , such as the moca cube copying test , to improve sensitivity when screening for pd - d .

over 170 million people , approximately 3% of the world s population , are infected with the hepatitis c virus ( hcv).(1 ) infection with this virus can result in hepatic fibrosis which can progress to cirrhosis and hepatocellular carcinoma . liver biopsy is the gold standard for the diagnosis and prognosis of hepatic fibrosis,(2 ) but this approach is costly , invasive , painful , and unreliable if the scarring in the liver is not homogeneous or biopsies under 10 mm are analyzed . current serum biomarkers only eliminate the need for biopsies in 26% of cases.(4 ) more reliable noninvasive markers to replace liver biopsy would benefit patients and practitioners alike . blood proteins whose expression levels change with increasing fibrosis could be used as less invasive biomarkers which are easily obtainable . such novel serological markers could be identified using proteomics to separate serum or plasma and identify the differentially expressed proteins . however , the wide dynamic range of protein concentrations which span over 10 orders of magnitude in serum and plasma , poses a significant problem for proteomic analysis . highly abundant proteins , especially albumin , immunoglobulins and transferrin , restrict protein load on gels for electrophoresis and limit the detection of low abundance proteins.(5 ) despite this we have previously used two - dimensional gel electrophoresis ( 2-de ) over the wide ph 310 range to identify several novel candidate serum biomarkers for liver fibrosis.(6 ) to address the problem posed by highly abundant proteins , here we use 2-de over a narrow ph 35.6 range since this lies outside the range of highly abundant albumin , transferrin and immunoglobulins . this enables the loading of four times more protein than in our previous fibrosis marker study , and considerably enhances representation of low abundance features . we present significantly improved gel - based separation of the acidic proteome and have identified low abundant features which were below the detection threshold in the study using the wide ph 310 range . we also examine the basic serum proteome for fibrosis markers using in - solution isoelectric focusing ( ief ) followed by sds - page and discuss the benefit of this approach over 2-de for basic high molecular weight proteins . using this approach we have identified the beta chains of both complement c3 and c4 as candidate fibrosis markers which were not observed previously by 2-de . the two proteomic approaches shown in this study can aid clinical biomarker discovery not only for hepatic fibrosis but many other diseases . one and 2 mg of pooled normal human serum ( sigma , dorset , uk ) were separated using 18 cm ph 310 , ph 56 , ph 35.6 nonlinear ( nl ) and ph 611 immobilized ph gradient ( ipg ) strips ( ge healthcare , bucks , u.k . ) . samples were made up to 375 l in ief rehydration buffer ( 5 m urea , 2 m thiourea , 2 mm tributyl phosphine , 65 mm dtt , 4% ( w / v ) chaps , 150 mm nondetergent sulfobetaine 256 ( ndsb-256 ) and 0.0012% ( w / v ) bromophenol blue ) . 2-de was performed as described earlier for the ph 310 gels.(6 ) gels covering the other ph ranges were also run in the same way except with 1.8% ( v / v ) ph 56 , ph 36 and ph 611 ampholytes ( servalyt , serva , heidelberg , germany ) for the ph 56 nl , ph 35.6 and ph 611 strips , respectively . samples were left overnight to rehydrate 18 cm ph 35.6 nl ipg drystrips ( ge healthcare , bucks , u.k . ) . strips were incubated in equilibration solution ( 4 m urea , 2 m thiourea , 50 mm tris - hcl ( ph 6.8 ) , 30% ( v / v ) glycerol , 2% ( w / v ) sds , 130 mm dtt , 0.002% ( w / v ) bromophenol blue ) for 15 min . proteins were separated by 916% ( w / v ) sds - page gradient gels using 20 ma per gel for 1 h , followed by 40 ma per gel for 4 h at 10 c . following electrophoresis , gels were fixed in 40% ( v / v ) ethanol and 10% ( v / v ) acetic acid and stained with the fluorescent dye ogt 1238.(7 ) although this dye is proprietary , it is similar to other commercial dyes such as sypro ruby or sypro orange which can be used as alternatives . gels were scanned using an apollo ii linear fluorescence scanner ( oxford glycosciences , abingdon , u.k . ) to obtain 16-bit images at 200 m resolution . this apollo ii scanner is custom built but any imager which can image fluorescently stained gels , such as the fuji las range of cameras , could be used as alternatives . features were detected using a custom version of the melanie ii software ( oxford glycosciences , abingdon , uk).(7 ) although customized , the software was only used to curate spots and determine the number of spots per gel which can be done with any commercially available software such as melanie 7 . serum ( 500 g ) was also resolved by 2-de using ph 310 nl ipg strips as previously described.(6 ) differentially expressed bands on the sds - page gels were assigned for mass spectrometric analysis . the bands on the sds - page gels were excised manually and dried in a speedvac . in - gel trypsin digestion was carried out manually according to the protocol of shevchenko and co - workers.(8 ) digested samples were lyophilized and dissolved in 0.1% ( v / v ) formic acid prior to mass spectrometric analysis . tryptic peptides were analyzed using a q - tof 1 mass spectrometer coupled to a caplc ( waters , hertfordshire , u.k . ) . peptides were concentrated and desalted on a 300 m i.d./5 mm c18 precolumn and resolved on a 75 m i.d./25 cm c18 pepmap analytical column ( lc packings , sunnyvale , ca ) with a 45 min 595% ( v / v ) acetonitrile gradient containing 0.1% ( v / v ) formic acid at a flow rate of 200 nl / min . ms to ms / ms switching was controlled in an automatic data - dependent fashion with a 1 s survey scan followed by three 1 s ms / ms scans . raw ms / ms spectra were smoothed and centered using proteinlynx global server 2.1.5 , spectra were not deisotoped . processed peak list ( .pkl ) files were searched against the swiss - prot database ( release 54.4 ) using mascot ( matrix science , london , u.k . ) . searches were restricted to the human ( 17 565 sequences ) and virus ( 11 132 sequences ) taxonomies . carbamidomethyl cysteine was defined as a fixed modification and oxidized methionine as a variable modification . data were searched allowing 0.5 da error to accommodate calibration drift and up to 2 missed tryptic cleavage sites . all data were checked for consistent error distribution and all positive identifications were checked manually . healthy control and cirrhotic blood samples were collected in serum separator tubes ( bd , oxford , u.k . ) . cirrhosis was determined using the ishak scoring method as previously described.(9 ) these scores along with the ages and gender are displayed in supporting table 1 , supporting information . collection of patient samples for this study was approved by the central oxford research ethics committee ( no . 98.137 ) and consent was obtained from each patient . sera were diluted in ief rehydration buffer with 1.8% ( v / v ) ph 310 carrier ampholytes and 0.002% ( w / v ) bromophenol blue to a final protein concentration of 298.5 g / ml . in - solution ief was performed as previously described using an ief fractionator ( invitrogen , paisley , u.k . ) with the following ph ranges in each fractionation chamber : ph 34.6 ; ph 4.65.4 ; ph 5.46.2 ; ph 6.27 ; ph 710 . the anode buffer was prepared with 7 m urea , 2 m thiourea and novex ief anode buffer ph 3.0 ( invitrogen ) . the cathode buffer was prepared with 7 m urea , 2 m thiourea and novex ief cathode buffer ph 10.4 ( invitrogen ) . anode and cathode buffers ( 17.5 ml each ) were loaded into the respective electrode reservoirs of the ief fractionator . fractionation was performed using 100 v for 20 min , 200 v for 80 min and 600 v for 80 min at 2 ma and 2 w. detergents and salts were removed from the samples by chloroformmethanol precipitation as previously described for in - solution ief(12 ) before being resolved by 412% ( w / v ) nupage bis - tris - hcl sds - page ( invitrogen ) according to the manufacturer s recommendation . the intensity of gel bands for healthy control were compared to cirrhotic serum using advanced image data analyzer software ( raytest , straubenhardt , germany ) . narrow range ipg strips with the ranges ph 611 , ph 56 and ph 35.6 along with wide range ph 310 ipg strips were used to separate 1 and 2 mg of normal human serum . these gels were compared with 500 g of normal human serum loaded on ph 310 strips , as used in our previous study,(6 ) ( figure 1 ) . the ph 35.6 range with a load of 2 mg showed the best separation with almost twice as many features as a ph 310 gel across the same ph 35.6 range ( supporting figure 1 , supporting information ) . a spot number count on the melanie ii software showed that the ph 35.6 range with a 2 mg load had 551 features whereas the ph 310 gel with a 500 g load had only 289 features across the same ph 35.6 range . the total spot number count on the 500 g ph 310 gel was 523 , less than the total feature count on the ph 35.6 gel . spots which were well separated in the 500 g ph 310 gel were merged together in the ph 310 gels with 1 and 2 mg of serum . gels were run with ( a ) ph 611 , ( b ) ph 56 , and ( c ) ph 35.6 nl ipg strips . in each case , 1 mg was separated using a narrow range ipg strip ( left ) , 2 mg was separated using the same narrow range ipg strip ( middle ) and 500 g separated using ph 310 nl ipg strips with the narrow ph range investigated highlighted within a dashed box ( right ) . healthy control and cirrhotic serum samples were separated by in - solution ief into five different ph ranges using an ief fractionator . figure 2 shows an sds - page gel with the five fractions from the ief fractionator where the band profile seen by sds - page resembles that of a ph 310 2-de gel since the fractions range from ph 3 to 10 . figure 3 shows each fraction resolved by sds - page highlighting the differentially expressed bands between the healthy control and cirrhotic samples . as with our previous 2-de analysis,(6 ) the following changes were observed in cirrhotic serum with respect to healthy control serum : a decrease in haptoglobin , an increase in alpha 2 macroglobulin , and an increase in igg ( both heavy and light chains ) . consistent with the 2-de results , other fragments of complement c3 and c4 were found to be more abundant in the serum of controls than cirrhotic patients . in the ph 710 fraction , differentially expressed bands at approximately 75 kda on the gel contained peptides derived from complement c3 and c4 , which in both cases span their -chains ( supporting figure 2 , supporting information ) . the protein score , sequence coverage and peptides identified for these complement proteins are shown in table 1 . in - solution ief and sds - page were repeated for additional samples followed by gel band densitometry ( supporting figure 3a , supporting information ) . this data shows that the higher molecular weight band , which contains complement c3 beta but predominantly c4 beta ( table 1 ) , is clearly expressed to a higher extent in controls compared to cirrhosis when analyzing multiple samples ( supporting figure 3b , upper panel ) . the lower molecular weight band containing complement c4 beta and predominately c3 beta ( table 1 ) is seen in both controls and cirrhosis but expressed to a higher extent in controls ( supporting figure 3b , lower panel ) . to validate these candidate cirrhosis markers , unfractionated control and cirrhosis samples were blotted for complement c3 beta and c4 beta ( supporting figure 3c ) . blot band densitometry showed a clear decrease for both complement c3 beta and c4 beta , the results being more consistent for complement c4 beta with lower standard deviation ( supporting figure 3d ) . the in - solution ief fractions from control serum were also separated by 2-de to confirm fractionation ( supporting figure 4 , supporting information ) . the results show that the ief fractionator does not give a clear - cut fractionation but is able to reproducibly enrich proteins in the ph range of the fraction . serum banding pattern for the ief fractionator fractions run by sds - page shows a similar profile to the spots seen by 2-de . ( a ) serum was separated by 916% 2-de with ph 310 nl ipg strips using a load of 500 g . ( b ) serum was separated using in - solution ief into five fractions ( f1 = ph 34.6 ; f2 = ph 4.65.4 ; f3 = ph 5.46.2 ; f4 = ph 6.27 ; f5 = ph 710 ) . m = molecular weight markers ( 225 , 150 , 100 , 75 , 50 , 35 , 25 , 15 , 10 kda ) . the five fractions from the ief fractionator span ph 3 to ph 10 and therefore the banding pattern seen by sds - page is similar to the spot profile seen using a ph 310 2-de gel . in - solution ief combined with sds - page allows improved representation of high molecular weight basic proteins . normal controls ( n ) and cirrhotic ( c ) serum samples were fractionated by in - solution ief . the resulting five fractions from the ief fractionator were then separated by 412% ( w / v ) sds - page alongside unfractionated serum . the dashed box region indicates the serum profile that is comparable to a ph 310 2-de gel ( see figure 3 ) . h = high molecular weight basic proteins that are not as well represented by 2-de . b ) differential analysis of the sds - page lanes comparing controls with cirrhotic serum for each of the fractions . u = unfractionated serum ; 1 = ph 34.6 ; 2 = ph 4.65.4 ; 3 = ph 5.46.2 ; 4 = ph 6.27 ; 5 = ph 710 ; m = molecular weight markers ; h - b = haptoglobin -chain ; h - a = haptoglobin -chain ; a2 m = 2 macroglobin ; c3/4b = -chains of complement c3 and c4 . bands 1 and 2 show the bands containing the complement proteins and the number of ms / ms peptide matches , percentage sequence coverage and protein score for these bands are shown in table 1 . the number of ms / ms peptide matches , percentage sequence coverage and protein score were determined by the mascot daemon search engine . individuals infected with hcv can develop hepatic fibrosis which can progress to liver cirrhosis and hepatocellular carcinoma . currently the most reliable way of assessing liver fibrosis is by biopsy and so there is an urgent need for less invasive serological biomarkers . in this study we use two novel proteomics approaches which can aid biomarker discovery for all diseases including liver fibrosis . the use of in - solution ief reveals the beta chains of both complement c3 and c4 to be decreased in cirrhosis . a major difficulty for discovering novel biomarkers in serum and plasma is the presence of highly abundant proteins which limits the detection of low abundant features and restricts the amount of total protein loaded onto gels.(5 ) both albumin and igg alone make up more than 75% of the total plasma / serum protein content.(14 ) to overcome this obstacle in finding biomarkers several groups have tried prefractionation strategies to deplete high abundance proteins from samples prior to electrophoresis and thus improve the representation of low abundant proteins . antibody - based immunoprecipitation methods appear to be most suitable for removal of highly abundant proteins and we have successfully performed this in the past to identify a novel biomarker for uveal melanoma by removing twelve of the more abundant serum proteins.(15 ) we achieved this using chicken igy antibodies since they offer broader host antigen binding and cleaner capture than igg methods due to the greater evolutionary distance between chickens and mammals . however , immunoprecipitation is expensive due to the vast amount of antibody required to deplete these highly abundant proteins . less expensive options for the removal of albumin include using cibacron blue - based prefractionation approaches . these dye - affinity methods have been compared alongside immunoprecipitation and are less efficient and less specific causing unwanted removal of a large number of nonalbumin proteins,(16 ) possibly including potential biomarkers . unlike the large amount of protein used in this study ( 2 mg ) , it is very challenging to load similar high levels of protein post depletion for multiple samples due to the low recovery rates during the removal of highly abundant proteins as well as losses during concentration . serum protein loads of 1 and 2 mg were investigated , the latter being the maximum load recommended by the manufacturer for preparative ief using 18 cm ipg strips . for the narrow ph ranges investigated , figure 1 shows improved representation of low abundant features for 2 mg serum compared to 1 mg serum . in the case for ph 310 gels , features which were well separated using a load of 500 g were merged together when using the higher 1 and 2 mg loads due to overloading indicating that this wide ph range is unsuitable for high protein loads . the ph 611 range showed no improvement over our previous ph 310 gels(6 ) in this alkaline region possibly due to the presence of highly abundant igg and transferrin . the reducing agent we used in this study , dithiothreitol , becomes negatively charged during ief and migrates toward the anode thereby decreasing the concentration of dithiothreitol in the basic end of the strip . these unreduced proteins have decreased solubility resulting in streaking in the basic region of the 2-de gel.(17 ) this particularly affects basic proteins which have a high molecular weight , since they have difficulty in entering the second dimension gel matrix from the ief strip.(18 ) the ph 56 range , although covering the main isoform of albumin , did display improved separation of proteins but with only a few additional features not previously seen in the wide range ph 310 analysis.(6 ) all of these additional features had already been observed in the ph 35.6 gels , therefore this ph range did not warrant complete analysis . the use of narrow range ipg strips in a range outside the isoelectric points of the main isoforms of the most abundant plasma / serum proteins would enable greater protein loads thereby increasing the representation of low abundant features . serum was separated using a wide ph 310 nl ipg strip ( figure 1 ) and the isoelectric point ranges of the main isoforms of albumin , igg and transferrin were determined using calibrated landmarks as ph 5.65.9 , 6.38.5 and 6.26.5 , respectively , which is consistent with previously reported 2-de data.(19 ) the ph range of these three most abundant plasma / serum proteins were above ph 5.6 which may explain why the ph 35.6 nl range chosen for analysis led to superior separation and highest feature number . some albumin bled over into the ph 35.6 range when using a serum load of 2 mg , although this was no worse than what we observed in our previous study.(6 ) we judged the gain in low abundance features with the higher protein load to outweigh this problem and the load of 2 mg was chosen for determining fibrosis biomarkers using these narrow range ipg strips . the ph 35.6 range with a load of 2 mg appeared to be the best narrow ph range for biomarker discovery . this ph range allowed four times more protein to be loaded ( 2 mg ) than in our previous study(6 ) ( 500 g ) , which allowed visualization of several new low abundant features . also the narrow ph range helped to increase the separation of the acidic plasma / serum proteome . the separation achieved in this ph range appeared to be better than previous large scale 2-de studies for identifying the human serum proteome(20 ) showing new features that were previously not observed by 2-de . therefore not only is this ph range better for biomarker discovery but it also may reveal new serum / plasma proteins that were previously not detected by 2-de . to prove that this ph range was suitable for biomarker discovery , plasma samples from healthy control individuals and patients with hcv induced cirrhosis were compared using 2-de over this range to identify novel biomarker candidates for hepatic fibrosis in hepatitis c patient.(21 ) using this ph 35.6 range 21 novel candidate fibrosis biomarkers were identified which were not seen in our previous study using the ph 310 range . this confirms that the ph 35.6 range helps in the discovery of clinical biomarkers for hepatic fibrosis and would be advantageous in determining novel serological markers for other diseases . the ph 35.6 range used in this study only covers the acidic proteome and therefore any biomarkers present in the alkaline region would be missed . the ph 611 range was investigated ( figure 1 ) but this range showed no improvement to the basic area compared to our previous study(6 ) and high molecular weight basic proteins are poorly resolved by 2-de . these difficulties with basic and high molecular weight proteins are not encountered with sds - page , a technique with lower resolution . to attain a higher resolution while taking advantage of the benefits of sds - page , we decided to fractionate serum samples by ph prior to electrophoresis . although the combination of in - solution ief and sds - page has previously been reported,(22 ) we indicate for the first time that this approach is beneficial for analyzing basic , high molecular weight proteins . sds - page analysis of unfractionated control and cirrhotic serum appeared to show no difference in differential band analysis due to the low resolution separation of this technique . however , by combining the in - solution ief method with the sds - page approach , the proteins were separated to an extent which allowed to discern differences . changes in complement c3 and c4 were observed in the high molecular weight basic fraction ( ph 710 ) . to confirm the reproducibility of this data , in - solution ief and sds - page were repeated for additional samples followed by gel band densitomtery ( supporting figure 3a , supporting information ) which showed that the changes in both bands containing c3 and c4 beta were consistent . these additional samples were not matched to any category such as age or sex ( supporting table 1 , supporting information ) . the point and advantage of this kind of marker finding exercise is that biomarkers should not be dependent on these categories and so we aimed to eliminate any group specific hits from the outset . we have previously identified 21 markers for liver fibrosis which were not dependent on age or sex.(21 ) the changes in complement c3 and c4 beta were not observed by 2-de analysis due to the problem with reduced solubility of basic proteins , demonstrating an advantage of the combined in - solution ief and sds - page approach over solely gel - based technologies . the theoretical pi of the -chain for c4 is ph 8.7 , which is consistent with the ph 710 range for this fraction . the theoretical pi of the -chain for c3 was determined to be ph 6.8 which is marginally outside the range of this fraction but this was expected since we found that the ief fractionator enriches proteins rather than providing a clear - cut fractionation for the ph range ; however , as proteins are enriched reproducibly this poses no problem . the differentially expressed band immediately below the 75 kda also contained sequences within the -chains of both c3 and c4 but these were fragments since the band was at a lower molecular weight . this band was seen in both control and cirrhosis samples but expressed to a higher extent in control samples , whereas the higher molecular weight band which was differentially expressed appeared only in control samples ( figure 3 and supporting figure 3a , supporting information ) . this lower molecular weight band contained predominantly complement c3 beta ( table 1 ) and showed a clear but less consistent change among multiple samples when validated by western blot ( supporting figure 3c and d ) . the more clearly changing higher molecular weight band contained predominantly complement c4 beta ( table 1 ) and showed a clear and consistent change among multiple samples when validated by western blot ( supporting figure 3c and d ) . this suggests that complement c4 beta may be a better biomarker for cirrhosis than complement c3 beta and would need to be validated using a larger number of samples . this study shows how two different proteomic approaches can aid in the discovery of disease biomarkers . to our knowledge this is the first time the ph 35.6 range has been used to separate serum by 2-de and we have shown that this ph range is useful for discovering novel biomarkers in diseases . we have also shown that the use of in - solution ief followed by sds - page improves the separation of the basic proteome thereby helping to identify disease biomarkers in the basic region of plasma and serum proteomes . using this approach we show that the beta chains of complement c3 and c4 decrease in serum from hepatitis c patients with cirrhosis , a change not observed previously by 2-de .

despite many shortcomings , liver biopsy is regarded as the gold standard for assessing liver fibrosis . a less invasive and equally or more reliable approach would constitute a major advancement in the field . proteomics can aid discovery of novel serological markers and these proteins can be measured in patient blood . a major challenge of discovering biomarkers in serum is the presence of highly abundant serum proteins , which restricts the levels of total protein loaded onto gels and limits the detection of low abundance features . to overcome this problem , we used two - dimensional gel electrophoresis ( 2-de ) over a narrow ph 35.6 range since this lies outside the range of highly abundant albumin , transferrin and immunoglobulins . in addition , we used in - solution isoelectric focusing followed by sds - page to find biomarkers in hepatitis c induced liver cirrhosis . using the ph 35.6 range for 2-de , we achieved improved representation of low abundance features and enhanced separation . we found in - solution isoelectric focusing to be beneficial for analyzing basic , high molecular weight proteins . using this method , the beta chains of both complement c3 and c4 were found to decrease in serum from hepatitis c patients with cirrhosis , a change not observed previously by 2-de . we present two proteomics approaches that can aid in the discovery of clinical biomarkers in various diseases and discuss how these approaches have helped to identify 23 novel biomarkers for hepatic fibrosis .

Introduction Experimental Section Results Discussion Conclusion

(1 ) infection with this virus can result in hepatic fibrosis which can progress to cirrhosis and hepatocellular carcinoma . liver biopsy is the gold standard for the diagnosis and prognosis of hepatic fibrosis,(2 ) but this approach is costly , invasive , painful , and unreliable if the scarring in the liver is not homogeneous or biopsies under 10 mm are analyzed . (4 ) more reliable noninvasive markers to replace liver biopsy would benefit patients and practitioners alike . blood proteins whose expression levels change with increasing fibrosis could be used as less invasive biomarkers which are easily obtainable . such novel serological markers could be identified using proteomics to separate serum or plasma and identify the differentially expressed proteins . however , the wide dynamic range of protein concentrations which span over 10 orders of magnitude in serum and plasma , poses a significant problem for proteomic analysis . highly abundant proteins , especially albumin , immunoglobulins and transferrin , restrict protein load on gels for electrophoresis and limit the detection of low abundance proteins. (5 ) despite this we have previously used two - dimensional gel electrophoresis ( 2-de ) over the wide ph 310 range to identify several novel candidate serum biomarkers for liver fibrosis. (6 ) to address the problem posed by highly abundant proteins , here we use 2-de over a narrow ph 35.6 range since this lies outside the range of highly abundant albumin , transferrin and immunoglobulins . this enables the loading of four times more protein than in our previous fibrosis marker study , and considerably enhances representation of low abundance features . we present significantly improved gel - based separation of the acidic proteome and have identified low abundant features which were below the detection threshold in the study using the wide ph 310 range . we also examine the basic serum proteome for fibrosis markers using in - solution isoelectric focusing ( ief ) followed by sds - page and discuss the benefit of this approach over 2-de for basic high molecular weight proteins . using this approach we have identified the beta chains of both complement c3 and c4 as candidate fibrosis markers which were not observed previously by 2-de . the two proteomic approaches shown in this study can aid clinical biomarker discovery not only for hepatic fibrosis but many other diseases . one and 2 mg of pooled normal human serum ( sigma , dorset , uk ) were separated using 18 cm ph 310 , ph 56 , ph 35.6 nonlinear ( nl ) and ph 611 immobilized ph gradient ( ipg ) strips ( ge healthcare , bucks , u.k . ) (6 ) gels covering the other ph ranges were also run in the same way except with 1.8% ( v / v ) ph 56 , ph 36 and ph 611 ampholytes ( servalyt , serva , heidelberg , germany ) for the ph 56 nl , ph 35.6 and ph 611 strips , respectively . samples were left overnight to rehydrate 18 cm ph 35.6 nl ipg drystrips ( ge healthcare , bucks , u.k . ) proteins were separated by 916% ( w / v ) sds - page gradient gels using 20 ma per gel for 1 h , followed by 40 ma per gel for 4 h at 10 c . this apollo ii scanner is custom built but any imager which can image fluorescently stained gels , such as the fuji las range of cameras , could be used as alternatives . (7 ) although customized , the software was only used to curate spots and determine the number of spots per gel which can be done with any commercially available software such as melanie 7 . (6 ) differentially expressed bands on the sds - page gels were assigned for mass spectrometric analysis . the bands on the sds - page gels were excised manually and dried in a speedvac . ms to ms / ms switching was controlled in an automatic data - dependent fashion with a 1 s survey scan followed by three 1 s ms / ms scans . healthy control and cirrhotic blood samples were collected in serum separator tubes ( bd , oxford , u.k . ) in - solution ief was performed as previously described using an ief fractionator ( invitrogen , paisley , u.k . ) fractionation was performed using 100 v for 20 min , 200 v for 80 min and 600 v for 80 min at 2 ma and 2 w. detergents and salts were removed from the samples by chloroformmethanol precipitation as previously described for in - solution ief(12 ) before being resolved by 412% ( w / v ) nupage bis - tris - hcl sds - page ( invitrogen ) according to the manufacturer s recommendation . narrow range ipg strips with the ranges ph 611 , ph 56 and ph 35.6 along with wide range ph 310 ipg strips were used to separate 1 and 2 mg of normal human serum . these gels were compared with 500 g of normal human serum loaded on ph 310 strips , as used in our previous study,(6 ) ( figure 1 ) . the ph 35.6 range with a load of 2 mg showed the best separation with almost twice as many features as a ph 310 gel across the same ph 35.6 range ( supporting figure 1 , supporting information ) . a spot number count on the melanie ii software showed that the ph 35.6 range with a 2 mg load had 551 features whereas the ph 310 gel with a 500 g load had only 289 features across the same ph 35.6 range . the total spot number count on the 500 g ph 310 gel was 523 , less than the total feature count on the ph 35.6 gel . spots which were well separated in the 500 g ph 310 gel were merged together in the ph 310 gels with 1 and 2 mg of serum . gels were run with ( a ) ph 611 , ( b ) ph 56 , and ( c ) ph 35.6 nl ipg strips . in each case , 1 mg was separated using a narrow range ipg strip ( left ) , 2 mg was separated using the same narrow range ipg strip ( middle ) and 500 g separated using ph 310 nl ipg strips with the narrow ph range investigated highlighted within a dashed box ( right ) . healthy control and cirrhotic serum samples were separated by in - solution ief into five different ph ranges using an ief fractionator . figure 2 shows an sds - page gel with the five fractions from the ief fractionator where the band profile seen by sds - page resembles that of a ph 310 2-de gel since the fractions range from ph 3 to 10 . figure 3 shows each fraction resolved by sds - page highlighting the differentially expressed bands between the healthy control and cirrhotic samples . consistent with the 2-de results , other fragments of complement c3 and c4 were found to be more abundant in the serum of controls than cirrhotic patients . in the ph 710 fraction , differentially expressed bands at approximately 75 kda on the gel contained peptides derived from complement c3 and c4 , which in both cases span their -chains ( supporting figure 2 , supporting information ) . in - solution ief and sds - page were repeated for additional samples followed by gel band densitometry ( supporting figure 3a , supporting information ) . this data shows that the higher molecular weight band , which contains complement c3 beta but predominantly c4 beta ( table 1 ) , is clearly expressed to a higher extent in controls compared to cirrhosis when analyzing multiple samples ( supporting figure 3b , upper panel ) . to validate these candidate cirrhosis markers , unfractionated control and cirrhosis samples were blotted for complement c3 beta and c4 beta ( supporting figure 3c ) . blot band densitometry showed a clear decrease for both complement c3 beta and c4 beta , the results being more consistent for complement c4 beta with lower standard deviation ( supporting figure 3d ) . the in - solution ief fractions from control serum were also separated by 2-de to confirm fractionation ( supporting figure 4 , supporting information ) . the results show that the ief fractionator does not give a clear - cut fractionation but is able to reproducibly enrich proteins in the ph range of the fraction . serum banding pattern for the ief fractionator fractions run by sds - page shows a similar profile to the spots seen by 2-de . ( b ) serum was separated using in - solution ief into five fractions ( f1 = ph 34.6 ; f2 = ph 4.65.4 ; f3 = ph 5.46.2 ; f4 = ph 6.27 ; f5 = ph 710 ) . the five fractions from the ief fractionator span ph 3 to ph 10 and therefore the banding pattern seen by sds - page is similar to the spot profile seen using a ph 310 2-de gel . in - solution ief combined with sds - page allows improved representation of high molecular weight basic proteins . normal controls ( n ) and cirrhotic ( c ) serum samples were fractionated by in - solution ief . the resulting five fractions from the ief fractionator were then separated by 412% ( w / v ) sds - page alongside unfractionated serum . h = high molecular weight basic proteins that are not as well represented by 2-de . b ) differential analysis of the sds - page lanes comparing controls with cirrhotic serum for each of the fractions . u = unfractionated serum ; 1 = ph 34.6 ; 2 = ph 4.65.4 ; 3 = ph 5.46.2 ; 4 = ph 6.27 ; 5 = ph 710 ; m = molecular weight markers ; h - b = haptoglobin -chain ; h - a = haptoglobin -chain ; a2 m = 2 macroglobin ; c3/4b = -chains of complement c3 and c4 . individuals infected with hcv can develop hepatic fibrosis which can progress to liver cirrhosis and hepatocellular carcinoma . currently the most reliable way of assessing liver fibrosis is by biopsy and so there is an urgent need for less invasive serological biomarkers . in this study we use two novel proteomics approaches which can aid biomarker discovery for all diseases including liver fibrosis . the use of in - solution ief reveals the beta chains of both complement c3 and c4 to be decreased in cirrhosis . a major difficulty for discovering novel biomarkers in serum and plasma is the presence of highly abundant proteins which limits the detection of low abundant features and restricts the amount of total protein loaded onto gels. (14 ) to overcome this obstacle in finding biomarkers several groups have tried prefractionation strategies to deplete high abundance proteins from samples prior to electrophoresis and thus improve the representation of low abundant proteins . antibody - based immunoprecipitation methods appear to be most suitable for removal of highly abundant proteins and we have successfully performed this in the past to identify a novel biomarker for uveal melanoma by removing twelve of the more abundant serum proteins. (15 ) we achieved this using chicken igy antibodies since they offer broader host antigen binding and cleaner capture than igg methods due to the greater evolutionary distance between chickens and mammals . however , immunoprecipitation is expensive due to the vast amount of antibody required to deplete these highly abundant proteins . unlike the large amount of protein used in this study ( 2 mg ) , it is very challenging to load similar high levels of protein post depletion for multiple samples due to the low recovery rates during the removal of highly abundant proteins as well as losses during concentration . serum protein loads of 1 and 2 mg were investigated , the latter being the maximum load recommended by the manufacturer for preparative ief using 18 cm ipg strips . for the narrow ph ranges investigated , figure 1 shows improved representation of low abundant features for 2 mg serum compared to 1 mg serum . in the case for ph 310 gels , features which were well separated using a load of 500 g were merged together when using the higher 1 and 2 mg loads due to overloading indicating that this wide ph range is unsuitable for high protein loads . the ph 611 range showed no improvement over our previous ph 310 gels(6 ) in this alkaline region possibly due to the presence of highly abundant igg and transferrin . the reducing agent we used in this study , dithiothreitol , becomes negatively charged during ief and migrates toward the anode thereby decreasing the concentration of dithiothreitol in the basic end of the strip . (17 ) this particularly affects basic proteins which have a high molecular weight , since they have difficulty in entering the second dimension gel matrix from the ief strip. (18 ) the ph 56 range , although covering the main isoform of albumin , did display improved separation of proteins but with only a few additional features not previously seen in the wide range ph 310 analysis. (6 ) all of these additional features had already been observed in the ph 35.6 gels , therefore this ph range did not warrant complete analysis . the use of narrow range ipg strips in a range outside the isoelectric points of the main isoforms of the most abundant plasma / serum proteins would enable greater protein loads thereby increasing the representation of low abundant features . serum was separated using a wide ph 310 nl ipg strip ( figure 1 ) and the isoelectric point ranges of the main isoforms of albumin , igg and transferrin were determined using calibrated landmarks as ph 5.65.9 , 6.38.5 and 6.26.5 , respectively , which is consistent with previously reported 2-de data. (19 ) the ph range of these three most abundant plasma / serum proteins were above ph 5.6 which may explain why the ph 35.6 nl range chosen for analysis led to superior separation and highest feature number . some albumin bled over into the ph 35.6 range when using a serum load of 2 mg , although this was no worse than what we observed in our previous study. (6 ) we judged the gain in low abundance features with the higher protein load to outweigh this problem and the load of 2 mg was chosen for determining fibrosis biomarkers using these narrow range ipg strips . the ph 35.6 range with a load of 2 mg appeared to be the best narrow ph range for biomarker discovery . this ph range allowed four times more protein to be loaded ( 2 mg ) than in our previous study(6 ) ( 500 g ) , which allowed visualization of several new low abundant features . also the narrow ph range helped to increase the separation of the acidic plasma / serum proteome . the separation achieved in this ph range appeared to be better than previous large scale 2-de studies for identifying the human serum proteome(20 ) showing new features that were previously not observed by 2-de . therefore not only is this ph range better for biomarker discovery but it also may reveal new serum / plasma proteins that were previously not detected by 2-de . to prove that this ph range was suitable for biomarker discovery , plasma samples from healthy control individuals and patients with hcv induced cirrhosis were compared using 2-de over this range to identify novel biomarker candidates for hepatic fibrosis in hepatitis c patient. (21 ) using this ph 35.6 range 21 novel candidate fibrosis biomarkers were identified which were not seen in our previous study using the ph 310 range . this confirms that the ph 35.6 range helps in the discovery of clinical biomarkers for hepatic fibrosis and would be advantageous in determining novel serological markers for other diseases . the ph 35.6 range used in this study only covers the acidic proteome and therefore any biomarkers present in the alkaline region would be missed . the ph 611 range was investigated ( figure 1 ) but this range showed no improvement to the basic area compared to our previous study(6 ) and high molecular weight basic proteins are poorly resolved by 2-de . these difficulties with basic and high molecular weight proteins are not encountered with sds - page , a technique with lower resolution . to attain a higher resolution while taking advantage of the benefits of sds - page , we decided to fractionate serum samples by ph prior to electrophoresis . although the combination of in - solution ief and sds - page has previously been reported,(22 ) we indicate for the first time that this approach is beneficial for analyzing basic , high molecular weight proteins . sds - page analysis of unfractionated control and cirrhotic serum appeared to show no difference in differential band analysis due to the low resolution separation of this technique . however , by combining the in - solution ief method with the sds - page approach , the proteins were separated to an extent which allowed to discern differences . changes in complement c3 and c4 were observed in the high molecular weight basic fraction ( ph 710 ) . to confirm the reproducibility of this data , in - solution ief and sds - page were repeated for additional samples followed by gel band densitomtery ( supporting figure 3a , supporting information ) which showed that the changes in both bands containing c3 and c4 beta were consistent . (21 ) the changes in complement c3 and c4 beta were not observed by 2-de analysis due to the problem with reduced solubility of basic proteins , demonstrating an advantage of the combined in - solution ief and sds - page approach over solely gel - based technologies . the theoretical pi of the -chain for c4 is ph 8.7 , which is consistent with the ph 710 range for this fraction . the theoretical pi of the -chain for c3 was determined to be ph 6.8 which is marginally outside the range of this fraction but this was expected since we found that the ief fractionator enriches proteins rather than providing a clear - cut fractionation for the ph range ; however , as proteins are enriched reproducibly this poses no problem . the differentially expressed band immediately below the 75 kda also contained sequences within the -chains of both c3 and c4 but these were fragments since the band was at a lower molecular weight . this band was seen in both control and cirrhosis samples but expressed to a higher extent in control samples , whereas the higher molecular weight band which was differentially expressed appeared only in control samples ( figure 3 and supporting figure 3a , supporting information ) . this lower molecular weight band contained predominantly complement c3 beta ( table 1 ) and showed a clear but less consistent change among multiple samples when validated by western blot ( supporting figure 3c and d ) . the more clearly changing higher molecular weight band contained predominantly complement c4 beta ( table 1 ) and showed a clear and consistent change among multiple samples when validated by western blot ( supporting figure 3c and d ) . this suggests that complement c4 beta may be a better biomarker for cirrhosis than complement c3 beta and would need to be validated using a larger number of samples . this study shows how two different proteomic approaches can aid in the discovery of disease biomarkers . to our knowledge this is the first time the ph 35.6 range has been used to separate serum by 2-de and we have shown that this ph range is useful for discovering novel biomarkers in diseases . we have also shown that the use of in - solution ief followed by sds - page improves the separation of the basic proteome thereby helping to identify disease biomarkers in the basic region of plasma and serum proteomes . using this approach we show that the beta chains of complement c3 and c4 decrease in serum from hepatitis c patients with cirrhosis , a change not observed previously by 2-de .

various lumbar fusion approaches have been used to treat lumbar degenerative disc diseases , aiming at reducing low back pain , radiculopathy , and disability . numerous comparative studies on the radiological and clinical outcomes of posterior lumbar interbody fusion ( plif ) , transforaminal lumbar interbody fusion ( tlif ) , and anterior lumbar interbody fusion ( alif ) have been published13,21,25,31 ) . tlif , which was first described by harms and rolinger9 ) has been widely used for its safety , good outcomes , and a high fusion rate , amongst a variety of lumbar interbody fusion approaches5,8,9,11 ) . surgeons are comfortable performing tlif because it utilizes a posterior approach , and tlif reduces dural retraction and enables direct neural decompression . however , this procedure carries risks for complications such as posterior spinal muscle injury , dural tears , and cerebrospinal fluid ( csf ) leakage15,16,26 ) . recently , mcafee et al.22 ) introduced direct lumbar interbody fusion ( dlif ) by using a transpsoas retroperitoneal approach , and ozgur et al.24 ) proposed minimally invasive dlif via a tubular system . dlif allows surgeons to avoid the shortcomings of plif and tlif , such as posterior element injury and dural tears , as well as those of alif , such as retrograde ejaculation due to great vessel injury and hypogastric sympathetic plexus injury2,27,28 ) . however , dlif can lead to psoas muscle injury and various types of nerve damage4,10,20 ) . until now , no comparative studies on the outcomes of dlif and other fusion procedures based on radiological and clinical approaches have been conducted . the aim of our study was to analyze the outcomes of dlif and tlif considering the radiological outcomes , functional disability , pain , fusion rates , and patients ' complications . this single - center study was conducted by 2 senior surgeons between january 2011 and december 2012 . tlif and dlif were performed by the surgeons to treat equivalent lumbar degenerative diseases including spinal stenosis , spondylolisthesis , and recurrent disc herniation . as dlif is not suitable for diseases with l5-s1 involvement , patients with diseases involving the l5-s1 level were excluded from the study . for the 2 groups of patients , the age , extent of the operation , operative levels , and bone mineral density ( bmd ) were examined . the cage height used in each procedure the tlif group underwent unilateral open tlif ( fusion material : autologous bone ) , pedicle screw fixation on the surgical side , and contralateral pedicle screw fixation via an interfascial approach . the tlif group was treated by using a single capstone cage ( medtronic , minneapolis , mn , usa ) . the dlif group underwent minimal invasive dlif [ fusion material : demineralized bone matrix ( dbm ) ] by using a transpsoas retroperitoneal approach and percutaneous pedicle screw fixation . dlif was performed using a dlif system ( medtronic , memphis , tn , usa ) and a percutaneous pedicle screw fixation system ( sextant ii or longitude system , medtronic , memphis , tn , usa ) . in addition , additional posterior decompression was performed for patients in the dlif group ( 33 of 81 patients ) who had severe central spinal stenosis or ruptured disc herniation . to compare patients with the same disease , patients who underwent posterior decompression in the dlif group were included in the study ( fig . pre- and post - operative clinical outcomes were compared between the groups by using the scores obtained with the visual analog scale ( vas ) and oswestry disability index ( odi ) . additionally , we assessed the length of hospital stay , operative time , and estimated blood loss ( ebl ) for each group . we measured the anterior and posterior disc heights , foraminal height , segmental sagittal and coronal angles , and lumbar lordosis on the preoperative and 12 months postoperative radiographs . the measurements were performed by using plain lateral radiographs taken with the patients in the neutral position . the segmental sagittal / coronal angles were defined as the cobb angle of vertebral bodies adjacent to the operative level . lumbar lordosis was defined as the angle between the upper endplates of l1 and s1 by using the cobb method . fusion was evaluated by using the bridwell et al.3 ) fusion grade system . according to this system , fusion grades are defined as follows : 1 ) grade 1 , complete remodeling with trabeculae across the disc space ; 2 ) grade 2 , intact graft with no lucent lines observed between the graft and the adjacent endplates ; 3 ) grade 3 , intact graft but a radiolucent line is present between the graft and an adjacent endplate ; and 4 ) grade 4 , lucency along an entire border of the graft or around a pedicle screw or the subsidence of the graft . on the basis of the classification system , we considered grades 1 and 2 as successful fusion . the measurements and evaluations on the basis of the radiographs were initially performed by one surgeon , and the 2 other surgeons reviewed and confirmed the results . we compared the radiological and clinical outcomes between the tlif and dlif groups by using an unpaired student 's t - test , mann - whitney u test , and the chi - squared test . we also compared the radiological and clinical outcomes pre- and post - surgery by using a paired t - test and wilcoxon signed - rank test . this single - center study was conducted by 2 senior surgeons between january 2011 and december 2012 . tlif and dlif were performed by the surgeons to treat equivalent lumbar degenerative diseases including spinal stenosis , spondylolisthesis , and recurrent disc herniation . as dlif is not suitable for diseases with l5-s1 involvement , patients with diseases involving the l5-s1 level were excluded from the study . for the 2 groups of patients , the age , extent of the operation , operative levels , and bone mineral density ( bmd ) were examined . the cage height used in each procedure the tlif group underwent unilateral open tlif ( fusion material : autologous bone ) , pedicle screw fixation on the surgical side , and contralateral pedicle screw fixation via an interfascial approach . the tlif group was treated by using a single capstone cage ( medtronic , minneapolis , mn , usa ) . the dlif group underwent minimal invasive dlif [ fusion material : demineralized bone matrix ( dbm ) ] by using a transpsoas retroperitoneal approach and percutaneous pedicle screw fixation . dlif was performed using a dlif system ( medtronic , memphis , tn , usa ) and a percutaneous pedicle screw fixation system ( sextant ii or longitude system , medtronic , memphis , tn , usa ) . in addition , additional posterior decompression was performed for patients in the dlif group ( 33 of 81 patients ) who had severe central spinal stenosis or ruptured disc herniation . to compare patients with the same disease , patients who underwent posterior decompression in the dlif group pre- and post - operative clinical outcomes were compared between the groups by using the scores obtained with the visual analog scale ( vas ) and oswestry disability index ( odi ) . additionally , we assessed the length of hospital stay , operative time , and estimated blood loss ( ebl ) for each group . we measured the anterior and posterior disc heights , foraminal height , segmental sagittal and coronal angles , and lumbar lordosis on the preoperative and 12 months postoperative radiographs . the measurements were performed by using plain lateral radiographs taken with the patients in the neutral position . the segmental sagittal / coronal angles were defined as the cobb angle of vertebral bodies adjacent to the operative level . lumbar lordosis was defined as the angle between the upper endplates of l1 and s1 by using the cobb method . fusion was evaluated by using the bridwell et al.3 ) fusion grade system . according to this system , fusion grades are defined as follows : 1 ) grade 1 , complete remodeling with trabeculae across the disc space ; 2 ) grade 2 , intact graft with no lucent lines observed between the graft and the adjacent endplates ; 3 ) grade 3 , intact graft but a radiolucent line is present between the graft and an adjacent endplate ; and 4 ) grade 4 , lucency along an entire border of the graft or around a pedicle screw or the subsidence of the graft . on the basis of the classification system , we considered grades 1 and 2 as successful fusion . the measurements and evaluations on the basis of the radiographs were initially performed by one surgeon , and the 2 other surgeons reviewed and confirmed the results . we compared the radiological and clinical outcomes between the tlif and dlif groups by using an unpaired student 's t - test , mann - whitney u test , and the chi - squared test . we also compared the radiological and clinical outcomes pre- and post - surgery by using a paired t - test and wilcoxon signed - rank test . the study included 81 patients who underwent dlif ( 106 levels ; 29 men and 52 women ) and 98 patients who underwent tlif ( 136 levels ; 41 men and 57 women ) to treat nearly identical lumbar degenerative diseases . the mean ages of the dlif and tlif groups were 60.8914.18 years and 62.7911.89 years , respectively . the mean bmd t - score was -0.761.78 in the dlif group and -1.161.31 in the tlif group . the preoperative diagnosis , extent of the operation , and operative levels are presented in table 1 . no statistically significant differences ( p>0.05 ) were detected between the 2 groups in terms of the age , sex , preoperative diagnosis , extent of the operation , operative levels , and bmd ( table 1 ) . the mean follow - up period was 16.55.8 months in the dlif group and 16.65.7 months in the tlif group . the most frequently used cage heights were 12 mm and 14 mm for both operative methods , with the cage heights being 10 - 16 mm in the dlif group and 8 - 14 mm in the tlif group . the mean cage heights were 12.941.30 mm and 12.451.59 mm in the dlif and tlif groups , respectively ( p=0.043 ) . the preoperative vas scores in the dlif and tlif groups were 6.431.18 and 6.530.80 , respectively , whereas the corresponding post - operative vas scores were 1.900.75 and 1.810.77 , respectively . in both groups , the postoperative vas scores revealed significant improvement in pain compared with the preoperative values ( p<0.001 ) . no between - group differences in the pre- and post - operative vas scores were noted ( p>0.05 ) . in addition , the variation of the vas scores pre- and post - surgery was not different between the groups ( p=0.180 ) . the preoperative odi values in the dlif and tlif groups were 39.7816.48% and 37.9511.46% , respectively , versus postoperative values of 11.145.74% and 11.854.05% , respectively . in both groups , the postoperative odi scores revealed significant improvements in pain compared to the preoperative values ( p<0.001 ) . no between - group differences were noted for the preoperative and postoperative odi values ( p>0.05 ) . in addition , the variation of the odi values pre- and post - surgery was not different between the groups ( p=0.147 ) ( table 2 ) . the mean operative times were 128.8333.23 min for 1-level surgery , 145.5932.30 min for 2-level surgery , and 183.7537.50 min for 3-level surgery in the dlif group , versus 138.9142.43 min , 194.6736.76 min , and 252.5022.54 min , respectively , in the tlif group . a significant difference between the groups was observed for 2- and 3-level surgery ( p<0.001 ) but not for 1-level surgery ( p>0.05 ) . the mean ebl values were 153.83104.75 ml , 173.5379.92 ml , and 200.0070.71 ml for 1- , 2- , and 3-level surgery , respectively , in the dlif group , compared to 215.47157.82 ml , 305.00149.34 ml , and 462.50165.90 ml , respectively , in the tlif group . the pre- and post - surgical anterior disc , posterior disc , and foraminal heights were not significantly different ( p>0.05 ) . however , the anterior disc height changed from 10.823.61 mm preoperatively to 15.832.58 mm postoperatively in the dlif group , and from 11.203.74 mm preoperatively to 13.063.20 mm postoperatively in the tlif group ; both changes were statistically significant ( p<0.001 ) . the difference between the pre- and post - operative anterior disc height was greater in the dlif group ( 5.003.09 mm vs. 2.022.65 mm ; p<0.001 ) . in addition , the pre- and post - operative changes in the posterior disc height were significantly different in both groups . the pre- and post - operative posterior disc heights were 7.712.68 mm and 11.752.40 mm , respectively , in the dlif group , compared to 7.462.36 mm and 8.852.23 mm , respectively , in the tlif group ( p<0.001 ) . however , the change of the posterior disc height was greater in the dlif group ( 4.042.60 mm vs. 1.392.01 mm ; p<0.001 ) ( table 3 ) . the foraminal height changed from 18.822.90 mm preoperatively to 25.372.41 mm postoperatively in the dlif group , and from 18.573.60 mm preoperatively to 20.573.58 mm postoperatively in the tlif group ( p<0.001 ) . the change of the foraminal height was significantly greater in the dlif group ( 6.552.92 mm vs. 2.001.96 mm ; p<0.001 ) ( table 3 ) . prior to surgery , the segmental sagittal / coronal angles and lumbar lordosis were not different between the groups ( p>0.05 ) . in the dlif group , the segmental coronal angle changed from 4.11 3.96 preoperatively to 1.171.29 postoperatively , whereas the value changed from 4.422.39 preoperatively to 3.431.55 postoperatively in the tlif group ( p<0.001 ) . the amount of variation in the segmental coronal angle was -2.933.18 in the dlif group , compared to -1.001.49 in the tlif group ( p<0.001 ) . the segmental sagittal angle significantly increased from 8.649.87 preoperatively to 9.948.47 postoperatively in the dlif group ( p=0.027 ) . however , lumbar lordosis did not change significantly post - operation in this group ( 40.1213.26 vs. 41.4711.49 ; the segmental sagittal angle slightly increased from 10.235.28 preoperatively to 10.445.28 postoperatively , whereas lumbar lordosis did not change from 41.1810.46 preoperatively to 41.3210.18 postoperatively ( p>0.05 ) ( table 4 ) . the fusion rate was 87.7% ( 71 of 81 patients ) at 12 months post - operation in the dlif group , compared to 98.1% ( 96 of 98 patients ) in the tlif group ( p=0.007 ) . in the dlif group , complications related to additional transpsoas retroperitoneal approaches occurred in 16 patients ( 19.6% ) , including psoas muscle ( 10 patients , 12.3% ) , lateral femoral cutaneous nerve ( 4 patients , 4.9% ) , and genitofemoral nerve symptoms ( 2 patients , 2.5% ) . most complications were temporary and disappeared within 2 months post - operation . in the tlif group , the preoperative vas scores in the dlif and tlif groups were 6.431.18 and 6.530.80 , respectively , whereas the corresponding post - operative vas scores were 1.900.75 and 1.810.77 , respectively . in both groups , the postoperative vas scores revealed significant improvement in pain compared with the preoperative values ( p<0.001 ) . no between - group differences in the pre- and post - operative vas scores were noted ( p>0.05 ) . in addition , the variation of the vas scores pre- and post - surgery was not different between the groups ( p=0.180 ) . the preoperative odi values in the dlif and tlif groups were 39.7816.48% and 37.9511.46% , respectively , versus postoperative values of 11.145.74% and 11.854.05% , respectively . in both groups , the postoperative odi scores revealed significant improvements in pain compared to the preoperative values ( p<0.001 ) . no between - group differences were noted for the preoperative and postoperative odi values ( p>0.05 ) . in addition , the variation of the odi values pre- and post - surgery was not different between the groups ( p=0.147 ) ( table 2 ) . the mean operative times were 128.8333.23 min for 1-level surgery , 145.5932.30 min for 2-level surgery , and 183.7537.50 min for 3-level surgery in the dlif group , versus 138.9142.43 min , 194.6736.76 min , and 252.5022.54 min , respectively , in the tlif group . a significant difference between the groups was observed for 2- and 3-level surgery ( p<0.001 ) but not for 1-level surgery ( p>0.05 ) . the mean ebl values were 153.83104.75 ml , 173.5379.92 ml , and 200.0070.71 ml for 1- , 2- , and 3-level surgery , respectively , in the dlif group , compared to 215.47157.82 ml , 305.00149.34 ml , and 462.50165.90 ml , respectively , in the tlif group . the pre- and post - surgical anterior disc , posterior disc , and foraminal heights were not significantly different ( p>0.05 ) . however , the anterior disc height changed from 10.823.61 mm preoperatively to 15.832.58 mm postoperatively in the dlif group , and from 11.203.74 mm preoperatively to 13.063.20 mm postoperatively in the tlif group ; both changes were statistically significant ( p<0.001 ) . the difference between the pre- and post - operative anterior disc height was greater in the dlif group ( 5.003.09 mm vs. 2.022.65 mm ; p<0.001 ) . in addition , the pre- and post - operative changes in the posterior disc height were significantly different in both groups . the pre- and post - operative posterior disc heights were 7.712.68 mm and 11.752.40 mm , respectively , in the dlif group , compared to 7.462.36 mm and 8.852.23 mm , respectively , in the tlif group ( p<0.001 ) . however , the change of the posterior disc height was greater in the dlif group ( 4.042.60 mm vs. 1.392.01 mm ; p<0.001 ) ( table 3 ) . the foraminal height changed from 18.822.90 mm preoperatively to 25.372.41 mm postoperatively in the dlif group , and from 18.573.60 mm preoperatively to 20.573.58 mm postoperatively in the tlif group ( p<0.001 ) . the change of the foraminal height was significantly greater in the dlif group ( 6.552.92 mm vs. 2.001.96 mm ; p<0.001 ) ( table 3 ) . prior to surgery , the segmental sagittal / coronal angles and lumbar lordosis were not different between the groups ( p>0.05 ) . in the dlif group , the segmental coronal angle changed from 4.11 3.96 preoperatively to 1.171.29 postoperatively , whereas the value changed from 4.422.39 preoperatively to 3.431.55 postoperatively in the tlif group ( p<0.001 ) . the amount of variation in the segmental coronal angle was -2.933.18 in the dlif group , compared to -1.001.49 in the tlif group ( p<0.001 ) . the segmental sagittal angle significantly increased from 8.649.87 preoperatively to 9.948.47 postoperatively in the dlif group ( p=0.027 ) . however , lumbar lordosis did not change significantly post - operation in this group ( 40.1213.26 vs. 41.4711.49 ; p=0.174 ) . in the tlif group , the segmental sagittal angle slightly increased from 10.235.28 preoperatively to 10.445.28 postoperatively , whereas lumbar lordosis did not change from 41.1810.46 preoperatively to 41.3210.18 postoperatively ( p>0.05 ) ( table 4 ) . the fusion rate was 87.7% ( 71 of 81 patients ) at 12 months post - operation in the dlif group , compared to 98.1% ( 96 of 98 patients ) in the tlif group ( p=0.007 ) . in the dlif group , complications related to additional transpsoas retroperitoneal approaches occurred in 16 patients ( 19.6% ) , including psoas muscle ( 10 patients , 12.3% ) , lateral femoral cutaneous nerve ( 4 patients , 4.9% ) , and genitofemoral nerve symptoms ( 2 patients , 2.5% ) . most complications were temporary and disappeared within 2 months post - operation . in the tlif group , tlif techniques have been used to treat various degenerative lumbar disorders over the last 3 decades . this operative method utilizes a posterior approach , which sufficiently exposes the disc space by resecting a single facet joint , reduces retraction of the thecal sac and nerve root , and preserves the contralateral structure . dlif , a different fusion method , is also widely used for degenerative lumbar disorders , and its application has been expanded . dlif avoids the risks of thecal sac injury , arachnoiditis , and csf fistula , and it has superior in terms of indirect decompression and sagittal and coronal restoration1,18,23 ) . by using a retroperitoneal space approach , it is possible to insert larger interbody cages supporting bilateral epiphyseal rings , reduce tissue trauma and blood loss , decrease the operative time , and preserve the posterior longitudinal ligament complex19,23 ) . however , the technique is associated with risks related to the transpsoas approach , including injuries to the psoas muscle , lateral femoral cutaneous nerve , geni - tofemoral nerve , and lumbosacral plexus4,10,20 ) . in addition , patients with l5-s1 involvement are not suitable candidates for dlif . an additional limiting factor is the requirement of additional posterior decompression when severe central spinal stenosis , uncontained disc herniation , or significant facet arthropathy is present23 ) . the dlif group displayed greater corrective force than the tlif group considering the intervertebral disc and foraminal heights . this result was predictable because of the differences between the groups in terms of surgical techniques and cage features . in dlif , there is no structure obstructing cage insertion , and the insertion of larger cages is possible because disc distraction can be performed in the disc midline . furthermore , as the cages are located on both sides of the ring apophysis , which is the strongest part of the vertebral body , disc distraction is effective and well preserved after cage insertion . on the other hand , in tlif , cage insertion should be performed more cautiously to avoid complications resulting from damage in adjacent areas , owing to the presence of the posterior structure and dural sac . the cage is located in concave parts of the endplate , which might be related with the ineffective increase of disc height after tlif cage insertion . compared with the tlif group , the dlif group displayed superior outcomes considering the correction of coronal balance . although there was no significant difference in the segmental sagittal angle between the groups , dlif was more effective in causing lordosis than tlif , as indicated by the pre- and post - operative difference in the segmental sagittal angle1 ) . there are a growing number of studies on the correction of sagittal and coronal balances in dlif1,14,17 ) . similar to our results , dlif has become applicable in correcting various degenerative lumbar deformities in adults as well6,12,30 ) . other studies have reported that the rate of bone fusion was 100% in patients who underwent posterolateral lumbar fusion with autologous bone , whereas in dbm , the fusion rate was 89.7%7 ) . as a result , it appears that the fusion rate of the dlif group who were treated with dbm was lower than that of the tlif group with autologous bone . in recent years , tlif has evolved from open tlif to minimally invasive tlif , and dlif has been more extensively used for deformity surgeries6,12,29,30 ) . on the basis of our results , tlif appears to be more appropriate for surgeries on short segments ; for the treatment of well - balanced spine diseases , ruptured disc herniation , or severe central spinal stenosis ; and for patients with l5-s1 involvement . dlif appears to be more appropriate for surgeries on long segments and for cases to restore or preserve sagittal and coronal angles . first , the follow - up period was relatively short for evaluating long - term clinical results . second , we did not examine the effect of the screw type or that of open or percutaneous screw systems . third , the effect of graft materials on the fusion rate also needs to be investigated for each group . we believe prospective long - term studies are necessary for a more comprehensive evaluation in the future . both dlif and tlif are less invasive , good surgical options for degenerative lumbar diseases . according to our data , dlif has higher potential in increasing neural foramina and correcting coronal balance , and involves a shorter operative time and reduced reduced ebl , in comparison with tlif . however , dlif displayed a lower fusion rate than tlif , and caused additional complications related to the transpsoas approach .

objectivethe use of direct lumbar interbody fusion ( dlif ) has gradually increased ; however , no studies have directly compared dlif and transforaminal lumbar interbody fusion ( tlif ) . we compared dlif and tlif on the basis of clinical and radiological outcomes.methodsa retrospective review was performed on the medical records and radiographs of 98 and 81 patients who underwent tlif and dlif between january 2011 and december 2012 . clinical outcomes were compared with a visual analog scale ( vas ) and the oswestry disability index ( odi ) . the preoperative and postoperative disc heights , segmental sagittal / coronal angles , and lumbar lordosis were measured on radiographs . fusion rates , operative time , estimated blood loss ( ebl ) , length of hospital stay , and complications were assessed.resultsdlif was superior to tlif regarding its ability to restore disc height , foraminal height , and coronal balance ( p<0.001 ) . as the extent of surgical level increased , dlif displayed significant advantages over tlif considering the operative time and ebl . however , fusion rates at 12 months post - operation were lower for dlif ( 87.8% ) than for tlif ( 98.1% ) ( p=0.007 ) . the changes of vas and odi between the tlif and dlif were not significantly different ( p>0.05).conclusionboth dlif and tlif are less invasive and thus good surgical options for treating degenerative lumber diseases . dlif has higher potential in increasing neural foramina and correcting coronal balance , and involves a shorter operative time and reduced ebl , in comparison with tlif . however , dlif displayed a lower fusion rate than tlif , and caused complications related to the transpsoas approach .

INTRODUCTION MATERIALS AND METHODS Patient data and inclusion criteria Surgical technique Clinical evaluations Radiographic measurements Statistical analysis RESULTS Clinical outcomes Radiological outcomes Complications DISCUSSION CONCLUSION

numerous comparative studies on the radiological and clinical outcomes of posterior lumbar interbody fusion ( plif ) , transforaminal lumbar interbody fusion ( tlif ) , and anterior lumbar interbody fusion ( alif ) have been published13,21,25,31 ) . tlif , which was first described by harms and rolinger9 ) has been widely used for its safety , good outcomes , and a high fusion rate , amongst a variety of lumbar interbody fusion approaches5,8,9,11 ) . recently , mcafee et al.22 ) introduced direct lumbar interbody fusion ( dlif ) by using a transpsoas retroperitoneal approach , and ozgur et al.24 ) proposed minimally invasive dlif via a tubular system . until now , no comparative studies on the outcomes of dlif and other fusion procedures based on radiological and clinical approaches have been conducted . the aim of our study was to analyze the outcomes of dlif and tlif considering the radiological outcomes , functional disability , pain , fusion rates , and patients ' complications . this single - center study was conducted by 2 senior surgeons between january 2011 and december 2012 . tlif and dlif were performed by the surgeons to treat equivalent lumbar degenerative diseases including spinal stenosis , spondylolisthesis , and recurrent disc herniation . the cage height used in each procedure the tlif group underwent unilateral open tlif ( fusion material : autologous bone ) , pedicle screw fixation on the surgical side , and contralateral pedicle screw fixation via an interfascial approach . pre- and post - operative clinical outcomes were compared between the groups by using the scores obtained with the visual analog scale ( vas ) and oswestry disability index ( odi ) . additionally , we assessed the length of hospital stay , operative time , and estimated blood loss ( ebl ) for each group . we measured the anterior and posterior disc heights , foraminal height , segmental sagittal and coronal angles , and lumbar lordosis on the preoperative and 12 months postoperative radiographs . the segmental sagittal / coronal angles were defined as the cobb angle of vertebral bodies adjacent to the operative level . according to this system , fusion grades are defined as follows : 1 ) grade 1 , complete remodeling with trabeculae across the disc space ; 2 ) grade 2 , intact graft with no lucent lines observed between the graft and the adjacent endplates ; 3 ) grade 3 , intact graft but a radiolucent line is present between the graft and an adjacent endplate ; and 4 ) grade 4 , lucency along an entire border of the graft or around a pedicle screw or the subsidence of the graft . on the basis of the classification system , we considered grades 1 and 2 as successful fusion . the measurements and evaluations on the basis of the radiographs were initially performed by one surgeon , and the 2 other surgeons reviewed and confirmed the results . we compared the radiological and clinical outcomes between the tlif and dlif groups by using an unpaired student 's t - test , mann - whitney u test , and the chi - squared test . this single - center study was conducted by 2 senior surgeons between january 2011 and december 2012 . tlif and dlif were performed by the surgeons to treat equivalent lumbar degenerative diseases including spinal stenosis , spondylolisthesis , and recurrent disc herniation . the cage height used in each procedure the tlif group underwent unilateral open tlif ( fusion material : autologous bone ) , pedicle screw fixation on the surgical side , and contralateral pedicle screw fixation via an interfascial approach . to compare patients with the same disease , patients who underwent posterior decompression in the dlif group pre- and post - operative clinical outcomes were compared between the groups by using the scores obtained with the visual analog scale ( vas ) and oswestry disability index ( odi ) . additionally , we assessed the length of hospital stay , operative time , and estimated blood loss ( ebl ) for each group . we measured the anterior and posterior disc heights , foraminal height , segmental sagittal and coronal angles , and lumbar lordosis on the preoperative and 12 months postoperative radiographs . the segmental sagittal / coronal angles were defined as the cobb angle of vertebral bodies adjacent to the operative level . lumbar lordosis was defined as the angle between the upper endplates of l1 and s1 by using the cobb method . according to this system , fusion grades are defined as follows : 1 ) grade 1 , complete remodeling with trabeculae across the disc space ; 2 ) grade 2 , intact graft with no lucent lines observed between the graft and the adjacent endplates ; 3 ) grade 3 , intact graft but a radiolucent line is present between the graft and an adjacent endplate ; and 4 ) grade 4 , lucency along an entire border of the graft or around a pedicle screw or the subsidence of the graft . on the basis of the classification system , we considered grades 1 and 2 as successful fusion . the measurements and evaluations on the basis of the radiographs were initially performed by one surgeon , and the 2 other surgeons reviewed and confirmed the results . we compared the radiological and clinical outcomes between the tlif and dlif groups by using an unpaired student 's t - test , mann - whitney u test , and the chi - squared test . the study included 81 patients who underwent dlif ( 106 levels ; 29 men and 52 women ) and 98 patients who underwent tlif ( 136 levels ; 41 men and 57 women ) to treat nearly identical lumbar degenerative diseases . the preoperative diagnosis , extent of the operation , and operative levels are presented in table 1 . no statistically significant differences ( p>0.05 ) were detected between the 2 groups in terms of the age , sex , preoperative diagnosis , extent of the operation , operative levels , and bmd ( table 1 ) . the mean cage heights were 12.941.30 mm and 12.451.59 mm in the dlif and tlif groups , respectively ( p=0.043 ) . the preoperative vas scores in the dlif and tlif groups were 6.431.18 and 6.530.80 , respectively , whereas the corresponding post - operative vas scores were 1.900.75 and 1.810.77 , respectively . in both groups , the postoperative vas scores revealed significant improvement in pain compared with the preoperative values ( p<0.001 ) . in addition , the variation of the vas scores pre- and post - surgery was not different between the groups ( p=0.180 ) . the preoperative odi values in the dlif and tlif groups were 39.7816.48% and 37.9511.46% , respectively , versus postoperative values of 11.145.74% and 11.854.05% , respectively . in both groups , the postoperative odi scores revealed significant improvements in pain compared to the preoperative values ( p<0.001 ) . no between - group differences were noted for the preoperative and postoperative odi values ( p>0.05 ) . in addition , the variation of the odi values pre- and post - surgery was not different between the groups ( p=0.147 ) ( table 2 ) . the mean operative times were 128.8333.23 min for 1-level surgery , 145.5932.30 min for 2-level surgery , and 183.7537.50 min for 3-level surgery in the dlif group , versus 138.9142.43 min , 194.6736.76 min , and 252.5022.54 min , respectively , in the tlif group . a significant difference between the groups was observed for 2- and 3-level surgery ( p<0.001 ) but not for 1-level surgery ( p>0.05 ) . the pre- and post - surgical anterior disc , posterior disc , and foraminal heights were not significantly different ( p>0.05 ) . however , the anterior disc height changed from 10.823.61 mm preoperatively to 15.832.58 mm postoperatively in the dlif group , and from 11.203.74 mm preoperatively to 13.063.20 mm postoperatively in the tlif group ; both changes were statistically significant ( p<0.001 ) . the difference between the pre- and post - operative anterior disc height was greater in the dlif group ( 5.003.09 mm vs. 2.022.65 mm ; p<0.001 ) . in addition , the pre- and post - operative changes in the posterior disc height were significantly different in both groups . the pre- and post - operative posterior disc heights were 7.712.68 mm and 11.752.40 mm , respectively , in the dlif group , compared to 7.462.36 mm and 8.852.23 mm , respectively , in the tlif group ( p<0.001 ) . however , the change of the posterior disc height was greater in the dlif group ( 4.042.60 mm vs. 1.392.01 mm ; p<0.001 ) ( table 3 ) . the foraminal height changed from 18.822.90 mm preoperatively to 25.372.41 mm postoperatively in the dlif group , and from 18.573.60 mm preoperatively to 20.573.58 mm postoperatively in the tlif group ( p<0.001 ) . the change of the foraminal height was significantly greater in the dlif group ( 6.552.92 mm vs. 2.001.96 mm ; p<0.001 ) ( table 3 ) . prior to surgery , the segmental sagittal / coronal angles and lumbar lordosis were not different between the groups ( p>0.05 ) . in the dlif group , the segmental coronal angle changed from 4.11 3.96 preoperatively to 1.171.29 postoperatively , whereas the value changed from 4.422.39 preoperatively to 3.431.55 postoperatively in the tlif group ( p<0.001 ) . the amount of variation in the segmental coronal angle was -2.933.18 in the dlif group , compared to -1.001.49 in the tlif group ( p<0.001 ) . however , lumbar lordosis did not change significantly post - operation in this group ( 40.1213.26 vs. 41.4711.49 ; the segmental sagittal angle slightly increased from 10.235.28 preoperatively to 10.445.28 postoperatively , whereas lumbar lordosis did not change from 41.1810.46 preoperatively to 41.3210.18 postoperatively ( p>0.05 ) ( table 4 ) . the fusion rate was 87.7% ( 71 of 81 patients ) at 12 months post - operation in the dlif group , compared to 98.1% ( 96 of 98 patients ) in the tlif group ( p=0.007 ) . in the dlif group , complications related to additional transpsoas retroperitoneal approaches occurred in 16 patients ( 19.6% ) , including psoas muscle ( 10 patients , 12.3% ) , lateral femoral cutaneous nerve ( 4 patients , 4.9% ) , and genitofemoral nerve symptoms ( 2 patients , 2.5% ) . most complications were temporary and disappeared within 2 months post - operation . in the tlif group , the preoperative vas scores in the dlif and tlif groups were 6.431.18 and 6.530.80 , respectively , whereas the corresponding post - operative vas scores were 1.900.75 and 1.810.77 , respectively . in both groups , the postoperative vas scores revealed significant improvement in pain compared with the preoperative values ( p<0.001 ) . in addition , the variation of the vas scores pre- and post - surgery was not different between the groups ( p=0.180 ) . in both groups , the postoperative odi scores revealed significant improvements in pain compared to the preoperative values ( p<0.001 ) . no between - group differences were noted for the preoperative and postoperative odi values ( p>0.05 ) . in addition , the variation of the odi values pre- and post - surgery was not different between the groups ( p=0.147 ) ( table 2 ) . the mean operative times were 128.8333.23 min for 1-level surgery , 145.5932.30 min for 2-level surgery , and 183.7537.50 min for 3-level surgery in the dlif group , versus 138.9142.43 min , 194.6736.76 min , and 252.5022.54 min , respectively , in the tlif group . a significant difference between the groups was observed for 2- and 3-level surgery ( p<0.001 ) but not for 1-level surgery ( p>0.05 ) . the mean ebl values were 153.83104.75 ml , 173.5379.92 ml , and 200.0070.71 ml for 1- , 2- , and 3-level surgery , respectively , in the dlif group , compared to 215.47157.82 ml , 305.00149.34 ml , and 462.50165.90 ml , respectively , in the tlif group . the pre- and post - surgical anterior disc , posterior disc , and foraminal heights were not significantly different ( p>0.05 ) . however , the anterior disc height changed from 10.823.61 mm preoperatively to 15.832.58 mm postoperatively in the dlif group , and from 11.203.74 mm preoperatively to 13.063.20 mm postoperatively in the tlif group ; both changes were statistically significant ( p<0.001 ) . the difference between the pre- and post - operative anterior disc height was greater in the dlif group ( 5.003.09 mm vs. 2.022.65 mm ; p<0.001 ) . in addition , the pre- and post - operative changes in the posterior disc height were significantly different in both groups . the pre- and post - operative posterior disc heights were 7.712.68 mm and 11.752.40 mm , respectively , in the dlif group , compared to 7.462.36 mm and 8.852.23 mm , respectively , in the tlif group ( p<0.001 ) . however , the change of the posterior disc height was greater in the dlif group ( 4.042.60 mm vs. 1.392.01 mm ; p<0.001 ) ( table 3 ) . the foraminal height changed from 18.822.90 mm preoperatively to 25.372.41 mm postoperatively in the dlif group , and from 18.573.60 mm preoperatively to 20.573.58 mm postoperatively in the tlif group ( p<0.001 ) . the change of the foraminal height was significantly greater in the dlif group ( 6.552.92 mm vs. 2.001.96 mm ; p<0.001 ) ( table 3 ) . prior to surgery , the segmental sagittal / coronal angles and lumbar lordosis were not different between the groups ( p>0.05 ) . in the dlif group , the segmental coronal angle changed from 4.11 3.96 preoperatively to 1.171.29 postoperatively , whereas the value changed from 4.422.39 preoperatively to 3.431.55 postoperatively in the tlif group ( p<0.001 ) . the amount of variation in the segmental coronal angle was -2.933.18 in the dlif group , compared to -1.001.49 in the tlif group ( p<0.001 ) . however , lumbar lordosis did not change significantly post - operation in this group ( 40.1213.26 vs. 41.4711.49 ; p=0.174 ) . in the tlif group , the segmental sagittal angle slightly increased from 10.235.28 preoperatively to 10.445.28 postoperatively , whereas lumbar lordosis did not change from 41.1810.46 preoperatively to 41.3210.18 postoperatively ( p>0.05 ) ( table 4 ) . the fusion rate was 87.7% ( 71 of 81 patients ) at 12 months post - operation in the dlif group , compared to 98.1% ( 96 of 98 patients ) in the tlif group ( p=0.007 ) . in the dlif group , complications related to additional transpsoas retroperitoneal approaches occurred in 16 patients ( 19.6% ) , including psoas muscle ( 10 patients , 12.3% ) , lateral femoral cutaneous nerve ( 4 patients , 4.9% ) , and genitofemoral nerve symptoms ( 2 patients , 2.5% ) . most complications were temporary and disappeared within 2 months post - operation . by using a retroperitoneal space approach , it is possible to insert larger interbody cages supporting bilateral epiphyseal rings , reduce tissue trauma and blood loss , decrease the operative time , and preserve the posterior longitudinal ligament complex19,23 ) . however , the technique is associated with risks related to the transpsoas approach , including injuries to the psoas muscle , lateral femoral cutaneous nerve , geni - tofemoral nerve , and lumbosacral plexus4,10,20 ) . on the other hand , in tlif , cage insertion should be performed more cautiously to avoid complications resulting from damage in adjacent areas , owing to the presence of the posterior structure and dural sac . compared with the tlif group , the dlif group displayed superior outcomes considering the correction of coronal balance . although there was no significant difference in the segmental sagittal angle between the groups , dlif was more effective in causing lordosis than tlif , as indicated by the pre- and post - operative difference in the segmental sagittal angle1 ) . other studies have reported that the rate of bone fusion was 100% in patients who underwent posterolateral lumbar fusion with autologous bone , whereas in dbm , the fusion rate was 89.7%7 ) . in recent years , tlif has evolved from open tlif to minimally invasive tlif , and dlif has been more extensively used for deformity surgeries6,12,29,30 ) . on the basis of our results , tlif appears to be more appropriate for surgeries on short segments ; for the treatment of well - balanced spine diseases , ruptured disc herniation , or severe central spinal stenosis ; and for patients with l5-s1 involvement . dlif appears to be more appropriate for surgeries on long segments and for cases to restore or preserve sagittal and coronal angles . both dlif and tlif are less invasive , good surgical options for degenerative lumbar diseases . according to our data , dlif has higher potential in increasing neural foramina and correcting coronal balance , and involves a shorter operative time and reduced reduced ebl , in comparison with tlif . however , dlif displayed a lower fusion rate than tlif , and caused additional complications related to the transpsoas approach .

physicians in primary care play a central role in the management of musculoskeletal disorders ( msds ) . in france , as in most industrialized countries , they represent one of the most frequent reasons for consultation . clinical guidelines emphasize the treatment of pain and inflammation in the acute phase with gradual recourse to stronger analgesia and the management of psychological impacts as the condition evolves toward chronicity . in this context , concerns have been raised regarding sub - optimal use of analgesics and psychotropic drugs ( antidepressants ) in the treatment of patients with chronic msds [ 2 , 3 ] . this issue has been examined by comparing guidelines with actual practice in benchmarking approaches [ 4 , 5 ] . the goal of this study was to describe physician s prescriptions for the management of acute and chronic msds in a large population of patients seen in primary care , and examine the pertinence of prescriptions against patient s self - assessed quality of life . the epi3-laser study was a nation - wide observational survey of a representative sample of general practitioners ( gps ) and their patients , conducted in france between march 2007 and july 2008 . its aims were to assess the burden of disease in general practice , considering physician and patients characteristics , co - morbidities and prescriptions with a specific focus on health - related quality of life . first , gps were randomly selected from the french national directory of physicians and invited to participate , which meant allowing a research assistant to be in the patients waiting room for a 1-day consultation session , collecting basic information on all patients consulting that day and recruiting volunteers to fill out a self - administered questionnaire . the second stage consisted of randomly sampling the 1-day of consultation for each participating physician to survey all patients attending the practice on that specific day . gps sampling was stratified to take into account the diversity of practices ( strict conventional medicine and use of homeopathy and other complementary medicines ) . all patients were eligible for inclusion to the exception of those whose health status or literacy level did not allow responding to a self - administered questionnaire . on the day selected for patients inclusion , a trained interviewer recruited on site , in the waiting room , all consecutive eligible patients up to a maximum of 15 . consenting patients completed a self - administered questionnaire that included information on age , gender , education , employment status and occupation , type of health insurance , hospitalization and medical visits in the previous 12 months , smoking , alcohol intake , physical activity , height and weight , and health related quality of life assessed by the short form 12 ( sf-12 ) questionnaire [ 68 ] . gps completed a medical questionnaire including the main reason for consultation and up to five other diagnoses present that day and for each , the duration of the health problem in its current episode . diagnoses were coded by a trained archivist using the 9th revision of the international classification of diseases . in this analysis adult patients 18 years and older with a msd as their main reason for consultation were included . msds were classified as spinal disorders ( sd ) with icd codes 720724 , and non - spinal msds ( ns - msd ) , such as osteoarthritis and tendonitis of the upper or lower limb , with icd codes 715 , 719 , 729 , 726728 , 782 . patients with a diagnosis of inflammatory , infectious or neoplastic joint disease as their main reason for consultation were excluded from the analyses . sd and ns - msd were classified as acute subacute or chronic using a 12-week ( 3 months ) cut - off for duration of symptoms in the current episode in accordance with consensus recommendations for research on sd . . characteristics of non - participating patients ( gender , age , length of time attending the gps practice , type of health insurance and main reason for consultation ) were used to calibrate the final sample so that it represented more closely the whole study population , using a procedure known in demographic studies as the calmar procedure . odds ratios were calculated for the comparison of prescriptions between chronic and non - chronic patients using mixed models accounting for a random physician effect ( glimmix and nlmixed procedures in sas ) . mean scores of the sf-12 mental and physical scales were adjusted for gender , age and co - morbidities using the analysis of covariance . tests for trend on proportions of medication users by quartile of the sf-12 subscales were performed using the cochrane armitage test for trend . the study was approved by the french national data - protection commission ( cnil ) and the french national council of physicians ( cnom ) . the epi3-laser study was a nation - wide observational survey of a representative sample of general practitioners ( gps ) and their patients , conducted in france between march 2007 and july 2008 . its aims were to assess the burden of disease in general practice , considering physician and patients characteristics , co - morbidities and prescriptions with a specific focus on health - related quality of life . first , gps were randomly selected from the french national directory of physicians and invited to participate , which meant allowing a research assistant to be in the patients waiting room for a 1-day consultation session , collecting basic information on all patients consulting that day and recruiting volunteers to fill out a self - administered questionnaire . the second stage consisted of randomly sampling the 1-day of consultation for each participating physician to survey all patients attending the practice on that specific day . gps sampling was stratified to take into account the diversity of practices ( strict conventional medicine and use of homeopathy and other complementary medicines ) . all patients were eligible for inclusion to the exception of those whose health status or literacy level did not allow responding to a self - administered questionnaire . on the day selected for patients inclusion , a trained interviewer recruited on site , in the waiting room , all consecutive eligible patients up to a maximum of 15 . consenting patients completed a self - administered questionnaire that included information on age , gender , education , employment status and occupation , type of health insurance , hospitalization and medical visits in the previous 12 months , smoking , alcohol intake , physical activity , height and weight , and health related quality of life assessed by the short form 12 ( sf-12 ) questionnaire [ 68 ] . gps completed a medical questionnaire including the main reason for consultation and up to five other diagnoses present that day and for each , the duration of the health problem in its current episode . diagnoses were coded by a trained archivist using the 9th revision of the international classification of diseases . in this analysis adult patients 18 years and older with a msd as their main reason for consultation were included . msds were classified as spinal disorders ( sd ) with icd codes 720724 , and non - spinal msds ( ns - msd ) , such as osteoarthritis and tendonitis of the upper or lower limb , with icd codes 715 , 719 , 729 , 726728 , 782 . patients with a diagnosis of inflammatory , infectious or neoplastic joint disease as their main reason for consultation were excluded from the analyses . sd and ns - msd were classified as acute subacute or chronic using a 12-week ( 3 months ) cut - off for duration of symptoms in the current episode in accordance with consensus recommendations for research on sd . characteristics of non - participating patients ( gender , age , length of time attending the gps practice , type of health insurance and main reason for consultation ) were used to calibrate the final sample so that it represented more closely the whole study population , using a procedure known in demographic studies as the calmar procedure . odds ratios were calculated for the comparison of prescriptions between chronic and non - chronic patients using mixed models accounting for a random physician effect ( glimmix and nlmixed procedures in sas ) . mean scores of the sf-12 mental and physical scales were adjusted for gender , age and co - morbidities using the analysis of covariance . tests for trend on proportions of medication users by quartile of the sf-12 subscales were performed using the cochrane armitage test for trend . the study was approved by the french national data - protection commission ( cnil ) and the french national council of physicians ( cnom ) . of the 17,206 gps randomly selected and invited by mail to participate , 825 agreed to participate in the study . their median age was 52 years , 23.5% were female , 51.8% worked in solo practice and 7.6% practiced fee - for - service in addition to the general health insurance regime . the median number of patients recruited at each physicians office was two with 11.2% recruiting five or more . of the 10,803 patients identified as potential participants , 2,151 ( 19.9% ) declined participation and 93 were excluded because of missing information leaving a final sample of 8,559 , of whom 665 ( 7.8% ) had a sd as their main reason for consultation , and 1,027 ( 12.0% ) a ns - msd ( table 1 ) . they were female in proportions of 59.0 and 61.6% for sd and ns - msd , respectively , and sd patients were younger by 8.2 years on average . the proportion who had their problem for 12 weeks or more at the time of consultation were respectively 43.0 and 58.5% for sd and ns - msd . factors associated with chronicity of sd were age , with the largest proportion observed over the age of 60 [ odds ratio ( or ) : 4.5 , 95% confidence interval ( ci ) : 2.19.6 ] , and not being employed ( or = 2.0 , 95% : 1.13.6 ) . for ns - msd , factors associated with chronicity were female gender ( or = 1.7 , 95% ci : 1.22.3 ) and age ( or for patients 60 and over = 2.3 , 95% ci : 1.24.4 ) . physician s characteristics ( sex , age and fee - for - service practice ) were not associated with chronicity.table 1socio - demographic characteristics of patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692)spinal disordersnon - spinal msd ( upper and lower limb)total12 weeks>12 weekstotal12 weeks>12 weeksn = 665n = 376n = 289n = 1027n = 404n = 623gender of patients ( % female)59.057.162.161.651.9 * 69.2*age of patients ( mean , sd)49.6 ( 14.1)47.0 * ( 14.3)53.7 * ( 13.2)57.9 ( 15.2)53.3 * ( 16.6)61.5 * ( 13.2)employment status ( % ) employed58.464.8 * 48.2 * 38.646.832.2 on unemployment benefits3.03.02.82.92.63.1 home maker3.22.25.03.52.74.0 student2.53.11.62.03.50.9 retired , other unemployed31.325.240.852.343.559.2education ( % completed high school)43.348.933.935.244.927.6familial status ( % ) living with a spouse69.369.868.664.966.663.4 living with children44.345.343.030.732.729.2body mass index ( % ) 02449.151.245.945.148.942.1 252937.335.440.434.333.934.6 30 and over11.911.612.319.717.121.8tobacco consumption ( % ) never smoked44.844.644.853.049.855.5 past smoker25.423.728.125.525.225.7 current smoker29.531.326.921.425.018.7alcohol consumption ( % ) never33.930.539.832.331.532.8 sometimes56.357.953.749.750.449.2 daily9.310.96.417.918.117.7physical exercise ( % ) 030 min per day59.261.156.058.854.662.1 31 min and over38.335.942.339.644.236.0physician visit previous 12 months yes96.095.397.196.495.097.5 * comparison 12 weeks , > 12 weeks statistically significant ( p < 0.05 ) in multiple logistic regression including all variables in the table socio - demographic characteristics of patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692 ) * comparison 12 weeks , > 12 weeks statistically significant ( p < 0.05 ) in multiple logistic regression including all variables in the table physicians declared about twice as much msd co - morbidities ( at least one secondary msd diagnosis ) for their chronic patients than their acute subacute patients ( table 2 ) . after controlling for age and gender , the odds ratio of having a msd co - morbidity in chronic sd patients was 2.4 ( 95% ci : 1.54.0 ) and in chronic ns - msd patients , 1.7 ( 95% ci : 1.12.7 ) . chronic patients also had about twice as much anxiety , depressive and sleeping disorders diagnoses than their acute subacute counterpart with odds ratios respectively in sd and ns - msd patients of 1.9 ( 95% ci : 1.22.9 ) and 2.0 ( 95% ci : 1.52.8 ) . differences between acute subacute and chronic patients for digestive co - morbidities were not statistically significant but an excess cardiovascular disorders was observed in chronic ns - msd patients ( or = 1.5 , 95% ci : 1.11.9 ) which was essentially explained by a four times higher number of hypertensive patients compared to their acute subacute counterpart ( 16 against 4).table 2co - morbidities and quality of life in patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692)co - morbidities present at the medical visitspinal disordersnon - spinal msd ( upper and lower limb)total12 weeks>12 weekstotal12 weeks>12 weeksn = 665n = 376n = 289n = 1027n = 404n = 623msd co - morbidities ( % ) 11.68.3 * 16.9 * 7.65.8 * 8.9*other co - morbidities ( % ) at least one32.326.8 * 41.1 * 56.140.9 * 68.0 * anxio - depressive disorders10.77.4 * 16.1 * 16.39.5 * 21.6 * sleeping disorders2.71.3 * 5.0 * 5.13.7 * 6.2 * cardiovascular respiratory19.318.021.137.225.4 * 46.4 * digestive disorders6.17.04.48.85.811.1quality of life sf-12 mental score mean ( sd)39.9 ( 1.2)40.8 ( 2.0)40.5 ( 2.0)40.6 ( 1.1)41.5 * ( 1.6)39.8 * ( 1.4 ) physical score mean ( sd)42.2 ( 1.3)42.0 * ( 2.2)39.6 * ( 2.2)42.7 ( 1.2)44.0 ( 1.6)43.1 ( 1.4 ) * comparison 12 and > 12 weeks : p < 0.01icd-9 codes for anxio - depressive disorders : 300316 , 799 ; sleeping disorders : 780 ; cardiovascular : 415426 , 428448 , 785 ; respiratory : 472474 , 476477 , 490496 ; digestive : 520537 , 540553 , 555558 , 560579 , 787 , 789mean adjusted for age , gender and presence of co - morbidities co - morbidities and quality of life in patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692 ) * comparison 12 and > 12 weeks : p < 0.01 icd-9 codes for anxio - depressive disorders : 300316 , 799 ; sleeping disorders : 780 ; cardiovascular : 415426 , 428448 , 785 ; respiratory : 472474 , 476477 , 490496 ; digestive : 520537 , 540553 , 555558 , 560579 , 787 , 789 mean adjusted for age , gender and presence of co - morbidities quality of life scores ( sf12 ) were almost identical between sd and ns - msd patients . chronic sd patients had a physical mean score adjusted for age , gender and co - morbidities , 2.4 points lower than acute subacute patients ( p = 0.001 ) , while their mental scores were almost identical . the reverse was observed in ns - msd patients where the difference was observed in the mental scale with a difference of 1.7 points lower among chronic patients ( p = 0.004 ) . chronic sd and ns - msd patients received half the analgesics , non - steroidal anti - inflammatory drugs ( nsaids ) and muscle relaxants prescriptions than their acute subacute counterpart after controlling for co - morbidities and other confounding factors [ table 3 , or = 0.4 ( 95% ci : 0.20.7 ) and 0.5 ( 95% ci : 0.30.6 ) , respectively , statistically significant ] . the reverse was observed for antidepressants , anxiolytics and hypnotics with almost twice more prescriptions in chronic sd patients ( or = 2.0 , 95% ci : 1.13.6 ) but not in ns - msd patients ( or = 1.3 , 95% ci : 0.82.1 ) . for non - pharmacological prescriptions , subacute than chronic patients but there was no statistical difference with referral to physiotherapy or to a specialist.table 3prescriptions at the medical visit in patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692)spinal disordersnon - spinal msd ( upper and lower limb)total12 weeks>12 weekstotal12 weeks>12 weeksn = 665n = 376n = 289n = 1027n = 404n = 623at least one ( % ) analgesics56.861.549.145.350.541.4 nsaid ( without aspirin)40.945.733.530.241.421.4 muscle relaxants31.138.020.05.87.14.8 anti - osteoarthritis drugs0.40.20.65.81.98.9>12 versus 12 weeks or ( 95% ci)*1.0 ( )0.4 ( 0.20.7)1.0 ( )0.5 ( 0.30.6)at least one ( % ) antidepressants7.54.612.18.94.312.5 anxiolytics5.03.96.99.05.811.6 hypnotics3.41.76.16.64.58.3>12 versus 12 weeks or ( 95% ci)*1.0 ( )2.0 ( 1.13.6)1.0 ( )1.3 ( 0.82.1)a least one ( % ) imaging ( x - rays , ct scan or mri)16.219.910.314.218.510.9 lab tests11.212.09.913.210.915.0>12 versus 12 weeks or ( 95% ci)*1.0 ( )0.4 ( 0.30.8)1.0 ( )0.7 ( 0.51.0)a least one ( % ) physiotherapy17.617.817.313.613.413.8 reference to a specialist9.16.613.217.615.119.7>12 versus 12 weeks or ( 95% ci)*1.0 ( )0.9 ( 0.61.4)1.0 ( )1.0 ( 0.71.4 ) * odds ratios and 95% confidence intervals derived from multivariate mixed regression models adjusting for all variables in tables 1 and 2 and for physician s effect prescriptions at the medical visit in patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692 ) * odds ratios and 95% confidence intervals derived from multivariate mixed regression models adjusting for all variables in tables 1 and 2 and for physician s effect frequencies of prescription of analgesics and of psychotropic drugs ( antidepressant , anxiolytic and hypnotic drugs combined ) in all chronic patients ( sd and ns - msd ) , were computed by sf-12 mental and physical scores ( table 4 ) . two statistically significant trends were found , one for psychotropic drugs increasing with a decreasing ( worsening ) mental score , and the other for analgesic drugs increasing with decreasing ( worsening ) physical score . similar trends were observed with physicians reporting of msd and anxio - depressive co - morbidities but reached statistical significance only in the former . it is noteworthy that among chronic patients in the lower decile of sf-12 mental score ( not shown in table 4 ) , 42.2% ( 38/91 ) were prescribed a psychotropic drug.table 4analgesic and psychotropic drugs prescription in patients with msds of more than 12 weeks duration by quality of life quartiles ( n = 911 ) * test for trend : p < 0.001antidepressants , anxiolytics and hypnotics analgesic and psychotropic drugs prescription in patients with msds of more than 12 weeks duration by quality of life quartiles ( n = 911 ) * test for trend : p < 0.001 antidepressants , anxiolytics and hypnotics physicians participation to this survey was low in part due to the important intrusive nature of the study into their practice . we found no equivalent in the scientific literature for a study of this size and the large population of patients was nevertheless representative of the french population consulting a gp . first , the distribution of gps individual characteristics differed from published national statistics only for gender with 23.5% female in the epi3 survey versus 39% in all of france for the year 2008 . this difference had potentially little impact on patients results as physicians characteristics were not associated with chronicity in our analyses . in addition , patients non participation was partially controlled by a weighting procedure based on socio - demographic information collected on non - respondents at recruitment . secondly , sf-12 scores observed in our study were not far away from those reported in three european population surveys of patients with msds , and score differences between acute and chronic patients were also similar [ 12 , 13 ] . finally , nsaids utilization was close to what has been observed in two other studies but our frequency of prescription of antidepressants was about half [ 214 ] . prescription of physiotherapy was also lower than what has been reported in another french survey . differences could be attributed to the cross - sectional nature of the surveys , differences in sources of information and type of insurance coverage of the patients . the impact of chronicity on quality of life , co - morbidities and prescriptions was similar between sd and ns - msd patients . our study showed that drug prescription in primary care was well correlated to physical and mental patient s self - assessed quality of life , using a standardized instrument independent from the clinician s evaluation . the neat gradient of prescription of psychotropic drugs with the mental scale and of analgesics with the physical scale of the sf-12 , provided evidence of the pertinence of physician s prescriptions . for instance , chronic msd patients were twice as likely to have anxio - depressive and sleeping disorders , a burden that has been raised in systematic reviews [ 16 , 17 ] . the concomitant presence of chronic pain and anxio - depressive disorders has been at the source of confusion in the scientific literature on efficacy of antidepressants against chronic pain [ 18 , 19 ] . our results indicated that clinicians seemed able to adapt their prescription to the pain and psychological components for their chronic pain patients . our results showed that over 50% of chronic patients with a very low mental score ( lower decile ) received no prescription for a psychotropic drug . this is not in contradiction , however , with recent evidence that indicates limited or no benefit of antidepressant drugs in populations with mild or moderate symptoms , which would account for a significant proportion of chronic pain patients . the prevalence of sleeping problems was low compared to what has been reported in a systematic review on that topic but data remains sparse on this topic . sleeping disorders have been associated to pain intensity but not to duration ( chronicity ) of symptoms which is the marker usually cited in clinical guidelines . anxio - depressive symptoms and sleeping quality should be a systematic part of the medical questionnaire with chronic msd patients because of their impact on quality of life . the high hypertension and digestive disorders observed in patients consulting for msds in primary care has been reported in another study and was partly explained by age . the higher , but not statistically significant , digestive co - morbidities observed in chronic compared to acute subacute ns - msd patients might explain the lower prescription of nsaids in that group . the main limitation of this study was the cross - sectional nature of data collection and analyses . associations found between quality of life and prescriptions for instance , can not be interpreted as causally linked . prescriptions of the day was a mix of new prescriptions and renewals and represented the patients status at one point in time . because patients responded to the sf-12 questionnaire before they saw their physician in consultation first , the scoring procedure used to derive mental and physical scores from responses to the sf-12 questionnaire , was not known from patients . secondly , the sf-12 questionnaire was included in a general questionnaire on health that did not focus on any specific health issue . no diagnostic criteria was suggested for reporting msds diagnoses ore anxio - depressive co - morbidities . this was a deliberate choice in order to describe clinical practice in real life situation . we feel confident that the large number of participating physicians and patients contributed to the overall representativity of clinical practice in primary care in france . in addition , msd patients were identified from a large sample of patients consulting for any reason in primary care , thus minimizing biases related to sampling msd patients directly . the main strength of the study was the combination of medical information on diagnoses and prescriptions on one hand , and patients information on quality of life on the other hand , both collected on the day of consultation insuring timely compatibility . in conclusion , important differences were observed in the clinical management of acute subacute and chronic msd french patients in primary care . anxiety and depressive disorders gps prescriptions of psychotropic and analgesic drugs seemed pertinent as they were consistent with a standardized measure of quality of life , independent from the clinicians judgement . this utilization of patients self - assessed quality of life provided a useful benchmarking approach to assess consistency between clinical practice and guidelines .

concerns have been raised regarding sub - optimal utilization of analgesics and psychotropic drugs in the treatment of patients with chronic musculoskeletal disorders ( msds ) and their associated co - morbidities . the objective of this study was to describe drug prescriptions for the management of spinal and non - spinal msds contrasted against a standardized measure of quality of life . a representative population sample of 1,756 msds patients [ 38.5% with spinal disorder ( sd ) and 61.5% with non - spinal msds ( ns - msd ) ] was drawn from the epi3-laser survey of 825 general practitioners ( gps ) in france . physicians recorded their diagnoses and prescriptions on that day . patients provided information on socio - demographics , lifestyle and quality of life using the short form 12 ( sf-12 ) questionnaire . chronicity of msds was defined as more than 12 weeks duration of the current episode . chronic sd and ns - msd patients were prescribed less analgesics and non - steroidal anti - inflammatory drugs than their non - chronic counterpart [ odds ratios ( or ) and 95% confidence intervals ( ci ) , respectively : 0.4 , 0.20.7 and 0.5 , 0.30.6 ] . they also had more anxio - depressive co - morbidities reported by their physicians ( sd : 16.1 vs.7.4% ; ns - msd : 21.6 vs. 9.5% ) who prescribed more antidepressants and anxiolytics with a difference that was statistically significant only for spinal disorder patients ( or , 95% ci : 2.0 , 1.13.6 ) . psychotropic drugs were more often prescribed in patients in the lower quartile of sf-12 mental score and prescriptions of analgesics in the lower quartile of sf-12 physical score ( p < 0.001 ) . in conclusion , anxiety and depressive disorders were commonly reported by gps among chronic msd patients . their prescriptions of psychotropic and analgesic drugs were consistent with patients self - rated mental and physical health .

Introduction Materials and methods Study design and population Statistical analysis Results Discussion Conclusion

physicians in primary care play a central role in the management of musculoskeletal disorders ( msds ) . in this context , concerns have been raised regarding sub - optimal use of analgesics and psychotropic drugs ( antidepressants ) in the treatment of patients with chronic msds [ 2 , 3 ] . the goal of this study was to describe physician s prescriptions for the management of acute and chronic msds in a large population of patients seen in primary care , and examine the pertinence of prescriptions against patient s self - assessed quality of life . the epi3-laser study was a nation - wide observational survey of a representative sample of general practitioners ( gps ) and their patients , conducted in france between march 2007 and july 2008 . its aims were to assess the burden of disease in general practice , considering physician and patients characteristics , co - morbidities and prescriptions with a specific focus on health - related quality of life . first , gps were randomly selected from the french national directory of physicians and invited to participate , which meant allowing a research assistant to be in the patients waiting room for a 1-day consultation session , collecting basic information on all patients consulting that day and recruiting volunteers to fill out a self - administered questionnaire . consenting patients completed a self - administered questionnaire that included information on age , gender , education , employment status and occupation , type of health insurance , hospitalization and medical visits in the previous 12 months , smoking , alcohol intake , physical activity , height and weight , and health related quality of life assessed by the short form 12 ( sf-12 ) questionnaire [ 68 ] . msds were classified as spinal disorders ( sd ) with icd codes 720724 , and non - spinal msds ( ns - msd ) , such as osteoarthritis and tendonitis of the upper or lower limb , with icd codes 715 , 719 , 729 , 726728 , 782 . sd and ns - msd were classified as acute subacute or chronic using a 12-week ( 3 months ) cut - off for duration of symptoms in the current episode in accordance with consensus recommendations for research on sd . odds ratios were calculated for the comparison of prescriptions between chronic and non - chronic patients using mixed models accounting for a random physician effect ( glimmix and nlmixed procedures in sas ) . mean scores of the sf-12 mental and physical scales were adjusted for gender , age and co - morbidities using the analysis of covariance . the epi3-laser study was a nation - wide observational survey of a representative sample of general practitioners ( gps ) and their patients , conducted in france between march 2007 and july 2008 . its aims were to assess the burden of disease in general practice , considering physician and patients characteristics , co - morbidities and prescriptions with a specific focus on health - related quality of life . consenting patients completed a self - administered questionnaire that included information on age , gender , education , employment status and occupation , type of health insurance , hospitalization and medical visits in the previous 12 months , smoking , alcohol intake , physical activity , height and weight , and health related quality of life assessed by the short form 12 ( sf-12 ) questionnaire [ 68 ] . msds were classified as spinal disorders ( sd ) with icd codes 720724 , and non - spinal msds ( ns - msd ) , such as osteoarthritis and tendonitis of the upper or lower limb , with icd codes 715 , 719 , 729 , 726728 , 782 . sd and ns - msd were classified as acute subacute or chronic using a 12-week ( 3 months ) cut - off for duration of symptoms in the current episode in accordance with consensus recommendations for research on sd . odds ratios were calculated for the comparison of prescriptions between chronic and non - chronic patients using mixed models accounting for a random physician effect ( glimmix and nlmixed procedures in sas ) . mean scores of the sf-12 mental and physical scales were adjusted for gender , age and co - morbidities using the analysis of covariance . they were female in proportions of 59.0 and 61.6% for sd and ns - msd , respectively , and sd patients were younger by 8.2 years on average . factors associated with chronicity of sd were age , with the largest proportion observed over the age of 60 [ odds ratio ( or ) : 4.5 , 95% confidence interval ( ci ) : 2.19.6 ] , and not being employed ( or = 2.0 , 95% : 1.13.6 ) . for ns - msd , factors associated with chronicity were female gender ( or = 1.7 , 95% ci : 1.22.3 ) and age ( or for patients 60 and over = 2.3 , 95% ci : 1.24.4 ) . physician s characteristics ( sex , age and fee - for - service practice ) were not associated with chronicity.table 1socio - demographic characteristics of patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692)spinal disordersnon - spinal msd ( upper and lower limb)total12 weeks>12 weekstotal12 weeks>12 weeksn = 665n = 376n = 289n = 1027n = 404n = 623gender of patients ( % female)59.057.162.161.651.9 * 69.2*age of patients ( mean , sd)49.6 ( 14.1)47.0 * ( 14.3)53.7 * ( 13.2)57.9 ( 15.2)53.3 * ( 16.6)61.5 * ( 13.2)employment status ( % ) employed58.464.8 * 48.2 * 38.646.832.2 on unemployment benefits3.03.02.82.92.63.1 home maker3.22.25.03.52.74.0 student2.53.11.62.03.50.9 retired , other unemployed31.325.240.852.343.559.2education ( % completed high school)43.348.933.935.244.927.6familial status ( % ) living with a spouse69.369.868.664.966.663.4 living with children44.345.343.030.732.729.2body mass index ( % ) 02449.151.245.945.148.942.1 252937.335.440.434.333.934.6 30 and over11.911.612.319.717.121.8tobacco consumption ( % ) never smoked44.844.644.853.049.855.5 past smoker25.423.728.125.525.225.7 current smoker29.531.326.921.425.018.7alcohol consumption ( % ) never33.930.539.832.331.532.8 sometimes56.357.953.749.750.449.2 daily9.310.96.417.918.117.7physical exercise ( % ) 030 min per day59.261.156.058.854.662.1 31 min and over38.335.942.339.644.236.0physician visit previous 12 months yes96.095.397.196.495.097.5 * comparison 12 weeks , > 12 weeks statistically significant ( p < 0.05 ) in multiple logistic regression including all variables in the table socio - demographic characteristics of patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692 ) * comparison 12 weeks , > 12 weeks statistically significant ( p < 0.05 ) in multiple logistic regression including all variables in the table physicians declared about twice as much msd co - morbidities ( at least one secondary msd diagnosis ) for their chronic patients than their acute subacute patients ( table 2 ) . after controlling for age and gender , the odds ratio of having a msd co - morbidity in chronic sd patients was 2.4 ( 95% ci : 1.54.0 ) and in chronic ns - msd patients , 1.7 ( 95% ci : 1.12.7 ) . chronic patients also had about twice as much anxiety , depressive and sleeping disorders diagnoses than their acute subacute counterpart with odds ratios respectively in sd and ns - msd patients of 1.9 ( 95% ci : 1.22.9 ) and 2.0 ( 95% ci : 1.52.8 ) . differences between acute subacute and chronic patients for digestive co - morbidities were not statistically significant but an excess cardiovascular disorders was observed in chronic ns - msd patients ( or = 1.5 , 95% ci : 1.11.9 ) which was essentially explained by a four times higher number of hypertensive patients compared to their acute subacute counterpart ( 16 against 4).table 2co - morbidities and quality of life in patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692)co - morbidities present at the medical visitspinal disordersnon - spinal msd ( upper and lower limb)total12 weeks>12 weekstotal12 weeks>12 weeksn = 665n = 376n = 289n = 1027n = 404n = 623msd co - morbidities ( % ) 11.68.3 * 16.9 * 7.65.8 * 8.9*other co - morbidities ( % ) at least one32.326.8 * 41.1 * 56.140.9 * 68.0 * anxio - depressive disorders10.77.4 * 16.1 * 16.39.5 * 21.6 * sleeping disorders2.71.3 * 5.0 * 5.13.7 * 6.2 * cardiovascular respiratory19.318.021.137.225.4 * 46.4 * digestive disorders6.17.04.48.85.811.1quality of life sf-12 mental score mean ( sd)39.9 ( 1.2)40.8 ( 2.0)40.5 ( 2.0)40.6 ( 1.1)41.5 * ( 1.6)39.8 * ( 1.4 ) physical score mean ( sd)42.2 ( 1.3)42.0 * ( 2.2)39.6 * ( 2.2)42.7 ( 1.2)44.0 ( 1.6)43.1 ( 1.4 ) * comparison 12 and > 12 weeks : p < 0.01icd-9 codes for anxio - depressive disorders : 300316 , 799 ; sleeping disorders : 780 ; cardiovascular : 415426 , 428448 , 785 ; respiratory : 472474 , 476477 , 490496 ; digestive : 520537 , 540553 , 555558 , 560579 , 787 , 789mean adjusted for age , gender and presence of co - morbidities co - morbidities and quality of life in patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692 ) * comparison 12 and > 12 weeks : p < 0.01 icd-9 codes for anxio - depressive disorders : 300316 , 799 ; sleeping disorders : 780 ; cardiovascular : 415426 , 428448 , 785 ; respiratory : 472474 , 476477 , 490496 ; digestive : 520537 , 540553 , 555558 , 560579 , 787 , 789 mean adjusted for age , gender and presence of co - morbidities quality of life scores ( sf12 ) were almost identical between sd and ns - msd patients . chronic sd patients had a physical mean score adjusted for age , gender and co - morbidities , 2.4 points lower than acute subacute patients ( p = 0.001 ) , while their mental scores were almost identical . the reverse was observed in ns - msd patients where the difference was observed in the mental scale with a difference of 1.7 points lower among chronic patients ( p = 0.004 ) . chronic sd and ns - msd patients received half the analgesics , non - steroidal anti - inflammatory drugs ( nsaids ) and muscle relaxants prescriptions than their acute subacute counterpart after controlling for co - morbidities and other confounding factors [ table 3 , or = 0.4 ( 95% ci : 0.20.7 ) and 0.5 ( 95% ci : 0.30.6 ) , respectively , statistically significant ] . the reverse was observed for antidepressants , anxiolytics and hypnotics with almost twice more prescriptions in chronic sd patients ( or = 2.0 , 95% ci : 1.13.6 ) but not in ns - msd patients ( or = 1.3 , 95% ci : 0.82.1 ) . for non - pharmacological prescriptions , subacute than chronic patients but there was no statistical difference with referral to physiotherapy or to a specialist.table 3prescriptions at the medical visit in patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692)spinal disordersnon - spinal msd ( upper and lower limb)total12 weeks>12 weekstotal12 weeks>12 weeksn = 665n = 376n = 289n = 1027n = 404n = 623at least one ( % ) analgesics56.861.549.145.350.541.4 nsaid ( without aspirin)40.945.733.530.241.421.4 muscle relaxants31.138.020.05.87.14.8 anti - osteoarthritis drugs0.40.20.65.81.98.9>12 versus 12 weeks or ( 95% ci)*1.0 ( )0.4 ( 0.20.7)1.0 ( )0.5 ( 0.30.6)at least one ( % ) antidepressants7.54.612.18.94.312.5 anxiolytics5.03.96.99.05.811.6 hypnotics3.41.76.16.64.58.3>12 versus 12 weeks or ( 95% ci)*1.0 ( )2.0 ( 1.13.6)1.0 ( )1.3 ( 0.82.1)a least one ( % ) imaging ( x - rays , ct scan or mri)16.219.910.314.218.510.9 lab tests11.212.09.913.210.915.0>12 versus 12 weeks or ( 95% ci)*1.0 ( )0.4 ( 0.30.8)1.0 ( )0.7 ( 0.51.0)a least one ( % ) physiotherapy17.617.817.313.613.413.8 reference to a specialist9.16.613.217.615.119.7>12 versus 12 weeks or ( 95% ci)*1.0 ( )0.9 ( 0.61.4)1.0 ( )1.0 ( 0.71.4 ) * odds ratios and 95% confidence intervals derived from multivariate mixed regression models adjusting for all variables in tables 1 and 2 and for physician s effect prescriptions at the medical visit in patients with musculoskeletal disorders ( msd ) in primary care ( n = 1,692 ) * odds ratios and 95% confidence intervals derived from multivariate mixed regression models adjusting for all variables in tables 1 and 2 and for physician s effect frequencies of prescription of analgesics and of psychotropic drugs ( antidepressant , anxiolytic and hypnotic drugs combined ) in all chronic patients ( sd and ns - msd ) , were computed by sf-12 mental and physical scores ( table 4 ) . two statistically significant trends were found , one for psychotropic drugs increasing with a decreasing ( worsening ) mental score , and the other for analgesic drugs increasing with decreasing ( worsening ) physical score . similar trends were observed with physicians reporting of msd and anxio - depressive co - morbidities but reached statistical significance only in the former . it is noteworthy that among chronic patients in the lower decile of sf-12 mental score ( not shown in table 4 ) , 42.2% ( 38/91 ) were prescribed a psychotropic drug.table 4analgesic and psychotropic drugs prescription in patients with msds of more than 12 weeks duration by quality of life quartiles ( n = 911 ) * test for trend : p < 0.001antidepressants , anxiolytics and hypnotics analgesic and psychotropic drugs prescription in patients with msds of more than 12 weeks duration by quality of life quartiles ( n = 911 ) * test for trend : p < 0.001 antidepressants , anxiolytics and hypnotics physicians participation to this survey was low in part due to the important intrusive nature of the study into their practice . the impact of chronicity on quality of life , co - morbidities and prescriptions was similar between sd and ns - msd patients . our study showed that drug prescription in primary care was well correlated to physical and mental patient s self - assessed quality of life , using a standardized instrument independent from the clinician s evaluation . for instance , chronic msd patients were twice as likely to have anxio - depressive and sleeping disorders , a burden that has been raised in systematic reviews [ 16 , 17 ] . anxio - depressive symptoms and sleeping quality should be a systematic part of the medical questionnaire with chronic msd patients because of their impact on quality of life . the higher , but not statistically significant , digestive co - morbidities observed in chronic compared to acute subacute ns - msd patients might explain the lower prescription of nsaids in that group . no diagnostic criteria was suggested for reporting msds diagnoses ore anxio - depressive co - morbidities . the main strength of the study was the combination of medical information on diagnoses and prescriptions on one hand , and patients information on quality of life on the other hand , both collected on the day of consultation insuring timely compatibility . in conclusion , important differences were observed in the clinical management of acute subacute and chronic msd french patients in primary care . anxiety and depressive disorders gps prescriptions of psychotropic and analgesic drugs seemed pertinent as they were consistent with a standardized measure of quality of life , independent from the clinicians judgement . this utilization of patients self - assessed quality of life provided a useful benchmarking approach to assess consistency between clinical practice and guidelines .

nanoparticles ( nps ) have attracted considerable interest as diagnostic and therapeutic tools for biomedicine , with magnetic nps offering unique opportunities for magnetic separation , targeted drug delivery , and hyperthermic ablation of cancer and as contrast agents in magnetic resonance imaging ( mri ) . mri is a powerful , noninvasive medical imaging technique where the signal originates predominately from water and lipids . medical diagnosis requires enhanced contrast between normal and pathological tissues , resulting in the development of exogenous mri contrast agents.(12 ) common t2-agents include superparamagnetic nanoparticles ( fe3o4 , -fe2o3 , mnfe2o4 , feco ) , the clinical mri applications of which range from imaging lymph - node to liver and spleen , as well as bone marrow and gastrointestinal tract . despite its widespread usage , superparamagnetic iron oxide ( spio ) has several disadvantages , including rapid clearance by phagocytic cells and hence limited trans - endothelia passage and tissue penetration . there is therefore a need for the design of biocompatible mri contrast agents with prolonged intravascular retention , improved tissue delineation , high chemical stability , and improved selectivity when targeting a tissue of interest . small nps avoid fast clearance by the reticulo - endothelial system and therefore should enable long blood circulation half - life , thereby improving their ability to reach specific targets . silica is often considered as the most attractive coating for engineered nps , forming an inert and biocompatible outer shell . not only does it create a protective shell against chemical degradation , but it also allows surface functionalization with functional alkoxysilanes.(7 ) an outer coating with functional organic molecules has many advantages , including steric and/or electrostatic repulsion between the nps , which can increase water solubility and stability in biological environment.(17 ) positively charged nps demonstrate increased cell internalization efficiency and biomedical molecules binding capability for multimodal imaging applications and drug delivery . characterized by high curie temperature and magneto - crystalline anisotropy , ironplatinum metallic alloy nps have emerged as promising candidates for a new generation of magnetic nanomaterials.(20 ) over the past decade , several chemical pathways have been developed to synthesize fept nps with controlled stoichiometry , size , and shape.(5 ) such synthetic routes include co - reduction of fe and pt precursors through both low - temperature emulsion protocols and the high - temperature polyol pathway , along with the thermal decomposition route . the latter approach often relies on pt(acac)2 , fe(co)5 , or collman s regeant precursors mixed with alkyl amines and acids solubilized in high - boiling - point solvents heated at temperatures of 300 c and above to control the growth , composition , and morphology of the nps . studies have demonstrated the biomedical potential of fept nps,(6 ) such as in magnetic separation and hyperthermic ablation and as t2 mri contrast agents . however , toxicity remains a major concern , preventing their use as a bioplatform for diagnosis and therapy . in this article , we describe the fabrication of fept nps specifically designed to enhance their magnetic properties for biomedical applications . we demonstrate that , in contrast with previous reports , fept nps can be made nontoxic , and we provide the first data on their cellular uptake mechanisms . we report a 6-fold increase in the fept - based t2 contrast properties compared to those of clinical iron oxide nps . the relationship between the mri contrast properties and the nps architecture is explored and rationalized as the basis for the design of nps as enhanced mri contrast agents . finally , we report the first observations of cellular and in vivo mr imaging with fept nps . our results opens the way for several applications of fept nps , such as regenerative medicine and stem cell therapy , in addition to enhanced mr diagnostic imaging , thus providing a platform to develop novel diagnostic and therapeutic agents . figure 1 displays the schematic structure of a clinical iron oxide np coated with dextran ( feridex , figure 1a1 ) and two fept nps : the face - centered - cubic ( fcc ) fept np surface functionalized with cysteamine ( fcc - fept - a , figure 1b1 ) and the fcc fept np coated with silica and surface - functionalized with ( 3-aminopropyl)triethoxysilane ( fcc - fept - silica - a , figure 1c1 ) . schematic representation ( 1 ) , tem images ( 2 ) , xrd patterns ( 3 ) , zfc - fc ( 4 ) , and hysteresis ( 5 ) curves of feridex ( a1a5 ) , fcc - fept - a nps ( b1b5 ) , and fcc - fept - silica - a nps ( c1c5 ) . in agreement with published literature,(42 ) transmission electron microscopy ( tem ) revealed that the commercial iron oxide nps samples ( figure 1a2 ) are composed of aggregated crystals approximately 5 nm in diameter , embedded in an organic coating . these aggregates form large particles of a size distribution ranging from 20 to > 50 nm . in contrast , the fcc - fept - a nps are well dispersed , with diameters of 5 nm ( figure 1b2 ) . the fcc - fept - silica - a nps are coated with a homogeneous silica shell of 17 nm in thickness , leading to a total diameter of 40 nm ( figure 1c2 ) . the powder x - ray diffraction ( xrd ) pattern of the feridex iron oxide confirms the presence of magnetite ( fe3o4 ) , the broad peaks at 38 , 45 , and 55 corresponding to the ( 220 ) , ( 311 ) , and ( 400 ) peaks , respectively ( figure 1a3 ) . figure 1b3,c3 shows peaks around 51 and 60 , characteristic of fcc - fept ( 111 ) and ( 200 ) peaks , respectively . the ( 111 ) peak position suggests that the nps composition includes between 40 and 45% of fe ( table 1 ) . for the silica - coated nps , the sio2 xrd characteristic peak the crystal size is 3.6 nm for both fcc - fept - a and fcc - fept - silica - a nps ( table 1 ) . dxrd is the crystalline grain size of the nps ; in fexpt1x , x is the composition based on the fept lattice constant ( si - figure 1);(43)tb is the blocking temperature ; ms is the saturation moment ; hc is the magnetic coercivity of the nps ; and r1 and r2 are the h relaxivity values for 1% agarose gel containing feridex , fcc - fept - a , and fcc - fept - silica - a nps . a range of other commercial mri contrast agent relaxivity values are presented in the supporting information ( si - table 2 ) . zero - field - cooling and field - cooling ( zfcfc ) of feridex samples highlight a 45 k blocking temperature ( tb , figure 1a4 ) . at 2 k , i.e. , lower than tb , feridex hysteresis loop measurements display ferrimagnetic behavior with a small coercivity value of 300 oe and a saturation moment ( ms ) of 101 emu / g of fe . at 300 k the ms value is 82 emu / g of fe , in agreement with the literature . in comparison , the fcc - fept - a nps have a higher tb of 90 k ( figure 1b4 ) . more importantly , when compared to feridex , the ms of fcc - fept - a nps is 2 times larger at 2 k and 35% stronger at 300 k ( figure 1b5 , table 1 ) . after silica coating , the blocking temperature is reduced to 50 k , and the zfc slope below tb is steeper ( figure 1c4 ) . at 2 k and 5 t , the magnetization of fcc - fept - silica - a nps is 5% lower than that of fcc - fept - a and 2 times larger than that of feridex . at 300 k and 5 t , the magnetization of fcc - fept - silica - a nps is 36% lower than that of fcc - fept - a nps and similar to that of feridex ( figure 1c5 , table 1 ) . cytotoxicity of the nps was evaluated in an a375 m cell line using fcc - fept - a nps and mts assay with various concentrations and incubation times.(43 ) both doseresponse and time course studies were performed ( figure 2a ) . as expected , and in contrast with bare iron oxide , feridex s dextran organic coating does not lead to any detectable cytotoxicity . at 120 g / ml , fcc - fept - a nps exhibited cytotoxicity that increases with incubation time . at a concentration of 60 g / ml , fcc - fept - a nps incubated with cells for 7 days showed a 15% loss of cell viability . significantly , there is no loss of cell viability at concentrations of 30 g / ml and below , even after prolonged incubation periods of up to 7 days . similar results were obtained with fcc - fept - a nps and both mcf7 and u2os tumor cell lines ( si - figure 5a , b).(43 ) viability of a375 m cells incubated with fcc - fept - a nps ( a ) . fe released from fcc - fept - a nps ( orange ) in ph 4.8 pbs ( b1 ) . fe released from fcc - fept - a nps ( orange ) and from feridex ( )(49 ) in solutions at ph 4.8 of rpmi-1640 cell media containing 20 mm sodium citrate . the incubation concentrations for fept and feridex(49 ) were 120 and 25 g / ml , respectively ( b2 ) . photomicrographs ( magnification 40 ) of prussian blue - stained a375 m cells cultured on coverslips in cell media ( c1 ) , media containing 30 g / ml of fcc - fept - a nps ( c2 ) , and media containing 30 g / ml of feridex ( c3 ) after overnight incubation . tem images of a375 m cells after 16 h incubation in media containing 30 g / ml of fcc - fept - a nps ( d1d3 ) . to confirm these results , the chemical stability of fcc - fept - a nps in an acidic environment was investigated by incubation of fcc - fept - a in ph 4.8 buffer solutions . the concentration of fept nps incubated in buffer solution is 120 g / ml , to match the highest nps concentration used in the cytotoxicity study ( figure 2a ) . in a ph 4.8 phosphate buffered saline ( pbs ) , cysteamine - coated fept nps show excellent chemical stability , with only 0.6% fe , i.e. < 0.1 g / ml , of iron released after 7 days of incubation ( figure 2b1 ) . as suggested by the recent literature , an in vitro lysosomal model , i.e. rpmi-1640 cell media containing 20 mm sodium citrate ( ph 4.8 ) , was also used to match the metabolic conditions . in these more extreme conditions ( figure 2b2 ) , more fe was released0.3 g / ml after 6 h and up to 3.3 g / ml after 7 days prussian blue staining experiments were carried out and showed that fept nps have greater stability compared to feridex ( figure 2c1c3).(43 ) indeed , photomicrographs obtained after overnight incubation show no stain for both blank control sample ( figure 2c1 ) and cells labeled with fcc - fept - a nps ( figure 2c2 ) . in contrast , feridex - labeled cells show significantly staining ( figure 2c3 ) characteristic of the release of fe in the cells . these results confirm that the cysteamine coating provides fcc - fept - a nps with high chemical stability . the cysteamine coating is positively charged , resulting in higher cellular uptake by human non - phagocytic a375 m tumor cells ( figure 2d1d3 ) compared to feridex . indeed , tem studies suggest a macropinocytosis cellular uptake mechanism by the large protrusions of the plasma membrane around np clusters ( figure 2d2 ) . the intracellular localization route is endosome , early lysosome , and late lysosome , which are basic cell biology phenomena . both endosomes and lysosomes are confined subcellular organelles , and , as both expected and commonly observed in other nps systems , the fept nps appear typically entrapped inside these subcellular vesicles ( figure 2d3 ) . the effectiveness of fcc - fept - a and fcc - fept - silica - a as mri contrast agents was investigated by measuring the dependence of the longitudinal relaxation rate ( t1 ) and transverse relaxation rate ( t2 ) of 1% agarose gels.(43 ) relaxivity ( ri ) quantifies the enhancement of either the longitudinal or the transverse relaxation rates of the protons in water . as expected at high magnetic field , the fept nps and feridex have a weak influence on the water h longitudinal relaxation rate ( table 1 and si - figure 7a ) . in contrast , fept nps have a significant effect upon the aqueous transverse relaxation rate ( figure 3a1 ) . the relaxivity r2 of the fcc - fept - silica - a nps is 40% larger than that of feridex , which is attributed to the stronger magnetic moment of the fcc - fept - silica - a nps ( si - figure 7b ) at the mri magnetic field ( 7 t).(43 ) importantly , the relaxivity r2 of the fcc - fept - a nps is more than 6 times larger than that of feridex . figure 3a2 displays t2-weighted mri images of various concentrations of fcc - fept - a nps in agarose gels . the fept nps produce an observable change in image contrast at concentrations as low as 6 g / ml , which is well within the safety range indicated by the cytotoxicity data ( figure 2a ) . similarly , signal intensity loss was observed in cells labeled with fcc - fept - a nps as the cell density increases from 10 10 to 1000 10 cells / ml ( figure 3b ) . t2 ( s ) vs [ fe ] ( mm ) of the water in 1% w / v agarose gel containing ( blue ) fcc - fept - a , ( green ) fcc - fept - silica - a , and ( orange ) feridex ( a1 ) . h longitudinal relaxation time t2-weighted mri images of 1% agarose gel / water solution containing fcc - fept - a nps with [ fe ] = 0.00 mm ( a ) , 0.02 mm ( cfept = 6 g / ml ) ( b ) , and 0.04 mm ( cfept = 12 g / ml ) ( c ) ( a2 ) . t2-weighted cellular imaging by mri ( 3d spin echo , te = 30 ms , tr = 1 s ) of a375 m cells loaded with fcc - fept - a nps with a cell density of 10 10 cells / ml ( a ) , 100 10 cells / ml ( b ) , and 1000 10 cells / ml ( c ) ( b ) . embryo injected in ovo with cell culture media ( 1 l ) containing no nps ( c ) and 20 g / ml fcc - fept - a ( d ) . 3d surface reconstruction of embryo eyes ( blue ) and blood vessels ( red ) showing the position in yellow of the transverse and coronal images ( c1,d1 ) ; dorsal view of transverse image of the embryo s head aligned through the eyes ( c2,d2 ) ; and anterior view of coronal image of the embryo s head aligned through the eyes ( c3,d3 ) . 2d slices from 128 128 128 3d rare-8 mri data set of day 4 quail embryo egg ( tr / te = 250/25 ms ) , field of view of 30 mm and pixel dimensions of 0.234 mm / pixel . labels : y , yolk ; a , albumen ; b , brain ; in - e , injected eye ; un - e , un - injected eye ; h , heart . scale bar indicates 1 mm . all the mri measurements were completed at 7.1 t. the in vivo potential of fept nps as mri contrast agents was demonstrated by injecting nps in cell culture media into the eyes of day 4 quail embryos in ovo . there is no difference in the contrast between the uninjected and the uppermost eye ( in - e ) which had been injected with a 1 l of culture medium only ( figure 3c2,c3 ) . however , when the injection medium contained 20 g / ml of fcc - fept - a nps , a very distinct hypointensity appears in the injected eye ( figure 3d2d3 ) . this thus demonstrates the capability of nontoxic fept nps as potent mri probes for cellular imaging , even at very low np concentrations . labeling the cells and these magnetic nps will allow , for instance , cell migration to be studied , with their biodistribution and behavior monitored in vivo , especially as we have demonstrated that the effect of contrast agents can be observed at non - cytotoxic concentrations lower than 30 g / ml . the major hurdle preventing the wider use of fept nps in biomedicine is their potential toxicity . in the literature , there are many inconsistencies in fept cytotoxicity , leaving the subject rather confused . a summary of the cytotoxicity data of fept nps is presented in table 2 . while it was reported that 24 h incubation of fept - cysteine nps had no significant toxicity at concentrations below 5 g / ml , these compounds resulted in 50% cell damage at 15.5 g / ml;(39 ) fept - fe3o4 nps showed no significant cell viability damage up to 3 days of incubation at 10 g / ml.(37 ) in contrast , fept - fe2o3 and fept - cos2 were reported to induce significant cell viability damage of over 50% even at concentrations of nps as low as 1.5 and a recent toxicity study with phospholipids - coated fcc - fept nps showed 50% cell viability damage above 1.25 g / ml fe concentration ( 7.8 g / ml fe40pt60 nps).(52 ) in this latter study , the high toxicity was attributed to fast fe release from phospholipids - coated fcc - fept nps . it was reported that 6% fe was already released after 6 h of incubation , and the percentage of fe released increased to 20% after 24 h of incubation in ph 4.8 pbs.(52 ) while iron is essential for almost all living organisms , it is toxic . it can act as a catalyst in the fenton reaction , which generates a wide range of free radical species , including hydroxyl radicals . these are among the most reactive free radical species known and have the ability to react with a wide range of cellular constituents , including pyrimidine bases of dna , and also to trigger lipid peroxidation in cellular membranes , leading to cell death.(53 ) listed are nps structure , precursors , and coating ; cells stands for the cell line ; [ nps ] is the nanoparticle concentration ; tinc is the incubation time ; cell damage is that after incubation . the fcc - fept - silica - a nps have good biocompatibility and , as expected , the silica coating forms an inert shell ( si - figure 5c).(43 ) in contrast to previously published results , our study demonstrates that cysteamine - coated fcc - fept nps also have very high chemical stability in ph 4.8 pbs . fe release after 24 h of incubation is only 0.3% , with only a slight increased to 0.6% after 7 days of incubation ( figure 2b1 ) . in an in vitro model ( i.e. , cell medium containing sodium citrate ) , fcc - fept - a nps were found to release as little fe as feridex ( figure 2b2 ) , with 5% fe found to be released after 24 h and 20% after 7 days . this confirms that the low ph in the endosome / lysosome environment is not sufficient to dissolve the nps and that the presence of endogenous citrate or similar metallic chelates is instrumental to the nps degradation by binding to solubilized fe atoms . the results presented herein are significant as they suggest that , when considering the fe release , the safety of fcc - fept - a nps is comparable to that of feridex under similar experimental conditions . the chemical stability of the fcc - fept - a nps is also supported by the prussian blue staining experiments ( figure 2c1c3 ) . the high chemical stability of cysteamine - coated fept nps certainly contributes to the nps noteworthy low cytotoxicity . it is quite possible that differences in the nps preparation , surface coating , ligand exchange , and extraction protocols could account for the spread of the cytotoxicity data reported in the literature . the toxicity may not necessarily be due to the material itself through the release of its constituent . in the present study , we used collman s reagent , na2fe(co)4 , to control the fept nps stoichiometry and to ensure the simultaneous presence of fe and pt species during the formation of fept alloy nps ; the use of fe(co)5 has been shown to form nps with a core and a shell rich in platinum and iron , respectively.(31 ) after ligand exchange , the extraction process was repeated up to six times to ensure removal of hazardous chemicals such as chloroform , oleic acid , and oleylamine , while the thiol group provides a strong bond between the cysteamine and the nps surface , preventing the ligands from becoming lethal . during the review of this publication , cysteamine - coated fept nps were reported to induce , after 24 h , very low cell damage on veron cells;(38 ) however , it is noticeable that the strong bonding to the surface of the nps , protecting them from the cells environment , can be contrasted with , for instance , a coating made with a bilayer structure of phospholipids and oleic acid / oleylamine.(52 ) indeed , even though this phospholipid approach provides aqueous solubility , it is also characterized by a weaker bonding strength and a more dynamic behavior , allowing the coating to slowly disperse in the cell . by doing so , the free ligands can alter the cell viability as well as the release of the np constituents as the surface of the particles is exposed to the cell , and the nps chemical integrity can be compromised further . such outcomes have been reported by xu et al . , although they elegantly focused on the opportunity this provided to use the nps as metal reservoirs to kill cancer cells.(52 ) while the very high chemical stability of fcc - fept - a nps was demonstrated in this report , cytotoxicity is observed at high concentrations above 30 g / ml fept and long incubation times . this could be explained by contributions from ( i ) the very small release of fe , which has been found to be < 1% after 168 h and ( ii ) the high concentration of positive surface charges , which could also interact with the more negatively charged cell membranes and induce membrane disruption.(54 ) in addition , ligands with strong anchoring groups can reduce the toxicity , as illustrated in this publication , and it is noticeable that the present results were obtained with commercially available ligands ( i.e. cysteamine ) , so one should expect enhanced viability from , for instance , custom - made dithiolate ligands , such as those developed recently for quantum dots . these could provide cell viability at np concentrations higher than 30 g / ml . in this context , the little consistency observed when considering fept nps cytotoxicity data available in the literature stresses that the measured toxicity should always be associated with the nanocolloids , i.e. inorganic nps + organic stabilizer . the interplay between the components will indeed contribute to the cytotoxicity results , with , for instance , a weak bonding to the nps surface increasing the probability of the nanocolloids being toxic . it then appears that , to be able to compare cytotoxicity data in a meaningful way , one must to develop and use standardized approaches including the correlation between ( i ) the evolution of the nanocolloids integrity , relying on both the inorganic nps and the organic stabilizers , and ( ii ) the cell viability . it should be noticed that , in the context of apparently toxic nps , the nanocolloids solution stability could easily be measured by , for instance , time - resolved transmission monitored sedimentation experiments , as while the ligands come off the nps surface , the nps tend to aggregate and fall out of solution . the cellular internalization of feridex is poor due to the weak binding of the neutral dextran coating to the plasma membrane , which limits the capability of cell internalization by the fluid - phase endocytosis pathway . enhancing cellular uptake of dextran - coated nps is possible with transfection agents and further surface functionalization but was not relevant to the present study.(44 ) in contrast , the amine - coated fept nps , fcc - fept - a and fcc - fept - silica - a , are efficiently internalized directly without requiring the use of any external mediation such as transfection agents or electroporation , illustrated with non - phagocytic human cells in figure 2d1 . the uptake appears to be mediated via macropinocytosis , a nonspecific endocytic process , which involves the internalization of large areas of plasma membrane together with significant amounts of fluid ( figure 2d2 ) . irregular macropinosome vesicles larger than 1 m are usually generated when membrane protrusions fuse back to the plasma membrane ( si - figure 6a ) . other pathways could be involved , e.g. , lipid raft - dependent mechanisms or the clathrin - dependent pathway ; however , we did not observe any evidence supporting their involvement . further studies are needed to clarify the exact cellular internalization mechanism . however , and as expected once inside the cells , the fept nps that are stabilized by coulombic interactions appear to be confined in specific subcellular organelles , most likely endosomes and lysosomes ( figure 2d3 ) . this is of significant importance when developing fept nps - based biomedical platforms and can be attributed to the interplay between the cationic surface of the nps and the negatively charged cell membrane . by considering a family of fept nps , we established that , despite the attractiveness of the silica coating this may be the result of a reduction of the magnetic effective volume and/or an alteration of the material . in support of this interpretation , the strong base environment associated with the silica coating can partially oxidize the fept surface , resulting in a thin layer of softer magnetic material like iron oxide or iron silicide , which because of its thinness is not visible by xrd or tem . the fcc - fept - a nps demonstrated a very high mri relaxivity r2 , which can be attributed to the combination of ( i ) an intrinsically high magnetic moment , ( ii ) a thin organic coating , and ( iii ) their small size , resulting in effective dispersion across a sample . while this combination created very effective t2 mri contrast agents even at g / ml concentrations , these results also underline the importance of engineering the interface of nps and demonstrate that a strong magnetic moment such as that of fept , combined with a thin coating layer having an open molecular structure allowing very close aqueous proximity , are essential parameters for the design of new generations of diagnostic and therapeutic mri contrast agents . finally , we demonstrate that fept nps can be successfully used as a potent cellular and in vivo mri probes . this is illustrated by both cell mri ( figure 3b ) and distinct hypointensity of 1 mm in size observed in the in vivo environment into which the fept nps were injected ( figure 3d and si - figure 8).(43 ) their more than 6 times higher relaxivity r2 allows using less nps than with iron oxide contrast agents such as feridex , which makes fept nps very promising contrast agents to identify small lesions such as lymph node metastases . in conclusion , we have presented the synthesis and characterization of a family of fept nps : fcc - fept - a nps and fcc - fept - silica - a nps . the reduction of magnetic properties observed with both squid magnetometry and mri relaxivity of silica - coated nps is attributed to the formation of very thin layers of oxides and silicides . in sharp contrast with some previous reports , our biocompatibility studies clearly demonstrate and explain the absence of cytotoxicity at concentrations below 30 g / ml , even after 7 days of incubation . another striking feature for cellular imaging is the ability of the fept nps to efficiently enter tumor cells without requiring any external mediation . the cellular uptake by non - phagocytic tumor cells occurs via an apparent macropinocytosis mechanism . the 7.1 t mri studies , in vitro and in vivo , have confirmed that both fcc - fept - a and fcc - fept - silica - a nps are stronger t2 contrast agents than commercial feridex , with fcc - fept - a t2 relaxivity ( r2 ) being more than 6 times larger . the superior capability of mri contrast enhancement associated with the fept nps makes them an ideal platform for the design of diagnostic and therapeutic agents for image - guided magnetic drug delivery and hyperthermic tumor ablation . the findings of this study underline the importance of engineering magnetic np interfaces combining intrinsically strong magnetic moment with coating layers . finally , our studies have also demonstrated that fept nps can be used successfully for cellular and in vivo imaging , thus confirming the potential of fept nps in regenerative medicine and stem cell therapy .

ironplatinum alloy nanoparticles ( fept nps ) are extremely promising candidates for the next generation of contrast agents for magnetic resonance ( mr ) diagnostic imaging and mr - guided interventions , including hyperthermic ablation of solid cancers . fept has high curie temperature , saturation magnetic moment , magneto - crystalline anisotropy , and chemical stability . we describe the synthesis and characterization of a family of biocompatible fept nps suitable for biomedical applications , showing and discussing that fept nps can exhibit low cytotoxicity . the importance of engineering the interface of strongly magnetic nps using a coating allowing free aqueous permeation is demonstrated to be an essential parameter in the design of new generations of diagnostic and therapeutic mri contrast agents . we report effective cell internalization of fept nps and demonstrate that they can be used for cellular imaging and in vivo mri applications . this opens the way for several future applications of fept nps , including regenerative medicine and stem cell therapy in addition to enhanced mr diagnostic imaging .

Introduction Results Discussion Conclusion

nanoparticles ( nps ) have attracted considerable interest as diagnostic and therapeutic tools for biomedicine , with magnetic nps offering unique opportunities for magnetic separation , targeted drug delivery , and hyperthermic ablation of cancer and as contrast agents in magnetic resonance imaging ( mri ) . medical diagnosis requires enhanced contrast between normal and pathological tissues , resulting in the development of exogenous mri contrast agents. (12 ) common t2-agents include superparamagnetic nanoparticles ( fe3o4 , -fe2o3 , mnfe2o4 , feco ) , the clinical mri applications of which range from imaging lymph - node to liver and spleen , as well as bone marrow and gastrointestinal tract . there is therefore a need for the design of biocompatible mri contrast agents with prolonged intravascular retention , improved tissue delineation , high chemical stability , and improved selectivity when targeting a tissue of interest . silica is often considered as the most attractive coating for engineered nps , forming an inert and biocompatible outer shell . (7 ) an outer coating with functional organic molecules has many advantages , including steric and/or electrostatic repulsion between the nps , which can increase water solubility and stability in biological environment. (17 ) positively charged nps demonstrate increased cell internalization efficiency and biomedical molecules binding capability for multimodal imaging applications and drug delivery . characterized by high curie temperature and magneto - crystalline anisotropy , ironplatinum metallic alloy nps have emerged as promising candidates for a new generation of magnetic nanomaterials. (20 ) over the past decade , several chemical pathways have been developed to synthesize fept nps with controlled stoichiometry , size , and shape. the latter approach often relies on pt(acac)2 , fe(co)5 , or collman s regeant precursors mixed with alkyl amines and acids solubilized in high - boiling - point solvents heated at temperatures of 300 c and above to control the growth , composition , and morphology of the nps . studies have demonstrated the biomedical potential of fept nps,(6 ) such as in magnetic separation and hyperthermic ablation and as t2 mri contrast agents . in this article , we describe the fabrication of fept nps specifically designed to enhance their magnetic properties for biomedical applications . we demonstrate that , in contrast with previous reports , fept nps can be made nontoxic , and we provide the first data on their cellular uptake mechanisms . we report a 6-fold increase in the fept - based t2 contrast properties compared to those of clinical iron oxide nps . the relationship between the mri contrast properties and the nps architecture is explored and rationalized as the basis for the design of nps as enhanced mri contrast agents . finally , we report the first observations of cellular and in vivo mr imaging with fept nps . our results opens the way for several applications of fept nps , such as regenerative medicine and stem cell therapy , in addition to enhanced mr diagnostic imaging , thus providing a platform to develop novel diagnostic and therapeutic agents . figure 1 displays the schematic structure of a clinical iron oxide np coated with dextran ( feridex , figure 1a1 ) and two fept nps : the face - centered - cubic ( fcc ) fept np surface functionalized with cysteamine ( fcc - fept - a , figure 1b1 ) and the fcc fept np coated with silica and surface - functionalized with ( 3-aminopropyl)triethoxysilane ( fcc - fept - silica - a , figure 1c1 ) . in agreement with published literature,(42 ) transmission electron microscopy ( tem ) revealed that the commercial iron oxide nps samples ( figure 1a2 ) are composed of aggregated crystals approximately 5 nm in diameter , embedded in an organic coating . the powder x - ray diffraction ( xrd ) pattern of the feridex iron oxide confirms the presence of magnetite ( fe3o4 ) , the broad peaks at 38 , 45 , and 55 corresponding to the ( 220 ) , ( 311 ) , and ( 400 ) peaks , respectively ( figure 1a3 ) . for the silica - coated nps , the sio2 xrd characteristic peak the crystal size is 3.6 nm for both fcc - fept - a and fcc - fept - silica - a nps ( table 1 ) . dxrd is the crystalline grain size of the nps ; in fexpt1x , x is the composition based on the fept lattice constant ( si - figure 1);(43)tb is the blocking temperature ; ms is the saturation moment ; hc is the magnetic coercivity of the nps ; and r1 and r2 are the h relaxivity values for 1% agarose gel containing feridex , fcc - fept - a , and fcc - fept - silica - a nps . a range of other commercial mri contrast agent relaxivity values are presented in the supporting information ( si - table 2 ) . cytotoxicity of the nps was evaluated in an a375 m cell line using fcc - fept - a nps and mts assay with various concentrations and incubation times. as expected , and in contrast with bare iron oxide , feridex s dextran organic coating does not lead to any detectable cytotoxicity . similar results were obtained with fcc - fept - a nps and both mcf7 and u2os tumor cell lines ( si - figure 5a , b). photomicrographs ( magnification 40 ) of prussian blue - stained a375 m cells cultured on coverslips in cell media ( c1 ) , media containing 30 g / ml of fcc - fept - a nps ( c2 ) , and media containing 30 g / ml of feridex ( c3 ) after overnight incubation . to confirm these results , the chemical stability of fcc - fept - a nps in an acidic environment was investigated by incubation of fcc - fept - a in ph 4.8 buffer solutions . the concentration of fept nps incubated in buffer solution is 120 g / ml , to match the highest nps concentration used in the cytotoxicity study ( figure 2a ) . in a ph 4.8 phosphate buffered saline ( pbs ) , cysteamine - coated fept nps show excellent chemical stability , with only 0.6% fe , i.e. in these more extreme conditions ( figure 2b2 ) , more fe was released0.3 g / ml after 6 h and up to 3.3 g / ml after 7 days prussian blue staining experiments were carried out and showed that fept nps have greater stability compared to feridex ( figure 2c1c3). these results confirm that the cysteamine coating provides fcc - fept - a nps with high chemical stability . the intracellular localization route is endosome , early lysosome , and late lysosome , which are basic cell biology phenomena . both endosomes and lysosomes are confined subcellular organelles , and , as both expected and commonly observed in other nps systems , the fept nps appear typically entrapped inside these subcellular vesicles ( figure 2d3 ) . the effectiveness of fcc - fept - a and fcc - fept - silica - a as mri contrast agents was investigated by measuring the dependence of the longitudinal relaxation rate ( t1 ) and transverse relaxation rate ( t2 ) of 1% agarose gels. as expected at high magnetic field , the fept nps and feridex have a weak influence on the water h longitudinal relaxation rate ( table 1 and si - figure 7a ) . the relaxivity r2 of the fcc - fept - silica - a nps is 40% larger than that of feridex , which is attributed to the stronger magnetic moment of the fcc - fept - silica - a nps ( si - figure 7b ) at the mri magnetic field ( 7 t). the fept nps produce an observable change in image contrast at concentrations as low as 6 g / ml , which is well within the safety range indicated by the cytotoxicity data ( figure 2a ) . t2 ( s ) vs [ fe ] ( mm ) of the water in 1% w / v agarose gel containing ( blue ) fcc - fept - a , ( green ) fcc - fept - silica - a , and ( orange ) feridex ( a1 ) . h longitudinal relaxation time t2-weighted mri images of 1% agarose gel / water solution containing fcc - fept - a nps with [ fe ] = 0.00 mm ( a ) , 0.02 mm ( cfept = 6 g / ml ) ( b ) , and 0.04 mm ( cfept = 12 g / ml ) ( c ) ( a2 ) . t2-weighted cellular imaging by mri ( 3d spin echo , te = 30 ms , tr = 1 s ) of a375 m cells loaded with fcc - fept - a nps with a cell density of 10 10 cells / ml ( a ) , 100 10 cells / ml ( b ) , and 1000 10 cells / ml ( c ) ( b ) . all the mri measurements were completed at 7.1 t. the in vivo potential of fept nps as mri contrast agents was demonstrated by injecting nps in cell culture media into the eyes of day 4 quail embryos in ovo . however , when the injection medium contained 20 g / ml of fcc - fept - a nps , a very distinct hypointensity appears in the injected eye ( figure 3d2d3 ) . this thus demonstrates the capability of nontoxic fept nps as potent mri probes for cellular imaging , even at very low np concentrations . labeling the cells and these magnetic nps will allow , for instance , cell migration to be studied , with their biodistribution and behavior monitored in vivo , especially as we have demonstrated that the effect of contrast agents can be observed at non - cytotoxic concentrations lower than 30 g / ml . the major hurdle preventing the wider use of fept nps in biomedicine is their potential toxicity . in the literature , there are many inconsistencies in fept cytotoxicity , leaving the subject rather confused . a summary of the cytotoxicity data of fept nps is presented in table 2 . while it was reported that 24 h incubation of fept - cysteine nps had no significant toxicity at concentrations below 5 g / ml , these compounds resulted in 50% cell damage at 15.5 g / ml;(39 ) fept - fe3o4 nps showed no significant cell viability damage up to 3 days of incubation at 10 g / ml. (52 ) in this latter study , the high toxicity was attributed to fast fe release from phospholipids - coated fcc - fept nps . it can act as a catalyst in the fenton reaction , which generates a wide range of free radical species , including hydroxyl radicals . these are among the most reactive free radical species known and have the ability to react with a wide range of cellular constituents , including pyrimidine bases of dna , and also to trigger lipid peroxidation in cellular membranes , leading to cell death. (53 ) listed are nps structure , precursors , and coating ; cells stands for the cell line ; [ nps ] is the nanoparticle concentration ; tinc is the incubation time ; cell damage is that after incubation . (43 ) in contrast to previously published results , our study demonstrates that cysteamine - coated fcc - fept nps also have very high chemical stability in ph 4.8 pbs . this confirms that the low ph in the endosome / lysosome environment is not sufficient to dissolve the nps and that the presence of endogenous citrate or similar metallic chelates is instrumental to the nps degradation by binding to solubilized fe atoms . the chemical stability of the fcc - fept - a nps is also supported by the prussian blue staining experiments ( figure 2c1c3 ) . the high chemical stability of cysteamine - coated fept nps certainly contributes to the nps noteworthy low cytotoxicity . it is quite possible that differences in the nps preparation , surface coating , ligand exchange , and extraction protocols could account for the spread of the cytotoxicity data reported in the literature . in the present study , we used collman s reagent , na2fe(co)4 , to control the fept nps stoichiometry and to ensure the simultaneous presence of fe and pt species during the formation of fept alloy nps ; the use of fe(co)5 has been shown to form nps with a core and a shell rich in platinum and iron , respectively. (31 ) after ligand exchange , the extraction process was repeated up to six times to ensure removal of hazardous chemicals such as chloroform , oleic acid , and oleylamine , while the thiol group provides a strong bond between the cysteamine and the nps surface , preventing the ligands from becoming lethal . during the review of this publication , cysteamine - coated fept nps were reported to induce , after 24 h , very low cell damage on veron cells;(38 ) however , it is noticeable that the strong bonding to the surface of the nps , protecting them from the cells environment , can be contrasted with , for instance , a coating made with a bilayer structure of phospholipids and oleic acid / oleylamine. by doing so , the free ligands can alter the cell viability as well as the release of the np constituents as the surface of the particles is exposed to the cell , and the nps chemical integrity can be compromised further . (52 ) while the very high chemical stability of fcc - fept - a nps was demonstrated in this report , cytotoxicity is observed at high concentrations above 30 g / ml fept and long incubation times . this could be explained by contributions from ( i ) the very small release of fe , which has been found to be < 1% after 168 h and ( ii ) the high concentration of positive surface charges , which could also interact with the more negatively charged cell membranes and induce membrane disruption. (54 ) in addition , ligands with strong anchoring groups can reduce the toxicity , as illustrated in this publication , and it is noticeable that the present results were obtained with commercially available ligands ( i.e. in this context , the little consistency observed when considering fept nps cytotoxicity data available in the literature stresses that the measured toxicity should always be associated with the nanocolloids , i.e. it then appears that , to be able to compare cytotoxicity data in a meaningful way , one must to develop and use standardized approaches including the correlation between ( i ) the evolution of the nanocolloids integrity , relying on both the inorganic nps and the organic stabilizers , and ( ii ) the cell viability . it should be noticed that , in the context of apparently toxic nps , the nanocolloids solution stability could easily be measured by , for instance , time - resolved transmission monitored sedimentation experiments , as while the ligands come off the nps surface , the nps tend to aggregate and fall out of solution . the cellular internalization of feridex is poor due to the weak binding of the neutral dextran coating to the plasma membrane , which limits the capability of cell internalization by the fluid - phase endocytosis pathway . (44 ) in contrast , the amine - coated fept nps , fcc - fept - a and fcc - fept - silica - a , are efficiently internalized directly without requiring the use of any external mediation such as transfection agents or electroporation , illustrated with non - phagocytic human cells in figure 2d1 . the uptake appears to be mediated via macropinocytosis , a nonspecific endocytic process , which involves the internalization of large areas of plasma membrane together with significant amounts of fluid ( figure 2d2 ) . however , and as expected once inside the cells , the fept nps that are stabilized by coulombic interactions appear to be confined in specific subcellular organelles , most likely endosomes and lysosomes ( figure 2d3 ) . this is of significant importance when developing fept nps - based biomedical platforms and can be attributed to the interplay between the cationic surface of the nps and the negatively charged cell membrane . by considering a family of fept nps , we established that , despite the attractiveness of the silica coating this may be the result of a reduction of the magnetic effective volume and/or an alteration of the material . the fcc - fept - a nps demonstrated a very high mri relaxivity r2 , which can be attributed to the combination of ( i ) an intrinsically high magnetic moment , ( ii ) a thin organic coating , and ( iii ) their small size , resulting in effective dispersion across a sample . while this combination created very effective t2 mri contrast agents even at g / ml concentrations , these results also underline the importance of engineering the interface of nps and demonstrate that a strong magnetic moment such as that of fept , combined with a thin coating layer having an open molecular structure allowing very close aqueous proximity , are essential parameters for the design of new generations of diagnostic and therapeutic mri contrast agents . finally , we demonstrate that fept nps can be successfully used as a potent cellular and in vivo mri probes . this is illustrated by both cell mri ( figure 3b ) and distinct hypointensity of 1 mm in size observed in the in vivo environment into which the fept nps were injected ( figure 3d and si - figure 8). (43 ) their more than 6 times higher relaxivity r2 allows using less nps than with iron oxide contrast agents such as feridex , which makes fept nps very promising contrast agents to identify small lesions such as lymph node metastases . in conclusion , we have presented the synthesis and characterization of a family of fept nps : fcc - fept - a nps and fcc - fept - silica - a nps . another striking feature for cellular imaging is the ability of the fept nps to efficiently enter tumor cells without requiring any external mediation . the 7.1 t mri studies , in vitro and in vivo , have confirmed that both fcc - fept - a and fcc - fept - silica - a nps are stronger t2 contrast agents than commercial feridex , with fcc - fept - a t2 relaxivity ( r2 ) being more than 6 times larger . the superior capability of mri contrast enhancement associated with the fept nps makes them an ideal platform for the design of diagnostic and therapeutic agents for image - guided magnetic drug delivery and hyperthermic tumor ablation . the findings of this study underline the importance of engineering magnetic np interfaces combining intrinsically strong magnetic moment with coating layers . finally , our studies have also demonstrated that fept nps can be used successfully for cellular and in vivo imaging , thus confirming the potential of fept nps in regenerative medicine and stem cell therapy .

brain - derived neurotrophic factor ( bdnf ) is a member of the neurotrophin family , a group of structurally related polypeptide growth factors . the family also includes nerve growth factor ( ngf ) , neurotrophin-3 ( nt-3 ) , and neurotrophin-4 ( nt-4/5 ) . other neurotrophins have also been identified , nt-6 and nt-7 ; however , these likely do not exist in mammals [ 24 ] . neurotrophins activate one or more of the high - affinity tropomyosin - receptor kinase ( trk ) receptor family [ 1 , 5 , 6 ] , as well as the low - affinity p75 neurotrophin receptor ( p75ntr ) . neurotrophins direct growth and differentiation in the developing nervous system [ 1 , 3 , 6 , 8 ] . levels of the different neurotrophins relate in a predictable pattern to stages of embryonic development . infusion of bdnf into the adult brain promotes neurogenesis [ 9 , 10 ] , and dendritic spine reorganization in the rat hippocampal formation . bdnf gene transfection triggers dendritic and axonal branching in dentate gyrus ( dg ) granule cell cultures while mice with a targeted gene deletion show substantial impairment in normal cerebellar development , among other developmental and behavioral deficits . trk receptors precipitate the activation of many signaling cascades , including phospholipase c ( plc ) , phosphoinositide 3-kinase ( pi3k ) , and ras [ 14 , 15 ] , which are involved in different aspects of neuronal survival and neurite outgrowth [ 1719 ] . the p75ntr is implicated in both pro- and antitrophic processes , such as neurite outgrowth and apoptosis [ 14 , 21 , 22 ] . bdnf regulates synaptic transmission and activity - dependent plasticity [ 23 , 24 ] and promotes long - term potentiation ( ltp ) . bdnf exerts presynaptic effects in a retrograde or paracrine fashion [ 27 , 28 ] and postsynaptic effects by modulating nmda receptor subunit expression . experimental reductions in bdnr or trkb activity significantly impair ltp [ 25 , 30 , 31 ] . previous research has demonstrated that exercise increases hippocampal bdnf mrna [ 32 , 33 ] and protein [ 34 , 35 ] . the effect of exercise to augment bdnf activity depends on camp - response - element binding protein ( creb ) activation , as creb suppression mitigates this effect . increases in hippocampal bdnf mrna have been detected after a period of running as short as 6 hours , to as long as two months , signifying a sensitive yet sustainable effect . physical activity leads to greater activation of cellular signaling pathways associated with survival and neurogenesis , and can enhance hippocampal ltp induction , all of which strongly implicate bdnf in their effects . this review will describe how bdnf and neurotrophins are involved in nervous system development , the signal transduction mechanisms associated with neurotrophin activity , and the modulating role bdnf plays in synaptic plasticity and ltp . the influence of exercise on bdnf activity , especially in the hippocampus , and the relationship between bdnf and excitotoxicity will also be examined . bdnf and nt-4/5 bind to trkb receptors [ 41 , 42 ] , as does nt-3 to a lesser extent . when a ligand binds to a trk receptor ( see figure 1 ) , the receptor dimerizes and autophosphorylates tyrosine residues to yield docking sites for the src homology 2 domain - containing adapter protein ( shc ) and phospholipase c- ( plc- ) , which are coupled to intracellular signaling cascades such as ras , pi3k [ 45 , 46 ] , and , as mentioned , plc- [ 14 , 15 , 47 , 48 ] . shc regulates protein interactions by binding , with a high - affinity , tyrosine - phosphorylated sites , and in this way regulates intracellular signaling such as ras . once shc is docked with the receptor and bound to the adapter protein grb2 , ras promotes activation of the mitogen - activated protein kinase ( mapk)/extracellular signal regulated kinase ( erk ) cascade , as well as the pi3k cascade [ 3 , 15 ] . mapk / erk is essential for neurogenesis and promotes survival by two ways , by induction of prosurvival genes and inhibition of proapoptotic proteins . ras also suppresses apoptosis via pi3k ; pi3k activates akt , which sequesters pro - apoptotic proteins in the cytoplasm away from their transcriptional targets . the pi3k cascade can be activated two ways by trk receptors , through ras as well as through adapter proteins shc , grb2 , and grb2-associated binder-1 ( gab1 ) [ 15 , 45 ] . in some neurons , bdnf phosphorylates the insulin receptor substrate , which can activate pi3k as well . vaillant and colleagues demonstrated that inhibition of any portion of the ras - pi3k - akt pathway significantly reduced survival of sympathetic neurons in culture in the presence of ngf . the adapter proteins shc and plc- are phosphorylated once they dock with the trk receptor [ 3 , 14 , 47 ] . once activated , plc- then catalyzes the breakdown of lipids to inositol 1,4,5 triphosphate ( ip3 ) , which promotes the release of calcium from intracellular stores , and a subsequent increase in intracellular calcium concentration and diacylglycerol ( dag ) occurs . dag regulates protein kinase c ( pkc- ) , which may be required for the mapk / erk signal , and both dag and mapk / erk have been implicated in neurite outgrowth [ 18 , 19 ] . interestingly , ltp is inhibited when plc- signaling is blocked , which may be related to reduced ip3 formation and subsequent reduction in intracellular calcium release . the other neurotrophin - responsive receptor , the p75ntr , can bind to each factor and regulate the trk receptor affinity for its ligand . the trk receptors and the p75ntr are often present in close proximity on the cellular membrane . while trkb responds to nt-3 , nt-4/5 , and bdnf , the receptor selectively binds only bdnf when colocalized with p75ntr . the largely presynaptic p75ntr serves dual roles in that it modulates trk receptor binding and is involved in prolonged ras - mediated activation of erk and neurite outgrowth , though p75ntr - induced neurite outgrowth ceases after prolonged p75ntr activation . the p75ntr also activates c - jun n - terminal kinase ( jnk ) and causes apoptosis in a variety of neurons [ 14 , 21 , 22 ] . although all trk receptors are assumed to behave in much the same fashion , differences do exist . kaplan and miller demonstrated , using trkb receptors with mutations at different binding sites , that trkb receptors use both pi3k and mapk cascades for cell survival while trka receptors depend mainly on pi3k . this demonstrates some comparative flexibility for trkb receptors with regard to pro - survival function . importantly , two truncated forms of trkb receptors exist that lack tyrosine kinase function and bind bdnf to negatively modulate trkb activity . given that bdnf - associated signaling modulates neuron survival and apoptosis , much attention has been focused on the role of bdnf ( and other neurotrophins ) in development . indeed , the critical involvement of trk receptors in neuronal survival [ 16 , 5052 ] , neurogenesis , and neurite outgrowth [ 12 , 18 , 19 ] implicates bdnf in developmental processes . throughout development , neurotrophins influence and guide neuronal differentiation in the central nervous system ( cns ) [ 6 , 8 ] and peripheral nervous system ( pns ) [ 1 , 3 ] . barnab - heider and miller demonstrated that cortical progenitor cells express bdnf and nt-3 and their associated trkb and trkc receptors , and that these neurotrophins were essential to the survival of the progenitor cells . bdnf mrna expression is lowest in the brains of early rat embryos and increases into adulthood . it demonstrates a reciprocal relationship with nt-3 mrna , which is at the highest levels in early embryos . maisonpierre and colleagues characterized distribution of bdnf , nt-3 , and ngf mrna during rat development and found that the amount of all three dramatically increased between embryonic days 11 and 12 , with transcripts widely distributed by embryonic day 13 . this timing corresponds directly to the onset of robust neurogenesis in the peripheral and central nervous systems [ 60 , 61 ] . examined bdnf protein concentration during postnatal development in rats and found that levels in the septum , cerebellum , and hippocampus increased over time and then stabilized ( although hippocampal protein levels took much longer to plateau ) while hypothalamic bdnf protein levels increased for a period following birth and then decreased . kim et al . further investigated bdnf - immunoreactivity ( ir ) in the rat forebrain and upper brainstem during postnatal development . the investigators defined three groups of bdnf cells : those that are detected early , increase in number , and remain stable during adulthood ( e.g. , piriform cortex , cingulate gyrus ) , those that increase with age then decrease in adulthood ( e.g. , basolateral nucleus of the amygdala ) , and those that appear briefly before dramatic reductions in adulthood ( e.g. , substantia nigra , various hypothalamic nuclei ) . in the hippocampus , bdnf - ir cells appeared in the pyramidal layer of ca2 and ca3 early on ( postnatal day 7 ) and staining increased through 28 days following parturition but then decreased , with comparatively reduced cell staining detected in 56-day - old adults . the bdnf - ir cell decrease detected in ca2 and ca3 in adulthood was completely absent in colchicine - treated adults , which instead showed an increase in bdnf - ir cells in these areas , possibly reflecting increased accumulation of bdnf in the cell body associated with inhibited axonal transport . bdnf - ir fibers were detected in the stratum lucidum layer of ca2 and ca3 and the polymorph layer of the dg also early on during the intial 28-day postnatal period , and these bdnf - ir fibers actually continued to increase into adulthood . bdnf serves a unique role among neurotrophins in development , especially with regard to the cerebellum . an examination of the cerebella of mice with a targeted bdnf gene deletion ( bndf mice ) revealed that bdnf is essential for normal development and function of the cerebellar cortex . the study showed reduced trk activation in purkinje cell bodies and dendrites in the mutant mice , indicating a lack of redundancy among neurotrophins with regard to bdnf in these cells . in bndf mice , there was a dramatic increase in cell death among developing granule cells , impaired development of the layers of the cerebellar cortex , and irregular foliation in the middle and posterior cerebellum ; defects were apparent in the declival sulcus ( absent ) , the intercrural fissure ( absent ) , the prepyramidal fissure ( nearly absent ) , and uvular sulcus ( nearly absent ) . bndf is crucial to postnatal survival , as most bndf mice die shortly after birth , but some do survive for a month or more [ 64 , 65 ] . mice that lack bdnf demonstrate severe deficiencies in pns development , especially with regard to afferent neurons , but exhibit comparatively mild deficiencies in cns development [ 64 , 66 ] . bndf mice show an abnormal gait with uncoordinated movements , and their stance is substantially wider ( distance between left and right paws ) , despite their smaller size compared to wildtype mice . this evidence further implicates the importance of bdnf not just to neuron development in the pns but also to cerebellar development and function . intraventricular bdnf application encourages neurogenesis in several parts of the rat brain , such as striatum , septum , thalamus , and hypothalamus , and infusion directly into the hippocampus increases the number of granule cells in the dg . danzer and colleagues transfected hippocampal cells in culture with either bdnf or ngf ; dg granule cells exhibited considerable axonal and dendritic branching following bdnf , but not ngf transfection . this effect was abolished with the application of a trk receptor tyrosine kinase inhibitor , demonstrating that bdnf and trk signaling promote neurogenesis both within and outside of the context of development . the involvement of p75ntr in refining trk receptor selectivity may serve to modulate targeting of growing neurons ( see figure 2 ) , with neurotrophic factors acting as markers . target tissues for growing pns neurons ( e.g. , skeletal muscle ) express neurotrophins , and bdnf is released postsynaptically to influence cns neurons in a retrograde fashion [ 27 , 69 ] . in a study of rat sympathetic neurons that largely express ngf and trka receptors , bdnf activated p75ntr - mediated apoptosis in conditions with low available ngf . for example , if a predominantly ngf / trka - associated neuron were to grow to network with a predominantly bdnf / trkb - associated neuron , the growing neuron would quickly become apoptotic . postsynaptic bdnf release and activation of the presynaptic p75ntr , coupled with the lack of ngf / trka signaling , would reduce the survivability of the growing neuron . thus p75ntr serves a regulatory role with regard to trk receptor behavior and neuronal proliferation . in summary , neurotrophins influence neuronal differentiation and are important to neuronal survival [ 16 , 5052 ] . bdnf mrna is present in relatively low levels early in life and generally increases through to adulthood [ 59 , 62 ] , especially in certain hippocampal neurons . studies of mice lacking bdnf demonstrate how it is essential for proper pns [ 64 , 66 ] and cerebellar development . bdnf is directly involved in the regulation of synaptic transmission and activity - dependent synaptic plasticity by both pre- and postsynaptic mechanisms [ 1 , 23 ] ( see figure 3 ) . these mechanisms underlie the critical role of bdnf in ltp [ 2426 , 29 , 65 ] . in a study with cultured hippocampal and cerebellar granule cells , caldeira and colleagues demonstrated that bdnf increased the levels of nmda receptor subunits in the plasma membrane of hippocampal cells . they recorded a correlated increase in intracellular calcium concentration and described an increased calcium entry through the additional nmda receptors . interestingly , hartmann and colleagues stimulated cultured hippocampal neurons presynaptically and detected a postsynaptic bdnf release , dependent upon calcium influx , thereby suggesting a bdnf - dependent positive feedback loop between presynaptic and postsynaptic signaling in the generation of ltp . . found that bdnf is essential for activity - induced potentiation of presynaptic vesicle cycling in cultured embryonic neocortical neurons of bdnf - knockout mice . this phenomenon was dependent upon nmda receptor activation , and was significantly reduced in bdnf - deficient neurons , indicating a possible retrograde messenger or paracrine role for bdnf . in support of these findings , previous work in bdnf knockout mice showed a pronounced impairment of vesicle docking in hippocampal synapses while direct application of bdnf reversed the deficits . estrogen promotes hippocampal plasticity , and in ovariectomized rats , the loss of estrogen reduces cytoskeletal reorganization in hippocampal dendritic spines and produces deficits in ltp . kramr and colleagues recently demonstrated that application of bdnf to hippocampal sections restored spine actin polymerization and ltp stability in these rats . backpropagating ( retrograde ) action potentials modulate short- and long - term changes to synaptic activity and also serve to relieve the mg block on nmda receptors . the extent of backpropagation in a neuron is dependent upon dendritic morphology , ion channel distribution , and synaptic input . importantly , application of an endoplasmic reticulum ca - atpase inhibitor stops this dendritic bdnf release , suggesting a calcium - dependent mechanism for retrograde release . these findings thus reveal several mechanisms for bdnf - mediated promotion of ltp in the hippocampus . bdnf is released in an activity - dependent manner , as demonstrated in both hippocampal neurons and peripheral neurons . while it may not be the only mediator of ltp , mice lacking bdnf demonstrated reduced tetanus - induced ltp , a deficit that disappeared with the return of bdnf function . as described above , bdnf works through both pre- and postsynaptic mechanisms to support ltp , and a simultaneous block of pre- and postsynaptic plc- activity , but not either by itself , is required to reduce ltp in mouse hippocampal cultures . interestingly , the involvement of trkb in ltp is not completely dependent on bdnf signaling ; nmda receptors can activate trkb by way of zinc influx , and adenosine 2a receptors can also influence trkb . in brief , bdnf is involved in trafficking nmda receptor subunits to the membrane , thereby increasing the potential for calcium influx . a sufficient local buildup of calcium causes postsynaptic bdnf release , which then leads to an increase in presynaptic vesicle cycling . exercise leads to substantial changes in bdnf and nmda receptor activity in the hippocampus , and these changes in large part underlie the effects of exercise on learning and synaptic plasticity . in rats with free access to activity wheels for one to six weeks , increases in hippocampal bdnf mrna [ 32 , 33 ] and bdnf protein [ 34 , 35 ] have been detected . berchtold , castello , and cotman demonstrated that hippocampal bdnf protein remained elevated for two weeks following three weeks of access to cage wheels ( protein levels were 186% of controls at the end of exercise , 137% one week followup , and 133% at two weeks ) . physical activity increases hippocampal bdnf expression after a relatively short amount of time , as voluntary wheel running induced bdnf mrna after only 6 hours of running . a substantial increase in bdnf mrna in the dg has also been described after 2 weeks , 3 weeks , 4 weeks , and 2 months of voluntary running . bdnf mrna and protein induction due to exercise varies over the life - span , however . younger mice ( 2 months ) exhibit a larger increase compared to older mice ( 15 and 24 months ) over four weeks of running . forced treadmill running for 30 minutes per day for six weeks increased hippocampal bdnf protein and brdu positive proliferating cells in younger rats ( 5 months ) , an effect that was less robust but still significant in older rats ( 24 months ) . exercise influences bdnf even under conditions of homeostatic perturbations such as stress or dysfunction in energy metabolism . in chronically stressed sprague - dawley rats , access to cage wheels for six weeks only during dark cycle ( active period ) yielded increased levels of bdnf protein in the striatum compared to sedentary rats . the characteristic lower levels of hippocampal bdnf protein and dendritic spine density in type ii diabetic mice ( db / db ) were significantly enhanced with free access to cage wheels . under a condition of food deprivation every other day , six weeks of wheel running still yielded significantly increased hippocampal bdnf protein compared to normally fed and food - deprived sedentary wistar rats , similar in magnitude to the levels observed in normally fed exercising rats . running is associated with a robust activation of survival pathways and vesicular proteins , effects that are linked to bdnf . shen and colleagues showed prolonged hippocampal mapk signaling following only a week of voluntary running . chen and russo - neustadt demonstrated a significant increase in hippocampal pi3k protein in rats after two weeks of wheel running . in another study , chen , and russo - neustadt found a similar consequence of running on akt protein , an effect dependent upon creb signaling ; in fact , the researchers showed that exercise - induced increases in hippocampal bdnf protein and mrna are also creb activation dependent , indicating the essential nature of creb signaling to exercise and bdnf - dependent cellular survival effects . these pathways are likely directly associated with the neurogenesis detected in the dg of exercising rats [ 80 , 81 ] . voluntary exercise selectively increases hippocampal synapsin i and synaptophysin mrna [ 35 , 82 ] , a phenomenon blocked by a recombinant human trkb - igg chimera , a highly specific antagonist of bdnf . synapsin i tethers vesicles to the cytoskeleton while synaptophysin may modulate vesicle recycling during high - intensity activity . indeed , this bdnf - mediated increase in synapsin following exercise may in part explain how exercise enhances ltp , along with the other mechanisms for bdnf - facilitated ltp described above . voluntary running also yielded better performance on the morris water maze test , an ltp - associated effect abolished with the selective blocking of bdnf . importantly , farmer and colleagues also identified an increase in nmda receptor subunit nr2b mrna in the dg following activity wheel running , which may represent yet another mechanism explaining how exercise promotes ltp and learning and memory . a complicated relationship exists between bdnf and epileptogenic hyperexcitability , but this relationship may be fundamentally characterized as a positive feedback loop . in general , enhanced bdnf signaling is associated with increased excitability , presumably through increased synaptic plasticity . for example , mrna and protein levels for bdnf are increased in the hippocampus after seizure [ 66 , 87 ] , and bdnf knockout mice demonstrate reduced seizure behavior in response to kindling . in transgenic mice with an overexpression of the truncated trkb receptor , the reduced bdnf signaling led to reduced seizure activity after systemic kainic acid administration . . demonstrated development of hyperexcitable circuits due to abnormal mossy fiber sprouting , in part caused by activity - induced bdnf release and trk receptor activity . this sprouting did not occur in the presence of a trk receptor tyrosine kinase inhibitor or anti - bdnf antibody . such abnormal remodeling is , in part , responsible for dentate hyperexcitability [ 89 , 90 ] . glutamatergic excitotoxicity , thought to result from excessive calcium influx via nmda receptors , is a major contributor to neuronal damage associated with anoxia , ischemia , and seizure . in cultured cerebellar granule cells , high glutamate concentrations cause immediate necrosis . following withdrawal to normal medium , hippocampal cell cultures , a high concentration of glutamate caused mainly necrosis while a lower concentration of glutamate led almost exclusively to delayed apoptosis . glazner and mattzon evaluated the effect of bdnf application on nmda receptor subunits , and found that nr1 and nr2a protein levels are increased whereas nr2b levels are decreased ; associated effects included increased calcium response to nmda activation , increased necrosis ( cell swelling ) , and decreased apoptosis ( cell shrinkage and chromatin fragmentation ) . bdnf - mediated enhancement of nmda receptor functions are consistent with the effects of exercise on increasing nmda receptor expression described above , further supporting the role of bdnf in exercise - enhanced synaptic plasticity . these results further illustrate how bdnf overexpression may lead to hyperexcitable circuitry through multiple mechanisms . bdnf thus encourages neurogenesis [ 9 , 10 ] , axonal , and dendritic sprouting , and nmda receptor - mediated transmission in a trk - b - dependent manner , which are generally considered to be beneficial effects on neuronal function . however , these same mechanisms may lead to hyperexcitable circuits in the dg [ 89 , 90 , 97 ] , which may eventually lead to spreading excitotoxicity . this condition can therefore become a self - sustaining cycle of sprouting and hyperexcitability , fueled in part by activity - dependent bdnf release [ 69 , 73 , 74 ] . in a kainic acid model of temporal lobe epilepsy utilizing transgenic mice , increased trkb signaling facilitated significantly shorter time to epileptogenesis and more severe epileptiform electroencephalogram activity compared to wild - type mice , and epileptogenesis was delayed in reduced trkb ( truncated form ) mice . based on this evidence for a prolonged state of hyperexcitability coupled with a shorter facilitation resulting from enhanced expression of bdnf or trkb signaling in the hippocampus , it follows that exercise - induced plasticity could leave the brain more vulnerable to epileptic seizures and excitotoxicity . microinjections of kainic acid into the hippocampi of anaesthetized rats produce more damage in subjects that were provided with running wheels for several weeks compared to sedentary controls . this enhanced excitotoxicity in exercising rats was interpreted by the investigators as a consequence of exercise - induced upregulation of hippocampal bdnf expression . on the other hand , a large body of evidence from both clinical studies in humans and basic research in animal models reveals that physical exercise produces a wide range of neuroprotective effects [ 100103 ] . for example , exercise in humans decreases risk of neurological disease and stroke , and it may improve recovery after brain trauma . the brain damage caused by experimental infarct is diminished in exercising rats compared to sedentary controls . exercise also protects against oxidative stress , and the neurotoxic effects of 6-ohda [ 104 , 105 ] and mptp . since neural hyperexcitability is a mechanism for the injury and death that occurs in all of these neurological insults and/or disorders [ 107 , 108 ] , determining how exercise protects against excitotoxicity should have broad implications for understanding its beneficial effects on the brain . nonetheless , these conditions are only indirectly linked to excitotoxicity , and a more direct examination of the influence of exercise on hyperexcitability is required to understand the mechanisms for its neuroprotective effects . in contrast to the findings of ramsden and colleagues described above , in which exercise led to increased excitotoxicty when kainic acid was injected into the hippocampus of anaesthetized rats , some evidence suggests that exercise may protect against hyperexcitability . experiments specifically designed to this hypothesis have revealed that exercise protects against kainic acid - induced seizures when studied in awake , freely behaving rats . reiss et al . reported that three weeks of voluntary exercise decreases the effects of kainic acid on seizure behaviors and hippocampal c - fos expression . further supporting the hypothesis that exercise diminishes hyperexcitability are similar reports of exercise protecting against experimentally induced seizures using other convulsant manipulations [ 110 , 111 ] . exercise also diminishes the excitotoxic effects of systemic domoic acid in mice and reduces the learning deficits caused by systemic kainic acid administration in rats . taken together , the conflicting findings of exercise effects on hyperexcitability and excitotoxicity suggest that an exercise - induced compensatory mechanism may counter the hyperexcitable state associated with upregulation of bdnf . importantly , this compensatory mechanism may only function in awake , freely behaving subjects . in the report by ramsden and colleagues , the exercise - associated increase in neurotoxicity caused by kainic acid injected directly into the hippocampi if such a mechanism depended on extrahippocampal circuitry , then it is reasonable to assume that it may not engage in an anaesthetized state . though it is likely that a variety of mechanisms exist to counter the hyperexcitable state in the hippocampus caused by bdnf , elucidation of such a mechanism related to exercise should be based on specific criteria . this mechanism should involve some neural factor that ( 1 ) is upregulated by exercise , ( 2 ) diminishes neural hyperexcitability acutely , and ( 3 ) exerts latent neuroprotective effects . research from this laboratory suggests that hippocampal afferents originating from the noradrenergic locus coeruleus may be a critical component of this mechanism . more specifically , experimental evidence points to the neuropeptide galanin as a critical factor in dampening hippocampal excitability . galanin is a 29 - 30 amino acid neurotransmitter and putative trophic factor that regulates neural activity in several brain structures , most notably the hypothalamus and hippocampus . retrograde tracing / double labeling experiments reveal that the hippocampus receives galaninergic innervation via projections from the locus coeruleus . at least three g - protein coupled galanin receptor subtypes have been confirmed , designated gal r1 - 3 . galaninergic transmission is predominantly inhibitory , producing hyperpolarization through increased k or decreased ca conductance . several lines of evidence reveal that galanin functions as an endogenous neuroprotective factor for hippocampal neurons [ 116118 ] . infusion of galanin into the hippocampus inhibits seizures provoked by a variety of methods , and the galanin receptor antagonists m-35- and m-40 block the antiseizure effects of galanin . transgenic galanin knock - out mice show increased susceptibility to kainic acid - induced seizures whereas transgenic mice overexpressing galanin display a resistance to these seizures . hippocampal electrophysiological activity is similarly exaggerated in the galanin knockouts and decreased in galanin overexpressers . in vitro studies in hippocampal cultures from transgenic mice previous work from this laboratory has demonstrated that either three weeks of voluntary wheel running or treadmill training increases galanin gene expression in locus coeruleus neurons in rats [ 120123 ] . no exercise - induced changes in neuropeptide - y or tyrosine hydroxylase were observed in these studies , suggesting that locus coeruleus neurons respond to exercise by specifically upregulating galanin . the critical role of galanin in mediating the protective effects of exercise against convulsant - induced seizures was demonstrated by the administration of the galanin receptor antagonist m-40 , which largely blocked the neuroprotection as measured by behavioral seizure severity . these results suggest that exercise - induced upregulation of galanin mediates the enhanced inhibitory tone that may counteract bndf - mediated hyperexcitability . to conclude , bdnf receptor activity encourages neurogenesis [ 9 , 10 ] , suppresses apoptosis , and modulates synaptic activity via a variety of signaling cascades [ 45 , 48 , 50 ] . these effects are beneficial with regard to development [ 1 , 8 ] and synaptic plasticity [ 25 , 26 , 29 , 65 ] , but can be harmful in conditions sensitive to hyperexcitability . dendritic bdnf release is activity - dependent , based on calcium influx [ 27 , 69 ] . this release causes an increase in presynaptic vesicle cycling , which further stimulates synaptic activity . the bdnf - induced increase in nmda receptor subunits creates more opportunity for calcium influx , supporting additional bdnf release . the increased trkb signaling boosts ip3 activity and intracellular calcium release , contributing to the enhancement of the cycle even further . these processes feed off of one another to overwhelm any modulatory effects of truncated trkb receptors ( which bind bdnf but lack tyrosine kinase function ) or p75ntr , the latter of which will act more to refine the actions of the increased available bdnf to yet further potentiate the cycle . since bdnf encourages neurogenesis [ 9 , 10 ] and neurite outgrowth , the effects of these processes are further compounded with the establishment of additional excitatory synapses ( see figure 4 ) . thus hyperexcitable circuits in the dg can develop due to excessive bdnf and nmda receptor activity [ 89 , 90 ] , activity that maintains a self - supporting cycle of neurotransmitter release in the hippocampus that can be overwhelmed to promote excitotoxic vulnerability . considering the maladaptive nature of this condition , selection for hippocampal circuitry that regulates this hyperexcitability would provide clear evolutionary benefits . furthermore , environmental stimuli that promote hippocampal bdnf expression , such as exercise , would be expected to engage this compensatory mechanism . evidence suggests that a circuit including galaninergic projections from the lc to the hippocampus provides this function [ 116 , 118 , 120 , 121 , 123 ] .

the present paper examines the nature and function of brain - derived neurotrophic factor ( bdnf ) in the hippocampal formation and the consequences of changes in its expression . the paper focuses on literature describing the role of bdnf in hippocampal development and neuroplasticity . bdnf expression is highly sensitive to developmental and environmental factors , and increased bdnf signaling enhances neurogenesis , neurite sprouting , electrophysiological activity , and other processes reflective of a general enhancement of hippocampal function . such increases in activity may mediate beneficial effects such as enhanced learning and memory . however , the increased activity also comes at a cost : bdnf plasticity renders the hippocampus more vulnerable to hyperexcitability and/or excitotoxic damage . exercise dramatically increases hippocampal bdnf levels and produces behavioral effects consistent with this phenomenon . in analyzing the literature regarding exercise - induced regulation of bdnf , this paper provides a theoretical model for how the potentially deleterious consequences of bdnf plasticity may be modulated by other endogenous factors . the peptide galanin may play such a role by regulating hippocampal excitability .

1. Brain-Derived Neurotrophic Factor Overview 2. Signal Transduction 3. Development 4. Synaptic Plasticity 5. Exercise 6. Excitotoxicity 7. Summary and Conclusion

brain - derived neurotrophic factor ( bdnf ) is a member of the neurotrophin family , a group of structurally related polypeptide growth factors . the family also includes nerve growth factor ( ngf ) , neurotrophin-3 ( nt-3 ) , and neurotrophin-4 ( nt-4/5 ) . other neurotrophins have also been identified , nt-6 and nt-7 ; however , these likely do not exist in mammals [ 24 ] . infusion of bdnf into the adult brain promotes neurogenesis [ 9 , 10 ] , and dendritic spine reorganization in the rat hippocampal formation . trk receptors precipitate the activation of many signaling cascades , including phospholipase c ( plc ) , phosphoinositide 3-kinase ( pi3k ) , and ras [ 14 , 15 ] , which are involved in different aspects of neuronal survival and neurite outgrowth [ 1719 ] . the p75ntr is implicated in both pro- and antitrophic processes , such as neurite outgrowth and apoptosis [ 14 , 21 , 22 ] . previous research has demonstrated that exercise increases hippocampal bdnf mrna [ 32 , 33 ] and protein [ 34 , 35 ] . increases in hippocampal bdnf mrna have been detected after a period of running as short as 6 hours , to as long as two months , signifying a sensitive yet sustainable effect . physical activity leads to greater activation of cellular signaling pathways associated with survival and neurogenesis , and can enhance hippocampal ltp induction , all of which strongly implicate bdnf in their effects . this review will describe how bdnf and neurotrophins are involved in nervous system development , the signal transduction mechanisms associated with neurotrophin activity , and the modulating role bdnf plays in synaptic plasticity and ltp . the influence of exercise on bdnf activity , especially in the hippocampus , and the relationship between bdnf and excitotoxicity will also be examined . when a ligand binds to a trk receptor ( see figure 1 ) , the receptor dimerizes and autophosphorylates tyrosine residues to yield docking sites for the src homology 2 domain - containing adapter protein ( shc ) and phospholipase c- ( plc- ) , which are coupled to intracellular signaling cascades such as ras , pi3k [ 45 , 46 ] , and , as mentioned , plc- [ 14 , 15 , 47 , 48 ] . shc regulates protein interactions by binding , with a high - affinity , tyrosine - phosphorylated sites , and in this way regulates intracellular signaling such as ras . ras also suppresses apoptosis via pi3k ; pi3k activates akt , which sequesters pro - apoptotic proteins in the cytoplasm away from their transcriptional targets . vaillant and colleagues demonstrated that inhibition of any portion of the ras - pi3k - akt pathway significantly reduced survival of sympathetic neurons in culture in the presence of ngf . dag regulates protein kinase c ( pkc- ) , which may be required for the mapk / erk signal , and both dag and mapk / erk have been implicated in neurite outgrowth [ 18 , 19 ] . interestingly , ltp is inhibited when plc- signaling is blocked , which may be related to reduced ip3 formation and subsequent reduction in intracellular calcium release . the other neurotrophin - responsive receptor , the p75ntr , can bind to each factor and regulate the trk receptor affinity for its ligand . while trkb responds to nt-3 , nt-4/5 , and bdnf , the receptor selectively binds only bdnf when colocalized with p75ntr . the largely presynaptic p75ntr serves dual roles in that it modulates trk receptor binding and is involved in prolonged ras - mediated activation of erk and neurite outgrowth , though p75ntr - induced neurite outgrowth ceases after prolonged p75ntr activation . given that bdnf - associated signaling modulates neuron survival and apoptosis , much attention has been focused on the role of bdnf ( and other neurotrophins ) in development . indeed , the critical involvement of trk receptors in neuronal survival [ 16 , 5052 ] , neurogenesis , and neurite outgrowth [ 12 , 18 , 19 ] implicates bdnf in developmental processes . barnab - heider and miller demonstrated that cortical progenitor cells express bdnf and nt-3 and their associated trkb and trkc receptors , and that these neurotrophins were essential to the survival of the progenitor cells . bdnf mrna expression is lowest in the brains of early rat embryos and increases into adulthood . maisonpierre and colleagues characterized distribution of bdnf , nt-3 , and ngf mrna during rat development and found that the amount of all three dramatically increased between embryonic days 11 and 12 , with transcripts widely distributed by embryonic day 13 . this timing corresponds directly to the onset of robust neurogenesis in the peripheral and central nervous systems [ 60 , 61 ] . examined bdnf protein concentration during postnatal development in rats and found that levels in the septum , cerebellum , and hippocampus increased over time and then stabilized ( although hippocampal protein levels took much longer to plateau ) while hypothalamic bdnf protein levels increased for a period following birth and then decreased . further investigated bdnf - immunoreactivity ( ir ) in the rat forebrain and upper brainstem during postnatal development . the investigators defined three groups of bdnf cells : those that are detected early , increase in number , and remain stable during adulthood ( e.g. in the hippocampus , bdnf - ir cells appeared in the pyramidal layer of ca2 and ca3 early on ( postnatal day 7 ) and staining increased through 28 days following parturition but then decreased , with comparatively reduced cell staining detected in 56-day - old adults . the bdnf - ir cell decrease detected in ca2 and ca3 in adulthood was completely absent in colchicine - treated adults , which instead showed an increase in bdnf - ir cells in these areas , possibly reflecting increased accumulation of bdnf in the cell body associated with inhibited axonal transport . bdnf - ir fibers were detected in the stratum lucidum layer of ca2 and ca3 and the polymorph layer of the dg also early on during the intial 28-day postnatal period , and these bdnf - ir fibers actually continued to increase into adulthood . an examination of the cerebella of mice with a targeted bdnf gene deletion ( bndf mice ) revealed that bdnf is essential for normal development and function of the cerebellar cortex . the study showed reduced trk activation in purkinje cell bodies and dendrites in the mutant mice , indicating a lack of redundancy among neurotrophins with regard to bdnf in these cells . in bndf mice , there was a dramatic increase in cell death among developing granule cells , impaired development of the layers of the cerebellar cortex , and irregular foliation in the middle and posterior cerebellum ; defects were apparent in the declival sulcus ( absent ) , the intercrural fissure ( absent ) , the prepyramidal fissure ( nearly absent ) , and uvular sulcus ( nearly absent ) . this evidence further implicates the importance of bdnf not just to neuron development in the pns but also to cerebellar development and function . intraventricular bdnf application encourages neurogenesis in several parts of the rat brain , such as striatum , septum , thalamus , and hypothalamus , and infusion directly into the hippocampus increases the number of granule cells in the dg . this effect was abolished with the application of a trk receptor tyrosine kinase inhibitor , demonstrating that bdnf and trk signaling promote neurogenesis both within and outside of the context of development . bdnf is directly involved in the regulation of synaptic transmission and activity - dependent synaptic plasticity by both pre- and postsynaptic mechanisms [ 1 , 23 ] ( see figure 3 ) . these mechanisms underlie the critical role of bdnf in ltp [ 2426 , 29 , 65 ] . in a study with cultured hippocampal and cerebellar granule cells , caldeira and colleagues demonstrated that bdnf increased the levels of nmda receptor subunits in the plasma membrane of hippocampal cells . found that bdnf is essential for activity - induced potentiation of presynaptic vesicle cycling in cultured embryonic neocortical neurons of bdnf - knockout mice . this phenomenon was dependent upon nmda receptor activation , and was significantly reduced in bdnf - deficient neurons , indicating a possible retrograde messenger or paracrine role for bdnf . in support of these findings , previous work in bdnf knockout mice showed a pronounced impairment of vesicle docking in hippocampal synapses while direct application of bdnf reversed the deficits . estrogen promotes hippocampal plasticity , and in ovariectomized rats , the loss of estrogen reduces cytoskeletal reorganization in hippocampal dendritic spines and produces deficits in ltp . kramr and colleagues recently demonstrated that application of bdnf to hippocampal sections restored spine actin polymerization and ltp stability in these rats . these findings thus reveal several mechanisms for bdnf - mediated promotion of ltp in the hippocampus . while it may not be the only mediator of ltp , mice lacking bdnf demonstrated reduced tetanus - induced ltp , a deficit that disappeared with the return of bdnf function . as described above , bdnf works through both pre- and postsynaptic mechanisms to support ltp , and a simultaneous block of pre- and postsynaptic plc- activity , but not either by itself , is required to reduce ltp in mouse hippocampal cultures . interestingly , the involvement of trkb in ltp is not completely dependent on bdnf signaling ; nmda receptors can activate trkb by way of zinc influx , and adenosine 2a receptors can also influence trkb . exercise leads to substantial changes in bdnf and nmda receptor activity in the hippocampus , and these changes in large part underlie the effects of exercise on learning and synaptic plasticity . in rats with free access to activity wheels for one to six weeks , increases in hippocampal bdnf mrna [ 32 , 33 ] and bdnf protein [ 34 , 35 ] have been detected . berchtold , castello , and cotman demonstrated that hippocampal bdnf protein remained elevated for two weeks following three weeks of access to cage wheels ( protein levels were 186% of controls at the end of exercise , 137% one week followup , and 133% at two weeks ) . physical activity increases hippocampal bdnf expression after a relatively short amount of time , as voluntary wheel running induced bdnf mrna after only 6 hours of running . a substantial increase in bdnf mrna in the dg has also been described after 2 weeks , 3 weeks , 4 weeks , and 2 months of voluntary running . in chronically stressed sprague - dawley rats , access to cage wheels for six weeks only during dark cycle ( active period ) yielded increased levels of bdnf protein in the striatum compared to sedentary rats . the characteristic lower levels of hippocampal bdnf protein and dendritic spine density in type ii diabetic mice ( db / db ) were significantly enhanced with free access to cage wheels . chen and russo - neustadt demonstrated a significant increase in hippocampal pi3k protein in rats after two weeks of wheel running . in another study , chen , and russo - neustadt found a similar consequence of running on akt protein , an effect dependent upon creb signaling ; in fact , the researchers showed that exercise - induced increases in hippocampal bdnf protein and mrna are also creb activation dependent , indicating the essential nature of creb signaling to exercise and bdnf - dependent cellular survival effects . voluntary exercise selectively increases hippocampal synapsin i and synaptophysin mrna [ 35 , 82 ] , a phenomenon blocked by a recombinant human trkb - igg chimera , a highly specific antagonist of bdnf . indeed , this bdnf - mediated increase in synapsin following exercise may in part explain how exercise enhances ltp , along with the other mechanisms for bdnf - facilitated ltp described above . voluntary running also yielded better performance on the morris water maze test , an ltp - associated effect abolished with the selective blocking of bdnf . importantly , farmer and colleagues also identified an increase in nmda receptor subunit nr2b mrna in the dg following activity wheel running , which may represent yet another mechanism explaining how exercise promotes ltp and learning and memory . in general , enhanced bdnf signaling is associated with increased excitability , presumably through increased synaptic plasticity . for example , mrna and protein levels for bdnf are increased in the hippocampus after seizure [ 66 , 87 ] , and bdnf knockout mice demonstrate reduced seizure behavior in response to kindling . in transgenic mice with an overexpression of the truncated trkb receptor , the reduced bdnf signaling led to reduced seizure activity after systemic kainic acid administration . demonstrated development of hyperexcitable circuits due to abnormal mossy fiber sprouting , in part caused by activity - induced bdnf release and trk receptor activity . this sprouting did not occur in the presence of a trk receptor tyrosine kinase inhibitor or anti - bdnf antibody . glazner and mattzon evaluated the effect of bdnf application on nmda receptor subunits , and found that nr1 and nr2a protein levels are increased whereas nr2b levels are decreased ; associated effects included increased calcium response to nmda activation , increased necrosis ( cell swelling ) , and decreased apoptosis ( cell shrinkage and chromatin fragmentation ) . bdnf - mediated enhancement of nmda receptor functions are consistent with the effects of exercise on increasing nmda receptor expression described above , further supporting the role of bdnf in exercise - enhanced synaptic plasticity . bdnf thus encourages neurogenesis [ 9 , 10 ] , axonal , and dendritic sprouting , and nmda receptor - mediated transmission in a trk - b - dependent manner , which are generally considered to be beneficial effects on neuronal function . however , these same mechanisms may lead to hyperexcitable circuits in the dg [ 89 , 90 , 97 ] , which may eventually lead to spreading excitotoxicity . based on this evidence for a prolonged state of hyperexcitability coupled with a shorter facilitation resulting from enhanced expression of bdnf or trkb signaling in the hippocampus , it follows that exercise - induced plasticity could leave the brain more vulnerable to epileptic seizures and excitotoxicity . this enhanced excitotoxicity in exercising rats was interpreted by the investigators as a consequence of exercise - induced upregulation of hippocampal bdnf expression . exercise also protects against oxidative stress , and the neurotoxic effects of 6-ohda [ 104 , 105 ] and mptp . nonetheless , these conditions are only indirectly linked to excitotoxicity , and a more direct examination of the influence of exercise on hyperexcitability is required to understand the mechanisms for its neuroprotective effects . taken together , the conflicting findings of exercise effects on hyperexcitability and excitotoxicity suggest that an exercise - induced compensatory mechanism may counter the hyperexcitable state associated with upregulation of bdnf . importantly , this compensatory mechanism may only function in awake , freely behaving subjects . in the report by ramsden and colleagues , the exercise - associated increase in neurotoxicity caused by kainic acid injected directly into the hippocampi if such a mechanism depended on extrahippocampal circuitry , then it is reasonable to assume that it may not engage in an anaesthetized state . though it is likely that a variety of mechanisms exist to counter the hyperexcitable state in the hippocampus caused by bdnf , elucidation of such a mechanism related to exercise should be based on specific criteria . research from this laboratory suggests that hippocampal afferents originating from the noradrenergic locus coeruleus may be a critical component of this mechanism . infusion of galanin into the hippocampus inhibits seizures provoked by a variety of methods , and the galanin receptor antagonists m-35- and m-40 block the antiseizure effects of galanin . transgenic galanin knock - out mice show increased susceptibility to kainic acid - induced seizures whereas transgenic mice overexpressing galanin display a resistance to these seizures . hippocampal electrophysiological activity is similarly exaggerated in the galanin knockouts and decreased in galanin overexpressers . in vitro studies in hippocampal cultures from transgenic mice previous work from this laboratory has demonstrated that either three weeks of voluntary wheel running or treadmill training increases galanin gene expression in locus coeruleus neurons in rats [ 120123 ] . no exercise - induced changes in neuropeptide - y or tyrosine hydroxylase were observed in these studies , suggesting that locus coeruleus neurons respond to exercise by specifically upregulating galanin . the critical role of galanin in mediating the protective effects of exercise against convulsant - induced seizures was demonstrated by the administration of the galanin receptor antagonist m-40 , which largely blocked the neuroprotection as measured by behavioral seizure severity . these results suggest that exercise - induced upregulation of galanin mediates the enhanced inhibitory tone that may counteract bndf - mediated hyperexcitability . to conclude , bdnf receptor activity encourages neurogenesis [ 9 , 10 ] , suppresses apoptosis , and modulates synaptic activity via a variety of signaling cascades [ 45 , 48 , 50 ] . these effects are beneficial with regard to development [ 1 , 8 ] and synaptic plasticity [ 25 , 26 , 29 , 65 ] , but can be harmful in conditions sensitive to hyperexcitability . the increased trkb signaling boosts ip3 activity and intracellular calcium release , contributing to the enhancement of the cycle even further . these processes feed off of one another to overwhelm any modulatory effects of truncated trkb receptors ( which bind bdnf but lack tyrosine kinase function ) or p75ntr , the latter of which will act more to refine the actions of the increased available bdnf to yet further potentiate the cycle . thus hyperexcitable circuits in the dg can develop due to excessive bdnf and nmda receptor activity [ 89 , 90 ] , activity that maintains a self - supporting cycle of neurotransmitter release in the hippocampus that can be overwhelmed to promote excitotoxic vulnerability . furthermore , environmental stimuli that promote hippocampal bdnf expression , such as exercise , would be expected to engage this compensatory mechanism . evidence suggests that a circuit including galaninergic projections from the lc to the hippocampus provides this function [ 116 , 118 , 120 , 121 , 123 ] .

cardiovascular disease ( cvd ) and diabetic nephropathy ( dn ) are the major causes of mortality in individuals with type 1 diabetes ( 13 ) , and a growing body of evidence from basic and clinical studies ( 47 ) supports the relationship between cvd and renal function in health and disease . cvd and dn have been proposed to be manifestations of the same underlying pathology and also exist as interrelated risk factors ( 8,9 ) . the increased mesangial matrix associated with dn is similar to the pathophysiology of coronary atherosclerosis ( 8) . while atherosclerosis tends to remain subclinical until adulthood in individuals with type 1 diabetes ( 10 ) , adolescents with type 1 diabetes demonstrate reduced peak exercise capacity and decreased cardiac function compared with nondiabetic adolescents ( 11 ) . although the mechanisms underlying the reduced peak exercise capacity are poorly understood , we have previously shown that insulin resistance and elevated ldl cholesterol ( ldl - c ) were associated with low peak volume of oxygen ( vo2peak ) ( 11 ) . elevated albumin - to - creatinine ratio ( acr ) and glomerular filtration rate ( gfr ) , which are early manifestations of dn , are also increasingly recognized in children and adolescents with type 1 diabetes ( 1,12 ) . increased gfr was shown to be associated with increased cardiovascular mortality in adults ( 13 ) . little is known , however , about the possible associations between early renal health and exercise capacity in adolescents with type 1 diabetes , and whether this relationship is independent of insulin sensitivity . accordingly , we sought to examine the associations between markers of renal health and peak exercise capacity in adolescents with type 1 diabetes . we hypothesized that cardiopulmonary fitness would be directly associated with acr and inversely related to estimated gfr ( egfr ) in adolescents with type 1 diabetes . pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements . control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado . sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels . screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) . type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement . the absence of diabetes was confirmed in control subjects by a 2-h , 75-g oral glucose tolerance test . inclusion criteria included tanner stage > 1 and sedentary status ( <3 h regular exercise / week ) to minimize pubertal and training effects . study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. , oral or inhaled steroids , metformin , thiazolidinediones , or atypical antipsychotic agents ) . pubertal development was assessed by a single pediatric endocrinologist using the criteria established by tanner and marshall for pubic hair and breast development ( 14 ) . all subsequent tests were performed after a 12-h fast , preceded by 3 days of restricted physical activity and a fixed - macronutrient , weight - maintenance diet ( 55% carbohydrates , 30% fat , 15% protein ) . participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study . the study was approved by the university of colorado denver institutional review board , and appropriate consent and assent were obtained . a 3-day pediatric physical activity recall questionnaire was used to estimate habitual physical activity ( 11,15 ) , reported as a 3-d average of daily metabolic equivalents . for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise . arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise . the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio . subjects were excluded if the peak respiratory exchange ratio was 1.1 . to determine vo2peak in all participants , a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . , st . paul , mn ) while breathing into the mouthpiece of the metabolic cart ( medgraphics cpx / d ; medical graphics corp . ) . vo2peak was reported in milliliters per kilogram per minute and milliliters per lean kilogram determined from dexa scan per minute , as previously reported ( 11,15 ) . blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu m min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) . samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) . the cystatin c and creatinine samples were analyzed in batches , reducing the effects of temporal systemic shifts on the assays . serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2 ( serum creatinine/96 ) ( serum cystatin c/1.2 ) ( weight/45 ) ( age/14 ) ( 19 ) . this equation has shown to have the best performance in estimating gfr in adolescents without chronic kidney disease ( 19 ) . spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated . analyses were performed in sas ( version 9.3 for windows ; sas institute , cary , nc ) . differences between continuous parametric variables were examined with the t test , continuous nonparametric variables were examined with the wilcoxon signed rank test , and dichotomous variables were examined with the test . egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups . pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level . pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements . control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado . sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels . screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) . type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement . the absence of diabetes was confirmed in control subjects by a 2-h , 75-g oral glucose tolerance test . inclusion criteria included tanner stage > 1 and sedentary status ( <3 h regular exercise / week ) to minimize pubertal and training effects . study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. , oral or inhaled steroids , metformin , thiazolidinediones , or atypical antipsychotic agents ) . pubertal development was assessed by a single pediatric endocrinologist using the criteria established by tanner and marshall for pubic hair and breast development ( 14 ) . all subsequent tests were performed after a 12-h fast , preceded by 3 days of restricted physical activity and a fixed - macronutrient , weight - maintenance diet ( 55% carbohydrates , 30% fat , 15% protein ) . participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study . the study was approved by the university of colorado denver institutional review board , and appropriate consent and assent were obtained . a 3-day pediatric physical activity recall questionnaire was used to estimate habitual physical activity ( 11,15 ) , reported as a 3-d average of daily metabolic equivalents . measurements were made during bicycle ergometer testing using a metabolic cart . for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise . arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise . the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio . subjects were excluded if the peak respiratory exchange ratio was 1.1 . to determine vo2peak in all participants , a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . , st . paul , mn ) while breathing into the mouthpiece of the metabolic cart ( medgraphics cpx / d ; medical graphics corp . ) . vo2peak was reported in milliliters per kilogram per minute and milliliters per lean kilogram determined from dexa scan per minute , as previously reported ( 11,15 ) . blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu m min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) . samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) . the cystatin c and creatinine samples were analyzed in batches , reducing the effects of temporal systemic shifts on the assays . serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2 ( serum creatinine/96 ) ( serum cystatin c/1.2 ) ( weight/45 ) ( age/14 ) ( 19 ) . this equation has shown to have the best performance in estimating gfr in adolescents without chronic kidney disease ( 19 ) . spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated . analyses were performed in sas ( version 9.3 for windows ; sas institute , cary , nc ) . differences between continuous parametric variables were examined with the t test , continuous nonparametric variables were examined with the wilcoxon signed rank test , and dichotomous variables were examined with the test . egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr ( low < 5.50 , middle range 5.509.13 , and high > 9.13 mg / g ) were stratified into tertiles . anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups . pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level . adolescents with type 1 diabetes had reduced peak exercise capacity compared with nondiabetic control subjects ( vo2peak 31.5 6.3 vs. 36.2 7.9 ml / kg min , p = 0.046 ; vo2peak / lean kg 43.7 7.0 vs. 51.0 8.6 ml / lean kg min , p = 0.007 ) , despite similar age , tanner stage , bmi , and habitual level of physical activity . when adjusting for egfr , the differences in tanner stage and sex - adjusted means for vo2peak / kg ( lsms se 32.3 1.0 vs. 34.2 1.5 , p = 0.30 ) and vo2peak / lean kg ( lsms se 49.0 2.1 vs. 45.0 1.4 , p = 0.09 ) between adolescents with and without type 1 diabetes lost significance . characteristics for adolescents with type 1 diabetes data are reported as lsms se , unless otherwise indicated . data are reported as the median ( q25q75 ) . in adolescents with type 1 diabetes , egfr correlated strongly with vo2peak ( r = 0.55 , r = 30.22% , p = 0.002 ) and vo2peak / lean kg ( r = 0.44 , r = 19.58% , p = 0.02 ) , and remained significantly associated with vo2peak and vo2peak / lean kg after adjusting for sex and tanner stage ( tables 2 and 3 ) . additional adjustments for insulin sensitivity , hba1c level , sbp , or ldl - c level did not attenuate the significance of the associations among egfr , vo2peak , and vo2peak / lean kg ; however , the association between egfr and vo2peak / lean kg became less significant ( p = 0.048 ; table 3 ) . in contrast , the associations between egfr and vo2peak / kg and vo2peak / lean kg in adolescent control subjects were not significant in fully adjusted models ( data not shown ) . in contrast to egfr , lnacr did not correlate with vo2peak ( se 1.42 1.49 , p = 0.35 ) and vo2peak / lean kg ( se 2.24 1.71 , p = 0.20 ) when adjusted for sex and tanner stage . associations between egfr and vo2peak / kg data are reported as se ; p value . age was not adjusted for because it is part of the bouvet egfr equation . * adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . associations between egfr and vo2peak / lean kg data are reported as se ; p value . adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . when stratifying egfrs into tertiles , subjects in the highest egfr tertile had significantly lower vo2peak ( lsm se 26.91 1.52 vs. 35.53 1.88 , p = 0.002 ) and vo2peak / lean kg ( lsm se 39.65 2.07 vs. 46.18 2.55 , p = 0.048 ) adjusted for sex and tanner stage , compared with those in the lowest egfr tertile ( fig . 1 ) . stratifying acr into tertiles demonstrated no differences in vo2peak or vo2peak / lean kg among the three groups ( data not shown ) . tanner stage and sex - adjusted means for vo2peak / kg and vo2peak / lean kg stratified by tertiles of egfr . our major observation in this study was that in adolescents with type 1 diabetes , a disease characterized by reduced exercise capacity , egfr was independently and negatively associated with cardiopulmonary fitness , independent of insulin sensitivity and other important risk factors . we previously demonstrated reduced peak exercise capacity , and cardiac and vascular function in otherwise healthy , nonobese adolescents with type 1 diabetes , compared with well - matched nondiabetic control subjects of similar bmi , pubertal stage , and habitual level of physical activity ( 11 ) . furthermore , we found that insulin sensitivity correlated strongly with vo2peak in youths with type 1 diabetes , as did ldl - c level to a lesser degree ( 11 ) . in this report , we present for the first time an independent relationship between renal health and cardiopulmonary fitness that is independent of insulin sensitivity , ldl - c level , and other important covariates . this association may represent a novel pathway of a cardiorenal connection in type 1 diabetes separate from atherosclerosis . type 1 diabetes is associated with shortened life span , and cardiac and vascular dysfunction , independent of coronary artery disease ( 20 ) . in fact , low fitness levels in adults with and without diabetes are associated with cvd mortality and decreased longevity ( 21,22 ) . control of conventional risk factors such as glucose level , cholesterol level , and blood pressure is important , but does not abolish the cardiovascular risk in subjects with type 1 diabetes ( 2,23 ) . although not without controversy , the finnish diabetic nephropathy study ( 24 ) and the pittsburgh epidemiology of diabetes complications study ( 3 ) demonstrated no increase in mortality in adults with type 1 diabetes compared with nondiabetic control subjects in the absence of dn . in contrast , the coronary artery calcification in type 1 diabetes study ( 9 ) demonstrated strong associations among egfr , acr , and progression of coronary artery calcification in adults with type 1 diabetes in the absence of established dn , suggesting that renal health may be associated with early cardiovascular pathophysiology that may take years to manifest clinically . the specific mechanisms underlying the association between renal and cardiovascular health , however , remain unclear , but increasing evidence supports the importance of shared risk factors and pathogenic pathways , including atherosclerosis ( 6,7,11,2528 ) . reduced exercise capacity and its association with renal health highlighted in this article , taken together with the established relationship between fitness and premature cvd mortality , may provide an alternative and novel pathway explaining the premature mortality of subjects with type 1 diabetes ( 21,22 ) . there is currently no generally accepted definition for renal hyperfiltration ( 29 ) , but it is increasingly recognized that elevated gfr is an early hemodynamic abnormality seen in diabetes that is linked with an increased risk of dn ( 1 ) . the meta - analysis from the chronic kidney disease prognosis consortium ( 30,31 ) demonstrated significantly greater all - cause mortality and cardiovascular mortality in individuals with the highest gfr values ( > 105 ml / min/1.73 m ) . similarly , the renal iohexol clearance survey in tromso 6 ( renis - t6 ) recently reported ( 13 ) significantly greater odds of both carotid atherosclerosis and left ventricular hypertrophy in participants in the highest gfr quartile ( > 101.2 ml / min/1.73 m ) . it is , however , also plausible that the mechanisms underlying the association between renal health and exercise capacity in adolescents with type 1 diabetes differ from the link seen with atherosclerosis . the mechanisms responsible for the relationship between renal health and exercise capacity are not yet clear . adults with type 1 diabetes and elevated gfr demonstrate cardiovascular dysfunction , including increases in arterial stiffness and altered flow - mediated dilatation ( 32,33 ) . we previously demonstrated reduced forearm blood flow in youths with type 1 diabetes ( 11 ) as evidence of vascular dysfunction , which correlated with reduced vo2peak . moreover , alterations in arterial stiffness have also been reported to be present in youths with type 1 diabetes ( 34 ) . elevated gfr is associated with endothelial dysfunction via cyclooxygenase-2 activity and nitric oxide synthase inhibition , and relatively higher blood pressures ( 3537 ) , both of which could directly impact exercise capacity . elevated gfr is also associated with increased carotid intimal medial thickness and left ventricular hypertrophy in nondiabetic individuals , further suggesting that increased gfr potentially reflects a generalized change in vascular function rather than simply reflecting intrarenal abnormalities ( 13 ) . endothelial dysfunction could also contribute to elevated gfr , thus the relationships could be bidirectional . we did not observe a significant association between acr and peak exercise capacity , potentially because increasing evidence supports the distinction of albuminuria as a risk factor for dn and not an early phenotype of dn ( 38 ) . furthermore , the prevalence of microalbuminuria ( n = 9 ) was low due to the young age of our population . . however , to minimize the effect of sample size , we used careful and detailed physiological measurements , including gold standard fitness testing and hyperinsulinemic - euglycemic clamp studies . we also chose groups similar in habitual physical activity , pubertal stage , sex , and bmi , and controlled for prestudy diet and physical activity . another limitation to the current study included the cross - sectional design , which prevents the determination of causality , and whether the association holds true longitudinally ; for that reason , the data should be viewed as hypothesis generating . the direction of the association between renal health and exercise capacity ( i.e. , whether renal health leads to reduced exercise capacity or vice versa ) is also difficult to determine with the available data . results from this study may also not be generalizable to older people with type 1 diabetes or to cohorts with a different ethnic distribution . finally , we used an estimate of gfr to assess renal function ; however , to estimate the gfr we used the bouvet equation , which was shown to have the best performance in estimating gfr in children and adolescents with normal to elevated gfrs ( 18 ) . the mean egfr in our lean nondiabetic control subjects is slightly lower than gfrs measured by insulin level in adolescents in some studies ( 39 ) , but is consistent with gfrs estimated by creatinine and cystatin c levels in adolescents in large - scale studies ( 40 ) . in conclusion , in adolescents with type 1 diabetes , a disease characterized by reduced peak exercise capacity compared with nondiabetic adolescents , there was a strong relationship between egfr and cardiopulmonary fitness , independent of directly measured insulin sensitivity and other important risk factors . this observation provides further evidence for the broad interactions observed between renal and cardiovascular function in type 1 diabetes . further research is needed to investigate the longitudinal relationships between renal and cardiopulmonary health in adolescents with type 1 diabetes , and to elucidate the specific mechanisms underlying this relationship as potential therapeutic targets .

objectivediabetic nephropathy and cardiovascular disease are strongly related in adults with type 1 diabetes , yet little is known about this relationship in adolescents prior to the onset of detectable clinical disease . we hypothesized that cardiopulmonary fitness would be directly associated with albumin - to - creatinine ratio ( acr ) and inversely related to estimated glomerular filtration rate ( egfr ) in adolescents with type 1 diabetes.research design and methodssixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects of similar pubertal stage and bmi had insulin sensitivity ( glucose infusion rate [ gir ] ) , measured by hyperinsulinemic - euglycemic clamp , and lean body mass , measured by dexa . cardiopulmonary fitness was measured by cycle ergometry to obtain peak volume of oxygen ( vo2peak ) , and renal function was measured by egfr using the bouvet equation ( measuring creatinine and cystatin c levels ) and acr.resultsadolescents ( 15.5 2.2 years of age ) with type 1 diabetes ( 6.3 3.8 years diabetes duration ) had reduced vo2peak ( 31.5 6.3 vs. 36.2 7.9 ml / kg min , p = 0.046 ) and vo2peak / lean kg ( 43.7 7.0 vs. 51.0 8.6 ml / lean kg min , p = 0.007 ) compared with nondiabetic control subjects . egfr was inversely associated with vo2peak and vo2peak / lean kg after adjusting for sex , tanner stage , gir , hba1c level , systolic blood pressure , and ldl cholesterol level ( se , vo2peak : 0.19 0.07 , p = 0.02 ; vo2peak / lean kg : 0.19 0.09 , p = 0.048 ) . moreover , participants in the highest tertile for egfr had significantly lower sex- and tanner - adjusted vo2peak and vo2peak / lean kg compared with participants in the lowest tertile.conclusionsadolescents with type 1 diabetes had reduced exercise capacity , which was strongly associated with renal health , independent of insulin sensitivity . future studies should examine the underlying interrelated pathophysiology in order to identify probable targets for treatment to reduce cardiovascular and renal complications .

Introduction Research Design and Methods Subjects Activity Questionnaires and Body Composition Exercise Testing Insulin Sensitivity Renal Measurements Statistical Analysis Results Conclusions

cardiovascular disease ( cvd ) and diabetic nephropathy ( dn ) are the major causes of mortality in individuals with type 1 diabetes ( 13 ) , and a growing body of evidence from basic and clinical studies ( 47 ) supports the relationship between cvd and renal function in health and disease . while atherosclerosis tends to remain subclinical until adulthood in individuals with type 1 diabetes ( 10 ) , adolescents with type 1 diabetes demonstrate reduced peak exercise capacity and decreased cardiac function compared with nondiabetic adolescents ( 11 ) . although the mechanisms underlying the reduced peak exercise capacity are poorly understood , we have previously shown that insulin resistance and elevated ldl cholesterol ( ldl - c ) were associated with low peak volume of oxygen ( vo2peak ) ( 11 ) . elevated albumin - to - creatinine ratio ( acr ) and glomerular filtration rate ( gfr ) , which are early manifestations of dn , are also increasingly recognized in children and adolescents with type 1 diabetes ( 1,12 ) . little is known , however , about the possible associations between early renal health and exercise capacity in adolescents with type 1 diabetes , and whether this relationship is independent of insulin sensitivity . accordingly , we sought to examine the associations between markers of renal health and peak exercise capacity in adolescents with type 1 diabetes . we hypothesized that cardiopulmonary fitness would be directly associated with acr and inversely related to estimated gfr ( egfr ) in adolescents with type 1 diabetes . pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements . sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels . screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) . type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement . study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study . for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise . arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise . the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio . blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu m min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) . samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) . serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2 ( serum creatinine/96 ) ( serum cystatin c/1.2 ) ( weight/45 ) ( age/14 ) ( 19 ) . egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups . pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level . pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements . control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado . sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels . screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) . type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement . study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study . for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise . arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise . the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio . to determine vo2peak in all participants , a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu m min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) . samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) . serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2 ( serum creatinine/96 ) ( serum cystatin c/1.2 ) ( weight/45 ) ( age/14 ) ( 19 ) . spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated . egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr ( low < 5.50 , middle range 5.509.13 , and high > 9.13 mg / g ) were stratified into tertiles . anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups . pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level . adolescents with type 1 diabetes had reduced peak exercise capacity compared with nondiabetic control subjects ( vo2peak 31.5 6.3 vs. 36.2 7.9 ml / kg min , p = 0.046 ; vo2peak / lean kg 43.7 7.0 vs. 51.0 8.6 ml / lean kg min , p = 0.007 ) , despite similar age , tanner stage , bmi , and habitual level of physical activity . when adjusting for egfr , the differences in tanner stage and sex - adjusted means for vo2peak / kg ( lsms se 32.3 1.0 vs. 34.2 1.5 , p = 0.30 ) and vo2peak / lean kg ( lsms se 49.0 2.1 vs. 45.0 1.4 , p = 0.09 ) between adolescents with and without type 1 diabetes lost significance . characteristics for adolescents with type 1 diabetes data are reported as lsms se , unless otherwise indicated . in adolescents with type 1 diabetes , egfr correlated strongly with vo2peak ( r = 0.55 , r = 30.22% , p = 0.002 ) and vo2peak / lean kg ( r = 0.44 , r = 19.58% , p = 0.02 ) , and remained significantly associated with vo2peak and vo2peak / lean kg after adjusting for sex and tanner stage ( tables 2 and 3 ) . additional adjustments for insulin sensitivity , hba1c level , sbp , or ldl - c level did not attenuate the significance of the associations among egfr , vo2peak , and vo2peak / lean kg ; however , the association between egfr and vo2peak / lean kg became less significant ( p = 0.048 ; table 3 ) . in contrast , the associations between egfr and vo2peak / kg and vo2peak / lean kg in adolescent control subjects were not significant in fully adjusted models ( data not shown ) . in contrast to egfr , lnacr did not correlate with vo2peak ( se 1.42 1.49 , p = 0.35 ) and vo2peak / lean kg ( se 2.24 1.71 , p = 0.20 ) when adjusted for sex and tanner stage . associations between egfr and vo2peak / kg data are reported as se ; p value . * adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . associations between egfr and vo2peak / lean kg data are reported as se ; p value . adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . when stratifying egfrs into tertiles , subjects in the highest egfr tertile had significantly lower vo2peak ( lsm se 26.91 1.52 vs. 35.53 1.88 , p = 0.002 ) and vo2peak / lean kg ( lsm se 39.65 2.07 vs. 46.18 2.55 , p = 0.048 ) adjusted for sex and tanner stage , compared with those in the lowest egfr tertile ( fig . stratifying acr into tertiles demonstrated no differences in vo2peak or vo2peak / lean kg among the three groups ( data not shown ) . tanner stage and sex - adjusted means for vo2peak / kg and vo2peak / lean kg stratified by tertiles of egfr . our major observation in this study was that in adolescents with type 1 diabetes , a disease characterized by reduced exercise capacity , egfr was independently and negatively associated with cardiopulmonary fitness , independent of insulin sensitivity and other important risk factors . we previously demonstrated reduced peak exercise capacity , and cardiac and vascular function in otherwise healthy , nonobese adolescents with type 1 diabetes , compared with well - matched nondiabetic control subjects of similar bmi , pubertal stage , and habitual level of physical activity ( 11 ) . furthermore , we found that insulin sensitivity correlated strongly with vo2peak in youths with type 1 diabetes , as did ldl - c level to a lesser degree ( 11 ) . in this report , we present for the first time an independent relationship between renal health and cardiopulmonary fitness that is independent of insulin sensitivity , ldl - c level , and other important covariates . this association may represent a novel pathway of a cardiorenal connection in type 1 diabetes separate from atherosclerosis . type 1 diabetes is associated with shortened life span , and cardiac and vascular dysfunction , independent of coronary artery disease ( 20 ) . in fact , low fitness levels in adults with and without diabetes are associated with cvd mortality and decreased longevity ( 21,22 ) . control of conventional risk factors such as glucose level , cholesterol level , and blood pressure is important , but does not abolish the cardiovascular risk in subjects with type 1 diabetes ( 2,23 ) . although not without controversy , the finnish diabetic nephropathy study ( 24 ) and the pittsburgh epidemiology of diabetes complications study ( 3 ) demonstrated no increase in mortality in adults with type 1 diabetes compared with nondiabetic control subjects in the absence of dn . in contrast , the coronary artery calcification in type 1 diabetes study ( 9 ) demonstrated strong associations among egfr , acr , and progression of coronary artery calcification in adults with type 1 diabetes in the absence of established dn , suggesting that renal health may be associated with early cardiovascular pathophysiology that may take years to manifest clinically . reduced exercise capacity and its association with renal health highlighted in this article , taken together with the established relationship between fitness and premature cvd mortality , may provide an alternative and novel pathway explaining the premature mortality of subjects with type 1 diabetes ( 21,22 ) . the meta - analysis from the chronic kidney disease prognosis consortium ( 30,31 ) demonstrated significantly greater all - cause mortality and cardiovascular mortality in individuals with the highest gfr values ( > 105 ml / min/1.73 m ) . similarly , the renal iohexol clearance survey in tromso 6 ( renis - t6 ) recently reported ( 13 ) significantly greater odds of both carotid atherosclerosis and left ventricular hypertrophy in participants in the highest gfr quartile ( > 101.2 ml / min/1.73 m ) . it is , however , also plausible that the mechanisms underlying the association between renal health and exercise capacity in adolescents with type 1 diabetes differ from the link seen with atherosclerosis . the mechanisms responsible for the relationship between renal health and exercise capacity are not yet clear . adults with type 1 diabetes and elevated gfr demonstrate cardiovascular dysfunction , including increases in arterial stiffness and altered flow - mediated dilatation ( 32,33 ) . we previously demonstrated reduced forearm blood flow in youths with type 1 diabetes ( 11 ) as evidence of vascular dysfunction , which correlated with reduced vo2peak . moreover , alterations in arterial stiffness have also been reported to be present in youths with type 1 diabetes ( 34 ) . elevated gfr is associated with endothelial dysfunction via cyclooxygenase-2 activity and nitric oxide synthase inhibition , and relatively higher blood pressures ( 3537 ) , both of which could directly impact exercise capacity . however , to minimize the effect of sample size , we used careful and detailed physiological measurements , including gold standard fitness testing and hyperinsulinemic - euglycemic clamp studies . we also chose groups similar in habitual physical activity , pubertal stage , sex , and bmi , and controlled for prestudy diet and physical activity . another limitation to the current study included the cross - sectional design , which prevents the determination of causality , and whether the association holds true longitudinally ; for that reason , the data should be viewed as hypothesis generating . , whether renal health leads to reduced exercise capacity or vice versa ) is also difficult to determine with the available data . results from this study may also not be generalizable to older people with type 1 diabetes or to cohorts with a different ethnic distribution . finally , we used an estimate of gfr to assess renal function ; however , to estimate the gfr we used the bouvet equation , which was shown to have the best performance in estimating gfr in children and adolescents with normal to elevated gfrs ( 18 ) . the mean egfr in our lean nondiabetic control subjects is slightly lower than gfrs measured by insulin level in adolescents in some studies ( 39 ) , but is consistent with gfrs estimated by creatinine and cystatin c levels in adolescents in large - scale studies ( 40 ) . in conclusion , in adolescents with type 1 diabetes , a disease characterized by reduced peak exercise capacity compared with nondiabetic adolescents , there was a strong relationship between egfr and cardiopulmonary fitness , independent of directly measured insulin sensitivity and other important risk factors . this observation provides further evidence for the broad interactions observed between renal and cardiovascular function in type 1 diabetes . further research is needed to investigate the longitudinal relationships between renal and cardiopulmonary health in adolescents with type 1 diabetes , and to elucidate the specific mechanisms underlying this relationship as potential therapeutic targets .

the growth in the number of cancer patients , together with the development of new palliative care services in romania , warrants the evaluation of nursing strategies meant to improve the level of comfort of patients who are suffering from advanced cancer . pain is one of the most frequent symptoms in patients with advanced cancer , and the use of opioids is a standard practice with patients who are in pain . oral opioids are the gold standard but several eastern european countries have access only to injectable morphine as medicine to treat severe pain . to maximize comfort , instead of using intramuscular or intravenous route , the subcutaneous administration with an indwelling subcutaneous butterfly needle has been proved to be painless , efficient and easy to use . for countries with limited resources it is a useful method of administering parenteral medication as it has a lower cost than intravenous pumps and it also gives patients and families the possibility , with some training , to administer the drugs themselves . the main objective of this study was to evaluate the optimal positioning of the subcutaneous ( sc ) butterfly , in accordance with its resistance in the insertion tissue , the local complications that occur and the evaluation of the time of resistance at the insertion site ( puncture ) with the daily frequency of injectable opioid administration . patients admitted to the hospice casa sperantei with moderate or severe cancer pain and who were receiving subcutaneously opioids , over the age of 18 , with normal body index ranging from 18.522.0 , were assigned randomly , after signing the informed consent , to one of two groups . in group one , the butterfly was positioned with the needle bevel up this was considered to be the control group as this modality of inserting the needle is considered standard practice ; in group two the butterfly was positioned with the needle bevel down - experimental group . the drugs used for pain relief were sc tramadol for moderate pain and sc morphine for severe pain . data from patients were collected between january and may 2011 , in a sample of 100 adult cancer patients . correlations between the frequency of administration of opioids and the occurrence of local complications , and between the occurrence of local complications and the time of resistance of the subcutaneous butterfly at the insertion site , were established . the hypothesis is that there is a significant positive relation between the frequency of injectable opioid administration and the occurrence of local complications , respectively a negative relation between the occurrence of complications and the time of resistance of the subcutaneous butterfly at the insertion place . in addition , we wanted to establish which of the two butterfly insertion positions ( butterfly with the needle bevel up or down ) had a longer time of resistance at the insertion site with respect to incidence of complications . the study was conducted between january and may 2011 , on a sample of 100 adult cancer patients who were all outpatients of hospice casa sperantei . the distribution of the study group according to gender shows a predominance of male over female gender . this is consistent with accessibility to medical care and the lower availability of clinicians in the romanian health care system . there was no statistical difference in the composition of the control and study group concerning gender and place of residence . concerning the age of the patients in the study and control group there were significantly fewer patients in the age group of 7079 years ( p=0.05 ) in the control group compared with the study group . the patients included in the study had most frequently broncho - pulmonary cancer ( 19% ) , colon cancer ( 15% ) and breast cancer ( 13% ) . there was no significant difference between the study and control group in terms of type of cancer . in the 100 cancer patients , the main cause of pain was visceral 47 patients ( 47% ) , somatic pain 43 ( 43% ) , and only 10 presented with neuropathic pain ( 10% ) ; the patients with moderate pain represented approximately two - thirds of the total number of patients and one - third suffered severe pain ( table iii ) . there was an even distribution of patients in the experimental and control group concerning type and intensity of pain . in the sample studied , the two opioids , tramadol hydrochloride and morphine , were administrated in different proportions ; tramadol , as a weak opioid , was administered for moderate pain and was given in almost two - thirds of the patients during the study period ( see table iv ) . morphine , given through the subcutaneous route , was the drug used for patients who had severe pain . there was no significant difference between study and control group in terms of type of medication received for pain . the two analgesics had a different frequency in administration : 2 , 3 or 4 times per day for tramadol hydrochloride and 2 , 3 , 4 or 6 times per day for morphine . this can be explained through the different pharmacokinetics of the two opioids , tramadol having a longer half time compared with morphine , it was an expected result to see fewer administrations per day compared to morphine . there was no difference between study and control group concerning the frequency of administration of the drugs . the most frequent complication encountered when administering analgesics in patients with cancer was subcutaneous tissue indurations ( 56% for the whole sample ) followed by redness and bleeding . the complications encountered are presented in table vi . there were also significant differences between the possibility of the occurrence of local complications among the patients in the two subgroups . the mean of local complications occurrence was significantly higher / significantly lower ( t=6.69 , p < .001 ) in the study group patients who had the sc butterfly positioned with the bevel down ( mean=1.24 , close to the first version , no complications ) compared with the control group - patients that had the sc butterfly positioned with the bevel up ( mean=1.80 , close to the second version , with complications ) . on average , the sc butterflies positioned with the bevel down caused fewer local complications , compared with the sc butterflies positioned with the bevel up . in daily practice , the sc butterfly positioning is done with the needle bevel facing up . evidence from this study comparing any significant difference in regard to the resistance of the sc butterflies and the occurrence of local complications in both modalities of positioning the bevel of the needle , could be used to support nurses in their daily practice . the design of the subset started with the separation of the study sample into two groups ; half of the patients had the sc butterfly inserted with the bevel up ( 50% ) control group and the other half with the bevel down ( 50% ) experimental group . we found significant differences between the resistance of the sc butterfly at the insertion site in the two subgroups ; the patients who had the sc butterfly inserted with the bevel up and the patients who had the sc butterfly inserted with the bevel down ( figure 1 ) . the mean of sc butterfly resistance at the place of insertion was significantly higher ( t=10.48 , p < .01 ) in the care of patients who had the sc butterfly positioned with the bevel down ( mean=2.88 , close to 3 the third version , meaning ( seven to nine days ) compared to the patients who had the sc butterfly positioned with the bevel up ( mean=2.00 , meaning four to six days ) . in order to determine if there is a significant relationship between the frequency of administration of subcutaneous analgesics and the occurrence of local complications , the frequencies of administration were two , three or four times per day for tramadol and two , three , four or six times per day for morphine . the incidence of local complications was evaluated using the terms : no complications and with complications. a positive correlation existed between the daily frequency of the administration of opioids and the occurrence of local complications at the administration site with a p - value of .049 when using tramadol . we expected to have an increase in the occurrence of complication with higher numbers of administration and our results support this assumption . the difference seen in the appearance of complications between morphine and tramadol might relate to better subcutaneous tolerance for morphine , or it might be the result of the small sample size in our study . the administration of opioids in cancer patients is a long - lasting process , and for this reason the use of a subcutaneous butterfly is a necessary procedure to enhance the comfort and the quality of life of the patient in countries where there are no oral opioids available . the resistance in time for maintaining the sc butterfly varied between one and 12 days ; in general , most of the patients had the sc butterfly maintained for four to six days ( 53% ) ( see table vii ) . our data supports the hypothesis that a significant negative correlation exists between the occurrence of local complications and the resistance in time for maintaining the subcutaneous butterfly at the insertion site with a p - value of .001 , r- 0.558 . the study was conducted between january and may 2011 , on a sample of 100 adult cancer patients who were all outpatients of hospice casa sperantei . the distribution of the study group according to gender shows a predominance of male over female gender . this is consistent with accessibility to medical care and the lower availability of clinicians in the romanian health care system . there was no statistical difference in the composition of the control and study group concerning gender and place of residence . concerning the age of the patients in the study and control group there were significantly fewer patients in the age group of 7079 years ( p=0.05 ) in the control group compared with the study group . the patients included in the study had most frequently broncho - pulmonary cancer ( 19% ) , colon cancer ( 15% ) and breast cancer ( 13% ) . there was no significant difference between the study and control group in terms of type of cancer . in the 100 cancer patients , the main cause of pain was visceral 47 patients ( 47% ) , somatic pain 43 ( 43% ) , and only 10 presented with neuropathic pain ( 10% ) ; the patients with moderate pain represented approximately two - thirds of the total number of patients and one - third suffered severe pain ( table iii ) . there was an even distribution of patients in the experimental and control group concerning type and intensity of pain . in the sample studied , the two opioids , tramadol hydrochloride and morphine , were administrated in different proportions ; tramadol , as a weak opioid , was administered for moderate pain and was given in almost two - thirds of the patients during the study period ( see table iv ) . morphine , given through the subcutaneous route , was the drug used for patients who had severe pain . there was no significant difference between study and control group in terms of type of medication received for pain . the two analgesics had a different frequency in administration : 2 , 3 or 4 times per day for tramadol hydrochloride and 2 , 3 , 4 or 6 times per day for morphine . this can be explained through the different pharmacokinetics of the two opioids , tramadol having a longer half time compared with morphine , it was an expected result to see fewer administrations per day compared to morphine . there was no difference between study and control group concerning the frequency of administration of the drugs . the most frequent complication encountered when administering analgesics in patients with cancer was subcutaneous tissue indurations ( 56% for the whole sample ) followed by redness and bleeding . the complications encountered are presented in table vi . there were also significant differences between the possibility of the occurrence of local complications among the patients in the two subgroups . the mean of local complications occurrence was significantly higher / significantly lower ( t=6.69 , p < .001 ) in the study group patients who had the sc butterfly positioned with the bevel down ( mean=1.24 , close to the first version , no complications ) compared with the control group - patients that had the sc butterfly positioned with the bevel up ( mean=1.80 , close to the second version , with complications ) . on average , the sc butterflies positioned with the bevel down caused fewer local complications , compared with the sc butterflies positioned with the bevel up . in daily practice , the sc butterfly positioning is done with the needle bevel facing up . evidence from this study comparing any significant difference in regard to the resistance of the sc butterflies and the occurrence of local complications in both modalities of positioning the bevel of the needle , could be used to support nurses in their daily practice . the design of the subset started with the separation of the study sample into two groups ; half of the patients had the sc butterfly inserted with the bevel up ( 50% ) control group and the other half with the bevel down ( 50% ) experimental group . we found significant differences between the resistance of the sc butterfly at the insertion site in the two subgroups ; the patients who had the sc butterfly inserted with the bevel up and the patients who had the sc butterfly inserted with the bevel down ( figure 1 ) . the mean of sc butterfly resistance at the place of insertion was significantly higher ( t=10.48 , p < .01 ) in the care of patients who had the sc butterfly positioned with the bevel down ( mean=2.88 , close to 3 the third version , meaning ( seven to nine days ) compared to the patients who had the sc butterfly positioned with the bevel up ( mean=2.00 , meaning in order to determine if there is a significant relationship between the frequency of administration of subcutaneous analgesics and the occurrence of local complications , the daily rate of administration of each drug was analyzed separately . the frequencies of administration were two , three or four times per day for tramadol and two , three , four or six times per day for morphine . the incidence of local complications was evaluated using the terms : no complications and with complications. a positive correlation existed between the daily frequency of the administration of opioids and the occurrence of local complications at the administration site with a p - value of .049 when using tramadol . we expected to have an increase in the occurrence of complication with higher numbers of administration and our results support this assumption . the difference seen in the appearance of complications between morphine and tramadol might relate to better subcutaneous tolerance for morphine , or it might be the result of the small sample size in our study . the administration of opioids in cancer patients is a long - lasting process , and for this reason the use of a subcutaneous butterfly is a necessary procedure to enhance the comfort and the quality of life of the patient in countries where there are no oral opioids available . the resistance in time for maintaining the sc butterfly varied between one and 12 days ; in general , most of the patients had the sc butterfly maintained for four to six days ( 53% ) ( see table vii ) . our data supports the hypothesis that a significant negative correlation exists between the occurrence of local complications and the resistance in time for maintaining the subcutaneous butterfly at the insertion site with a p - value of .001 , r- 0.558 . in order to determine if there is a significant relationship between the frequency of administration of subcutaneous analgesics and the occurrence of local complications , the daily rate of administration of each drug was analyzed separately . the frequencies of administration were two , three or four times per day for tramadol and two , three , four or six times per day for morphine . the incidence of local complications was evaluated using the terms : no complications and with complications. a positive correlation existed between the daily frequency of the administration of opioids and the occurrence of local complications at the administration site with a p - value of .049 when using tramadol . we expected to have an increase in the occurrence of complication with higher numbers of administration and our results support this assumption . the difference seen in the appearance of complications between morphine and tramadol might relate to better subcutaneous tolerance for morphine , or it might be the result of the small sample size in our study . the administration of opioids in cancer patients is a long - lasting process , and for this reason the use of a subcutaneous butterfly is a necessary procedure to enhance the comfort and the quality of life of the patient in countries where there are no oral opioids available . the resistance in time for maintaining the sc butterfly varied between one and 12 days ; in general , most of the patients had the sc butterfly maintained for four to six days ( 53% ) ( see table vii ) . our data supports the hypothesis that a significant negative correlation exists between the occurrence of local complications and the resistance in time for maintaining the subcutaneous butterfly at the insertion site with a p - value of .001 , r- 0.558 . the development of practical research studies that aim at improving current nursing techniques by translating research findings into practice has become a focus of many interdisciplinary studies concerning the care of cancer patients . these modalities of care aim at improving the quality and consistency of nursing decision - making , particularly for cancer patients who experience pain . our major finding concerning the nursing practice is related to the positioning of the butterfly , the needle with the bevel down . this placement position is associated with a longer resistance in time at the site of insertion and causes less local complications compared to the sc butterflies positioned with the bevel up . these findings are a challenge for the actual practice of placing the butterfly with the bevel up . a limitation of our study is the fact that we did not study the correlation between complications , position of butterfly and the expertise of the nurses carrying out the procedure . a previous canadian study suggests that the rate of local side effects seems to have decreased over time as the team s expertise in the use of intermittent subcutaneous opioid delivery increased . we did not measure the expertise of the nurses inserting the butterfly and this could be the one limitation factor that we have not taken into consideration . in contrast with other previous data , no site infections and accidental needle pulling were evidenced . because primary health care is underdeveloped in romania there is a low number of district nurses and professional care - takers for home care services . in romania , home - based palliative care services rely heavily on the input and involvement of the family in the care of patients . in our study , after the butterfly was placed by a hospice nurse the family was taught to administer the medication through the butterfly . although we provided some training for family members we had no control over the subsequent process . that said , data concerning the duration of the butterfly at the site of insertion were comparable with data from other studies that show a 6.5 days to eight - day overall duration of the sites for intermittent infusions and for continuous infusions of opioids ( seven days ) . in our study , the most frequent injectable opioid administered to cancer patients with pain was tramadol hydrochloride . this can be explained by the fact that 65% of the patients included in our study had moderate pain and tramadol is a mild opioid adequate to treat moderate pain . another explanation is the regulation concerning prescribing procedures for opioids : tramadol is not a scheduled drug in romania , it is available on normal prescription and physicians are less reluctant to prescribe it . this practice is different from other countries where the most frequently used opioid was hydromorphone , which has also been reported to be well tolerated when used subcutaneously , both as continuous and intermittent infusion fudin , 2000 ) . we do not have available in romania injectable oxycodone and methadone , which are also reported to be used and well tolerated in subcutaneous administration . the most encountered frequency of administration was three times per day for tramadol hydrochloride and four times per day for morphine . as the rate or frequency of the rate of administration of sc injectable opioids increases , the chances of local complications occurrence also increase . however , our data also showed administration of tramadol twice a day and of morphine twice , three times or four times a day . this is not standard practice for good pain management in romania unless there is a degree of kidney failure . it was not the purpose of our study to look at the prescribing patterns ; we have not recorded parameters concerning the renal function for the patients in our study , therefore we can not judge the appropriateness of the prescribing patterns in this study . in our study , the most frequent injectable opioid administered to cancer patients with pain was tramadol hydrochloride . this can be explained by the fact that 65% of the patients included in our study had moderate pain and tramadol is a mild opioid adequate to treat moderate pain . another explanation is the regulation concerning prescribing procedures for opioids : tramadol is not a scheduled drug in romania , it is available on normal prescription and physicians are less reluctant to prescribe it . this practice is different from other countries where the most frequently used opioid was hydromorphone , which has also been reported to be well tolerated when used subcutaneously , both as continuous and intermittent infusion fudin , 2000 ) . we do not have available in romania injectable oxycodone and methadone , which are also reported to be used and well tolerated in subcutaneous administration . the most encountered frequency of administration was three times per day for tramadol hydrochloride and four times per day for morphine . as the rate or frequency of the rate of administration of sc injectable opioids increases , the chances of local complications occurrence also increase . however , our data also showed administration of tramadol twice a day and of morphine twice , three times or four times a day . this is not standard practice for good pain management in romania unless there is a degree of kidney failure . it was not the purpose of our study to look at the prescribing patterns ; we have not recorded parameters concerning the renal function for the patients in our study , therefore we can not judge the appropriateness of the prescribing patterns in this study . practical research studies are needed to examine and improve current nursing techniques and strategies that are employed in everyday clinical practice . the article challenges the standard nursing practice regarding the insertion of the subcutaneous butterfly with the bevel up . according to our findings the position of the needle with the bevel down ensures a better resistance of the subcutaneous butterfly and fewer side effects . the results of this pilot study need to be proved in a larger study and with better observation given to potential limitation factors such as the number of administrations , types of drugs , nutrition status of the patient , and experience of the nurses performing the procedure .

background and aimsthe increasing number of cancer patients , together with the development of new palliative care services in romania , warrants the evaluation of nursing strategies meant to improve the level of comfort of patients who are suffering from advanced cancer.the main objective of the study was to evaluate the optimal positioning of the subcutaneous ( sc ) butterfly , in accordance with its resistance in the insertion tissue , the local complications that may occur , and the evaluation of the time of resistance at the insertion site ( puncture ) with the daily frequency of injectable opioid administration.methodsa prospective experimental pilot study was designed and conducted between january and may 2011 . patients admitted to the hospice casa sperantei ( brasov , romania ) with moderate or severe cancer pain , who were receiving subcutaneously opioids , over the age of 18 , with normal body index ranging from 18.5 22.0 , were assigned randomly to one of two groups , after signing the informed consent . in group one , the butterfly was positioned with the needle bevel up this was considered to be the control group as this modality of inserting the needle is considered standard practice ; in group two the butterfly was positioned with the needle bevel down experimental group . the drugs used for pain relief were sc tramadol for moderate pain and sc morphine for severe pain.resultsour research supported the hypothesis that the occurrence of local complications coincides with the decrease of sc butterfly resistance in time at the place of insertion , and the sc butterfly has a higher rate of resistance in time at the insertion site if the frequency of injectable opioids administration is lower ( twice per day).conclusionthe positioning of the butterflies with the bevel down ( experimental group ) is associated with a longer resistance in time at the site of insertion , and causes fewer local complications compared to the sc butterflies positioned with the bevel up ( control group ) .

Background and aims Methods Results Characteristics of the patients in the study Pain characteristics in the study population Use of analgesics in the study population Complications with subcutaneous administration of analgesics Positioning of the butterfly Analysis performed on the whole sample of patients Correlation between complications and frequency of administration Resistance in time at the injection site Discussion This is a confirmation that administration of pain medication via subcutaneously butterfly by family members is safe practice Conclusion

the growth in the number of cancer patients , together with the development of new palliative care services in romania , warrants the evaluation of nursing strategies meant to improve the level of comfort of patients who are suffering from advanced cancer . pain is one of the most frequent symptoms in patients with advanced cancer , and the use of opioids is a standard practice with patients who are in pain . to maximize comfort , instead of using intramuscular or intravenous route , the subcutaneous administration with an indwelling subcutaneous butterfly needle has been proved to be painless , efficient and easy to use . for countries with limited resources it is a useful method of administering parenteral medication as it has a lower cost than intravenous pumps and it also gives patients and families the possibility , with some training , to administer the drugs themselves . the main objective of this study was to evaluate the optimal positioning of the subcutaneous ( sc ) butterfly , in accordance with its resistance in the insertion tissue , the local complications that occur and the evaluation of the time of resistance at the insertion site ( puncture ) with the daily frequency of injectable opioid administration . patients admitted to the hospice casa sperantei with moderate or severe cancer pain and who were receiving subcutaneously opioids , over the age of 18 , with normal body index ranging from 18.522.0 , were assigned randomly , after signing the informed consent , to one of two groups . in group one , the butterfly was positioned with the needle bevel up this was considered to be the control group as this modality of inserting the needle is considered standard practice ; in group two the butterfly was positioned with the needle bevel down - experimental group . the drugs used for pain relief were sc tramadol for moderate pain and sc morphine for severe pain . data from patients were collected between january and may 2011 , in a sample of 100 adult cancer patients . correlations between the frequency of administration of opioids and the occurrence of local complications , and between the occurrence of local complications and the time of resistance of the subcutaneous butterfly at the insertion site , were established . the hypothesis is that there is a significant positive relation between the frequency of injectable opioid administration and the occurrence of local complications , respectively a negative relation between the occurrence of complications and the time of resistance of the subcutaneous butterfly at the insertion place . in addition , we wanted to establish which of the two butterfly insertion positions ( butterfly with the needle bevel up or down ) had a longer time of resistance at the insertion site with respect to incidence of complications . the study was conducted between january and may 2011 , on a sample of 100 adult cancer patients who were all outpatients of hospice casa sperantei . this is consistent with accessibility to medical care and the lower availability of clinicians in the romanian health care system . there was no statistical difference in the composition of the control and study group concerning gender and place of residence . concerning the age of the patients in the study and control group there were significantly fewer patients in the age group of 7079 years ( p=0.05 ) in the control group compared with the study group . the patients included in the study had most frequently broncho - pulmonary cancer ( 19% ) , colon cancer ( 15% ) and breast cancer ( 13% ) . there was no significant difference between the study and control group in terms of type of cancer . in the 100 cancer patients , the main cause of pain was visceral 47 patients ( 47% ) , somatic pain 43 ( 43% ) , and only 10 presented with neuropathic pain ( 10% ) ; the patients with moderate pain represented approximately two - thirds of the total number of patients and one - third suffered severe pain ( table iii ) . there was an even distribution of patients in the experimental and control group concerning type and intensity of pain . in the sample studied , the two opioids , tramadol hydrochloride and morphine , were administrated in different proportions ; tramadol , as a weak opioid , was administered for moderate pain and was given in almost two - thirds of the patients during the study period ( see table iv ) . morphine , given through the subcutaneous route , was the drug used for patients who had severe pain . there was no significant difference between study and control group in terms of type of medication received for pain . this can be explained through the different pharmacokinetics of the two opioids , tramadol having a longer half time compared with morphine , it was an expected result to see fewer administrations per day compared to morphine . there was no difference between study and control group concerning the frequency of administration of the drugs . there were also significant differences between the possibility of the occurrence of local complications among the patients in the two subgroups . the mean of local complications occurrence was significantly higher / significantly lower ( t=6.69 , p < .001 ) in the study group patients who had the sc butterfly positioned with the bevel down ( mean=1.24 , close to the first version , no complications ) compared with the control group - patients that had the sc butterfly positioned with the bevel up ( mean=1.80 , close to the second version , with complications ) . on average , the sc butterflies positioned with the bevel down caused fewer local complications , compared with the sc butterflies positioned with the bevel up . in daily practice , the sc butterfly positioning is done with the needle bevel facing up . evidence from this study comparing any significant difference in regard to the resistance of the sc butterflies and the occurrence of local complications in both modalities of positioning the bevel of the needle , could be used to support nurses in their daily practice . the design of the subset started with the separation of the study sample into two groups ; half of the patients had the sc butterfly inserted with the bevel up ( 50% ) control group and the other half with the bevel down ( 50% ) experimental group . we found significant differences between the resistance of the sc butterfly at the insertion site in the two subgroups ; the patients who had the sc butterfly inserted with the bevel up and the patients who had the sc butterfly inserted with the bevel down ( figure 1 ) . the mean of sc butterfly resistance at the place of insertion was significantly higher ( t=10.48 , p < .01 ) in the care of patients who had the sc butterfly positioned with the bevel down ( mean=2.88 , close to 3 the third version , meaning ( seven to nine days ) compared to the patients who had the sc butterfly positioned with the bevel up ( mean=2.00 , meaning four to six days ) . in order to determine if there is a significant relationship between the frequency of administration of subcutaneous analgesics and the occurrence of local complications , the frequencies of administration were two , three or four times per day for tramadol and two , three , four or six times per day for morphine . the incidence of local complications was evaluated using the terms : no complications and with complications. a positive correlation existed between the daily frequency of the administration of opioids and the occurrence of local complications at the administration site with a p - value of .049 when using tramadol . we expected to have an increase in the occurrence of complication with higher numbers of administration and our results support this assumption . the difference seen in the appearance of complications between morphine and tramadol might relate to better subcutaneous tolerance for morphine , or it might be the result of the small sample size in our study . the administration of opioids in cancer patients is a long - lasting process , and for this reason the use of a subcutaneous butterfly is a necessary procedure to enhance the comfort and the quality of life of the patient in countries where there are no oral opioids available . the resistance in time for maintaining the sc butterfly varied between one and 12 days ; in general , most of the patients had the sc butterfly maintained for four to six days ( 53% ) ( see table vii ) . our data supports the hypothesis that a significant negative correlation exists between the occurrence of local complications and the resistance in time for maintaining the subcutaneous butterfly at the insertion site with a p - value of .001 , r- 0.558 . the study was conducted between january and may 2011 , on a sample of 100 adult cancer patients who were all outpatients of hospice casa sperantei . the distribution of the study group according to gender shows a predominance of male over female gender . this is consistent with accessibility to medical care and the lower availability of clinicians in the romanian health care system . there was no statistical difference in the composition of the control and study group concerning gender and place of residence . concerning the age of the patients in the study and control group there were significantly fewer patients in the age group of 7079 years ( p=0.05 ) in the control group compared with the study group . the patients included in the study had most frequently broncho - pulmonary cancer ( 19% ) , colon cancer ( 15% ) and breast cancer ( 13% ) . there was no significant difference between the study and control group in terms of type of cancer . in the 100 cancer patients , the main cause of pain was visceral 47 patients ( 47% ) , somatic pain 43 ( 43% ) , and only 10 presented with neuropathic pain ( 10% ) ; the patients with moderate pain represented approximately two - thirds of the total number of patients and one - third suffered severe pain ( table iii ) . there was an even distribution of patients in the experimental and control group concerning type and intensity of pain . in the sample studied , the two opioids , tramadol hydrochloride and morphine , were administrated in different proportions ; tramadol , as a weak opioid , was administered for moderate pain and was given in almost two - thirds of the patients during the study period ( see table iv ) . morphine , given through the subcutaneous route , was the drug used for patients who had severe pain . there was no significant difference between study and control group in terms of type of medication received for pain . this can be explained through the different pharmacokinetics of the two opioids , tramadol having a longer half time compared with morphine , it was an expected result to see fewer administrations per day compared to morphine . there was no difference between study and control group concerning the frequency of administration of the drugs . there were also significant differences between the possibility of the occurrence of local complications among the patients in the two subgroups . the mean of local complications occurrence was significantly higher / significantly lower ( t=6.69 , p < .001 ) in the study group patients who had the sc butterfly positioned with the bevel down ( mean=1.24 , close to the first version , no complications ) compared with the control group - patients that had the sc butterfly positioned with the bevel up ( mean=1.80 , close to the second version , with complications ) . on average , the sc butterflies positioned with the bevel down caused fewer local complications , compared with the sc butterflies positioned with the bevel up . in daily practice , the sc butterfly positioning is done with the needle bevel facing up . evidence from this study comparing any significant difference in regard to the resistance of the sc butterflies and the occurrence of local complications in both modalities of positioning the bevel of the needle , could be used to support nurses in their daily practice . the design of the subset started with the separation of the study sample into two groups ; half of the patients had the sc butterfly inserted with the bevel up ( 50% ) control group and the other half with the bevel down ( 50% ) experimental group . we found significant differences between the resistance of the sc butterfly at the insertion site in the two subgroups ; the patients who had the sc butterfly inserted with the bevel up and the patients who had the sc butterfly inserted with the bevel down ( figure 1 ) . the mean of sc butterfly resistance at the place of insertion was significantly higher ( t=10.48 , p < .01 ) in the care of patients who had the sc butterfly positioned with the bevel down ( mean=2.88 , close to 3 the third version , meaning ( seven to nine days ) compared to the patients who had the sc butterfly positioned with the bevel up ( mean=2.00 , meaning in order to determine if there is a significant relationship between the frequency of administration of subcutaneous analgesics and the occurrence of local complications , the daily rate of administration of each drug was analyzed separately . the incidence of local complications was evaluated using the terms : no complications and with complications. a positive correlation existed between the daily frequency of the administration of opioids and the occurrence of local complications at the administration site with a p - value of .049 when using tramadol . we expected to have an increase in the occurrence of complication with higher numbers of administration and our results support this assumption . the difference seen in the appearance of complications between morphine and tramadol might relate to better subcutaneous tolerance for morphine , or it might be the result of the small sample size in our study . the administration of opioids in cancer patients is a long - lasting process , and for this reason the use of a subcutaneous butterfly is a necessary procedure to enhance the comfort and the quality of life of the patient in countries where there are no oral opioids available . the resistance in time for maintaining the sc butterfly varied between one and 12 days ; in general , most of the patients had the sc butterfly maintained for four to six days ( 53% ) ( see table vii ) . our data supports the hypothesis that a significant negative correlation exists between the occurrence of local complications and the resistance in time for maintaining the subcutaneous butterfly at the insertion site with a p - value of .001 , r- 0.558 . in order to determine if there is a significant relationship between the frequency of administration of subcutaneous analgesics and the occurrence of local complications , the daily rate of administration of each drug was analyzed separately . the incidence of local complications was evaluated using the terms : no complications and with complications. a positive correlation existed between the daily frequency of the administration of opioids and the occurrence of local complications at the administration site with a p - value of .049 when using tramadol . we expected to have an increase in the occurrence of complication with higher numbers of administration and our results support this assumption . the difference seen in the appearance of complications between morphine and tramadol might relate to better subcutaneous tolerance for morphine , or it might be the result of the small sample size in our study . the administration of opioids in cancer patients is a long - lasting process , and for this reason the use of a subcutaneous butterfly is a necessary procedure to enhance the comfort and the quality of life of the patient in countries where there are no oral opioids available . the resistance in time for maintaining the sc butterfly varied between one and 12 days ; in general , most of the patients had the sc butterfly maintained for four to six days ( 53% ) ( see table vii ) . our data supports the hypothesis that a significant negative correlation exists between the occurrence of local complications and the resistance in time for maintaining the subcutaneous butterfly at the insertion site with a p - value of .001 , r- 0.558 . the development of practical research studies that aim at improving current nursing techniques by translating research findings into practice has become a focus of many interdisciplinary studies concerning the care of cancer patients . these modalities of care aim at improving the quality and consistency of nursing decision - making , particularly for cancer patients who experience pain . our major finding concerning the nursing practice is related to the positioning of the butterfly , the needle with the bevel down . this placement position is associated with a longer resistance in time at the site of insertion and causes less local complications compared to the sc butterflies positioned with the bevel up . these findings are a challenge for the actual practice of placing the butterfly with the bevel up . a limitation of our study is the fact that we did not study the correlation between complications , position of butterfly and the expertise of the nurses carrying out the procedure . a previous canadian study suggests that the rate of local side effects seems to have decreased over time as the team s expertise in the use of intermittent subcutaneous opioid delivery increased . we did not measure the expertise of the nurses inserting the butterfly and this could be the one limitation factor that we have not taken into consideration . because primary health care is underdeveloped in romania there is a low number of district nurses and professional care - takers for home care services . in romania , home - based palliative care services rely heavily on the input and involvement of the family in the care of patients . in our study , after the butterfly was placed by a hospice nurse the family was taught to administer the medication through the butterfly . that said , data concerning the duration of the butterfly at the site of insertion were comparable with data from other studies that show a 6.5 days to eight - day overall duration of the sites for intermittent infusions and for continuous infusions of opioids ( seven days ) . in our study , the most frequent injectable opioid administered to cancer patients with pain was tramadol hydrochloride . this can be explained by the fact that 65% of the patients included in our study had moderate pain and tramadol is a mild opioid adequate to treat moderate pain . another explanation is the regulation concerning prescribing procedures for opioids : tramadol is not a scheduled drug in romania , it is available on normal prescription and physicians are less reluctant to prescribe it . we do not have available in romania injectable oxycodone and methadone , which are also reported to be used and well tolerated in subcutaneous administration . as the rate or frequency of the rate of administration of sc injectable opioids increases , the chances of local complications occurrence also increase . this is not standard practice for good pain management in romania unless there is a degree of kidney failure . it was not the purpose of our study to look at the prescribing patterns ; we have not recorded parameters concerning the renal function for the patients in our study , therefore we can not judge the appropriateness of the prescribing patterns in this study . in our study , the most frequent injectable opioid administered to cancer patients with pain was tramadol hydrochloride . this can be explained by the fact that 65% of the patients included in our study had moderate pain and tramadol is a mild opioid adequate to treat moderate pain . another explanation is the regulation concerning prescribing procedures for opioids : tramadol is not a scheduled drug in romania , it is available on normal prescription and physicians are less reluctant to prescribe it . we do not have available in romania injectable oxycodone and methadone , which are also reported to be used and well tolerated in subcutaneous administration . as the rate or frequency of the rate of administration of sc injectable opioids increases , the chances of local complications occurrence also increase . this is not standard practice for good pain management in romania unless there is a degree of kidney failure . it was not the purpose of our study to look at the prescribing patterns ; we have not recorded parameters concerning the renal function for the patients in our study , therefore we can not judge the appropriateness of the prescribing patterns in this study . the article challenges the standard nursing practice regarding the insertion of the subcutaneous butterfly with the bevel up . according to our findings the position of the needle with the bevel down ensures a better resistance of the subcutaneous butterfly and fewer side effects . the results of this pilot study need to be proved in a larger study and with better observation given to potential limitation factors such as the number of administrations , types of drugs , nutrition status of the patient , and experience of the nurses performing the procedure .

until recently , conceptual and empirical research on psychopathology has tended to focus on symptomatology and on grouping symptoms into syndromes . so , for example , studies of anxiety or depression focus on the neurophysiological , behavioral , or cognitive components of the symptoms of anxiety and depression . in the last 20 years , however , evolutionary approaches to psychopathology have emerged that focus on evolved strategies and their complex regulators . this directs attention to the evolved functions of motives and emotions , and , importantly for this paper , how they can suppress and coregulate each other . the evolutionary model starts with the fact that a range of emotional and motivational systems evolved because they helped meet the challenges of survival and gene replication in competitive environments . brains and minds therefore monitor their social contexts and change their relationship to them with regard to risks , opportunities , and potential supports . it is now recognized that one of the most important strategic adaptations for primates , and especially humans , is sociality ; we are basically wired to seek helpful connections with others . it is our human motivation for connecting , relating , and communicating that has driven social intelligence , from toolmaking to science , and more recently , the proliferation of communication technologies , social media on a global scale , and emotion regulation . within this drive for connection and relating , humans play out a complex variety of motivations and interacting styles with each other ( eg , they can be supportive , caring , and desirous , or indifferent , exploitative , or hostile ) . these multifaceted and multifarious motivations may compete for expression across relationships and even within any one relationship . in addition , we have minds with internal relationships with ourselves , so we can feel friendly , indifferent , or hostile to ourselves . what has become clear over the last 20 years is that certain motivational and emotional processes , which are linked to phenotypic and strategic orientations , are more strongly linked to physical and mental health than others . in brief , threat focus and competitive self - focus may be less conducive to wellbeing than a prosocial focus on self and others . in fact , prosocial relationships have major impacts on a range of physiological systems , including genetic expression . fredrickson al found that eudemonic well - being ( positive emotion associated with meaning and helping others ) , in contrast to self - focused pleasure and hedonic well - being , was linked to better physiological profiles involving proinflammatory genes . hence , facilitating affiliative and prosocial processing , rather than ( just ) reducing threat processing or enhancing self - focused competitiveness , becomes the target for therapeutic and preventative interventions . the sections below explore the ways that prosocial behavior is linked to motives and emotions that alleviate and help prevent mental health difficulties . obvious nonsocial ones are harm avoidance , food , and shelter - seeking . in the social domain , for example , sexual behavior involves a ( courting ) dance between two partners , and at any point , miscommunication can result in one partner attacking or taking flight from the other . social motives that require specialist competencies for processing potentially rapidly changing , dynamic , and reciprocal interactional sequences , have been referred to as social mentalities . central to this paper is the issue that different motives and social mentalities organize the brain in very different ways . given that many of our evolved motivational systems are competing for expression and are sensitive to social contexts , this raises the issue of how different motivational systems are related to health , and to vulnerability to psychopathology . to explore this we would ideally have a nosology of motives , but there are no agreed evolution - derived nosologies for social motives and mentalities for example , among the most common social ones are : competition , cooperation , and alliance building ; care providing , care seeking , and sexual . various blends of these would include desires for power , achievement , connection , belonging , socializing , sex , and so on . we can note how different social mentalities create different patterns in a range of biopsychosocial processes , by contrasting two of them such as competitive and caring . individuals who are orientated through competitive motives are highly focused on rank and power issues , shame / pride , are very sensitive to social comparison , vulnerable to envy , and are self - focused and self - monitoring . in contrast , caring motivations do not utilize these social processing competencies , but instead are highly sensitive to signals of distress and needs , and recruit sympathetic and caring competencies . seeing someone injured in a competitive conflict could be experienced as positive and rewarding , whereas the same outcome in a caring relationship would be experienced as threatening and distressing . individuals engaged in competitive interactions will be ( neuro)physiologically and psychologically organized in different ways , compared with caring and supportive interactions . simon - thomas et al conducted an fmri study to explore neurophysiological differences in compassion / caring versus pride activation . they found that : compassion induction was associated with activation in the midbrain periaqueductal gray ( pag ) , a region that is activated during pain and the perception of others ' pain , and that has been implicated in parental nurturance behaviors . pride induction engaged the posterior medial cortex , a region that has been associated with self - referent processing , ( p. 635 ) such studies seek to identify different neurological patterns associated with different motivational systems . the bottom line of this is that prosocial motives that are associated with taking an interest in caring for others , in contrast to self - focused ( competitive ) motives to get ahead or avoid being inferior , shamed , and rejected , are associated with improved well - being and reduce vulnerabilities to psychopathology . derived from social mentality theory , mcewan et al showed that in students and a depressed population , depression was linked to thwarted competitive motives ( feeling inferior , unable to compete in the world , feeling like a loser ) but was not related to caring motives ( being kind , helpful , and trustworthy ) ; depressed patients do not feel inferior in caring domains . so it is not negative self - evaluation in general , but a sense of self within a particular motivational system that is an issue for depression . in addition , depressed people can be fearful of being open to receiving compassion from others , as well as blocking self - compassion . in regard to motivational systems underpinning prosocial motivation and emotion , there appear to be two main forms . the other relates to alliance - building , cooperation , and friendship - network formations . bailey distinguishes these two domains in terms of : ( i ) genetic kin - like forms of relating focusing on intimacy and closeness ; and ( ii ) psychological kinships based on similarity of values and interests with cooperative potential . the simplest form of relating arises in dyads where two individuals come together for various reasons such as caring , helping , sharing , or sexual engagement . working together , with a number of others , and feeling part of a group , from me - ness to we - ness , expands out into issues of group identity and group belonging . it is well - known that kin - like , intimate and close , prosocial , loving , and caring early attachment relationships provide a wealth of resources which shape physiological systems and set phenotypes for increased health and well - being , whereas neglect and/or abuse do the opposite . in addition , there is now considerable evidence that alliances and friendships also play vital roles in physical and mental health , while , in contrast , loneliness and a lack of cooperating alliances ( friendships ) are highly detrimental to well-being.indeed , studies of loneliness show it to be associated with a range of physiological problems , including adverse effects on telomere length . in many forms of psychological difficulty feeling alone , different , and separate(d ) from others , is a very common experience . in addition , shame and self - criticism not only constitute negative self - experiences , in contrast to prosocial , liking , and helpful relationships with oneself , they also interfere with people 's abilities and confidence to develop supportive , affiliative relationships with others . people can be fearful of others being compassionate towards them because of what ( they fear ) might be discovered about them if other get too close , and the risk of rejection . such fears disrupt the potential benefits of prosocial relationships and are associated with depression and anxiety . researchers have long sought to derive an evolution - based , functional classification of emotions and identify their universal regulators , and roles in clinical problems . panskepp delineated seven types of evolved functionally specific emotions that can also blend and operate together . these include : ( i ) emotions for seeking rewards / resources ; ( ii ) emotions linked to lust which are particularly ( but not only ) focused on sexual stimuli ; ( iii ) emotions linked to caring and affection ; ( iv ) emotions linked to loss and feelings of grief ; ( v ) threat emotions of rage ; and ( vi ) fear ; ( vii ) emotions that are linked to play and give a sense of joyfulness in activities . derived from the work of depue and morrone - strupinsky , ledoux , panksepp , and others , based on clinical observation , and focusing on more general rather than specific functions , gilbert highlighted three core evolved functions of emotions , loosely identified as : emotions that serve the functions of threat detection and generating defensive and safety strategies emotions that serve the functions of detecting , energizing and seeking / acquiring resources for survival and reproduction emotions that serve the function of contentment , satisfaction , calming , settling , and allowing rest and digest . systems , it is more accurate to see them as rooted in patterns of ( neuro ) physiological activation that are constantly blending and coregulating . for example , a threat to people we love raises anxiety , or anger to those who are threatening ; spending time with friends and loved ones can be activating , enjoyable , and exciting ; being in the presence of caring others helps us feel safe and content , and when we are distressed , can be soothing and calming . the reasons why social relationships that signal either threat or positive support / help , have such powerful psychophysiologically regulating impacts , are related to our evolution as socially interacting mammals ; indeed there is general agreement that it was our sociality that drove human intelligence . the three - circle model of emotion is linked with attachment theory . gilbert bowlby , and mikulincer and shaver highlighted the fact that early attachments provide the young with proximity maintenance , a secure base and safe haven that are fundamental for development . a secure base provides the encouragement and confidence to go out and explore the world ; a safe haven provides a source to return to for protection , soothing , and calming should the infant become distressed . these functions continue throughout life and we turn to others friends , partners and lovers to provide them . importantly , signals indicating a presence or absence of a secure base and safe haven are linked to all three systems . the value of this way of thinking can be seen when working with trauma , particularly in veterans . during their training , and while on missions , military personnel will experience elevated threat . they are then subsequently calmed and soothed in the context of being with their buddies . indeed , the military deliberately fosters close interconnectedness . in essence , the secure base and safe haven become ( re)wired from family and home to signals of the presence of buddies . when they return home with these rewired systems , a sense of connectedness and soothing may ( for some ) no longer be stimulated by wife and children , or even old friends . the removal of a sense of buddy - connectedness and safeness signals can activate the threat - vigilance systems ; typical of major loss / separation . so veterans can experience high levels of threat from the sudden removal of important safeness signals , with a yearning to return to be with their ( safe haven ) buddies . however , they are so expecting ( hoping ) to feel safe and well when they come hope that they can find these inner experiences very distressing and confusing . some believe they ought to feel safe and secure ( or excited ) back with their families and are not aware that all three systems have been ( re)wired and so it will take time for them to be wired back into civilian , social contexts . explaining the three - circle model and the possible process of rewiring according to context , to them and their families , can be very helpful and deshaming . trying to understand trauma only through focusing on threat processing will be limited without also discussing the notions of secure base , safe haven , and how the parasympathetic system is linked to the functioning of the soothing system . indeed , understanding the importance of the buddy system has stimulated work on the value of recruiting buddies and fellow veterans in the treatment of trauma . there are a number of key physiology adaptations that have facilitated enhanced affiliative and care - focused relating . amongst evolved challenges for mammalian sociality are : ( i ) for close proximity not to trigger fear / flight or anger / fight ; and ( ii ) provide advantages that support survival . maclean highlighted the fact that parents needed to stop treating their offspring as just another meal ( as some fish do ) . in addition , play became a key source for mammalian interaction , especially where offspring are living close together . porges suggests that the evolution of the myelinated vagus nerve was one part of the solution to such challenges . he has been at the forefront of suggesting that the myelinated parasympathetic system provides the physiological bases to put a brake on predatory , fight - and - flight behavior when individuals are in close proximity , and instead facilitate soothing and calming in the context of close relationships . this provides physiological infrastructures for the development of caring and cooperation ; to enable individuals to help each other . although some of the details of polyvagal theory have been questioned , there is good evidence now that this branch of the parasympathetic system is associated with prosocial behavior and well - being ( see kogan et al for a major review ) . a common way to explore the sympathetic - parasympathetic balance is with measures of heart rate variability there is now good evidence that heart rate variability is an indicator for a range of physical or mental health difficulties and is strongly linked to the quality of social relationships . another key evolved physiological factor in prosociality is the hormone oxytocin , a nine - amino - acid neuropeptide hormone , produced in the hypothalamus . oxytocin plays a fundamental role in a range of physiological processes throughout the body and is a key hormone in prosocial and affiliative behaviors . oxytocin was central to the evolution of the mammalian caring / attachment behavior and now supports conspecific recognition , monogamous bonding , kin - attachment and bonding , increases trust , improves competencies in mind reading tasks , increases feelings of liking others , and reduces activation in the amygdala to threat . importantly , it plays a significant role in threat regulation in general , there being oxytocin receptors in the amygdala . however , oxytocin is not a be nice to all hormone . it is also linked with greater hostility to outsiders and maternal aggression to potential threats to their infants . from an evolutionary point of view , oxytocin guides people to be selective in the choice of targets on which they focus prosocial behavior . a central argument of this section is that understanding how affiliative and prosocial systems work is key to understanding threat processing and psychopathology ; one can not understand psychopathology by analyzing threat or symptoms alone . it 's important then to recognize that the ( neuro)physiological mechanisms that support prosocial and affiliative behavior have been identified as problematic in many people with psychological difficulties . given parasympathetic functioning , and in particular heart rate variability , is linked to prosociality and mental well - being , kemp and quintana have provided a major overview of the link between poor heart rate variability and a range of psychological and physical difficulties . low resting heart rate variability has knock - on effects to cardiovascular and immune systems , as well as mood regulation . not only is there a direct connection between feeling socially connected and heart rate variability ; gillie and thayer review the evidence that parasympathetic tone is very important for executive control and the integration of frontal cortical systems with deeper brain systems . they describe how executive control is compromised in people with post - traumatic stress disorder , and identify difficulties in parasympathetic regulation as a potential source . austin et al found that people with borderline difficulties ( reflected in difficulties in regulating emotions , a fragile sense of self , and problems in interpersonal relationships ) do not differ in terms of sympathetic activation to threat compared with controls , but differ significantly in the parasympathetic responses to potential helpfulness actually showing a more fight - flight profile in situations of helpfulness . in a study looking at people 's response to compassion , which involved imagining receiving kindness and compassion from others , rockliff et al found that , when trying to imagine a compassionate other , low self - criticism and secure attachment were associated with improved / increased heart rate variability , but high self - criticism and insecure attachment were associated with a worsening of heart rate variability . in an fmri study exploring the neurophysiological patterns of self - criticism and self - reassurance to threatening events , longe et al found that high and low self - critics differed fundamentally in their neurophysiological profiles . for low self - critics , self - reassurance was associated with brain areas for calming . however , for those with higher self - criticism , efforts to be self - reassuring were associated with threat areas , such as the amygdala . hence , for some individuals , efforts to be compassionate , reassuring , and kind to oneself activate threat systems . so , there are many studies suggesting that some of the core physiological systems for prosocial emotion , thinking , and behavior are compromised in people with mental health problems . people are not able to use the parasympathetic - based soothing system for affect regulation and executive control . looking at another major physiological system for prosociality , oxytocin , yuen this may account for some of the depressed person 's feelings of social disconnection , being cut off and different from others , separated , isolated , and lonely . stanley and siever suggested that a number of the interpersonal and emotion regulation difficulties associated with borderline personality disorder are indications of problems in oxytocin . ebert et al developed a game to measure interpersonal trust and explored the impact of oxytocin ( ot ) in controls and people with borderline personality difficulties . rockliff et al explored the impact of nasal oxytocin and placebo on the ability to generate and feel reassured by compassionate images . while oxytocin increased the experiences of compassion for many , some individuals with high self - criticism actually felt worse . given that compassionate and prosocial behavior have such powerful influences on a range of physiological , psychological , and social processes , it follows that training people to cultivate compassion motives and emotions may be therapeutic . this raises the question about the focus for compassion , because compassion can be explored in relation to compassion we have for others , the way we respond to the compassion from others and self - compassion . there is increasing evidence that forms of meditation practices involving imagining compassion for others creates beneficial changes in the frontal cortex and immune system , as well as feelings of well - being . hutcherson et al found that a brief loving - kindness meditation increases feelings of social connectedness and affiliation towards strangers . fredrickson et al found that six 60-minute weekly group sessions with home practice based on a cd of loving kindness meditation ( compassion directed to self , then others , then strangers ) increases positive emotions , mindfulness , feelings of purpose in life , and social support , and decreases illness symptoms compared with a control group . weng et al found that compassion training increases people 's prosocial behavior and neurophysiological responses to suffering in others . hoge et al found that women with experience of loving - kindness meditation had longer relative telomere length than controls . the beneficial effects of compassion cultivation are not just linked to meditations but to values , and to the ways we live our lives . psychotherapy has also begun to focus on compassion and prosocial cultivation as therapeutic targets in their own right . focusing on the experience and development of compassion has been found to reduce depression , anxiety , and self - criticism in people presenting to a community mental health team , in people with long - term mental health problems , and people in a high - security psychiatric setting . compassion - focused therapy ( cft ) has been shown to be helpful for people with psychosis ; and for people with emotional difficulties and personality disorders . ashworth et al found cft to be a valuable addition in helping people with acquired brain injury . some researchers have focused on a particular and specific kind of self - compassion that involves cultivation of mindfulness ( rather than attentional absorption in difficulties ) , a sense of common humanity ( rather than a sense of shame and isolation ) , and non - judgement ( rather than self - criticalness ) . recent trials in nonclinical populations have shown this to be beneficial to well - being . for depressed people , kuyken et al found that in a mindfulness trial , self - compassion was the significant mediator between mindfulness , change , and recovery from depression . a recent meta - analysis of compassion focused interventions found good evidence of effectiveness . although compassion cultivation training can be helpful , therapists need to be aware that they can run into serious obstacles along the way . as noted above , there are a range of physiological systems that may be compromised and make compassion processing difficult . a series of studies suggest that some patients have negative beliefs about compassion and generally being kind and supportive to oneself . these include ideas about not deserving it , no trusting it , or seeing it as a weakness or an indulgence . in addition , when some individuals , especially those from a neglectful and abusive background , start to experience compassion , this can ignite a powerful grief process . importantly too , early trauma can create body memories that can block compassion and make experiencing caring and affiliation from others frightening . we should not be surprised by the power of prosocial motives and emotions to create contexts for health , because supportive , affiliative , and helpful relationships provide major benefits for survival and reproduction . the mammalian and human brain and body are highly adapted to be regulated through social relationships . moreover , it has been known for a long time that secure attachment and ongoing support throughout life provide major buffers against stress and vulnerability . despite this , it is comparatively recently that researchers have begun to look at people 's psychological and physiological capacities and competencies for prosocial processing as a way of helping to stabilize affect regulation and sense of self . most pharmacological models ignore these complexities and focus mostly only on threat the physiologies of processing , eg , of anxiety or depression , rather than their regulators , such as social contexts , sense of self - identity , shame , trauma memories , or prosocial competencies and motives . as we understand more about the regulating processes underpinning prosocial relating , we will be better able to develop therapies for cultivating them , and not simply rely on trying to tone down the threat system , be it with drugs , various forms of exposure , or cognitive reappraisals .

this paper argues that studies of mental health and wellbeing can be contextualized within an evolutionary approach that highlights the coregulating processes of emotions and motives . in particular , it suggests that , although many mental health symptoms are commonly linked to threat processing , attention also needs to be directed to the major regulators of threat processing , ie , prosocial and affiliative interactions with self and others . given that human sociality has been a central driver for a whole range of human adaptations , a better understanding of the effects of prosocial interactions on health is required , and should be integrated into psychiatric formulations and interventions . insight into the coregulating processes of motives and emotions , especially prosocial ones , offers improved ways of understanding mental health difficulties and their prevention and relief .

Introduction Evolution and motivation Types of social affiliation Evolution and emotion Some (neuro)physiological mediators of prosocial motives and emotions and mental health Problems with the neurophysiology of prosocial relating to self and others Training in compassion and prosocial behavior Conclusion

until recently , conceptual and empirical research on psychopathology has tended to focus on symptomatology and on grouping symptoms into syndromes . so , for example , studies of anxiety or depression focus on the neurophysiological , behavioral , or cognitive components of the symptoms of anxiety and depression . in the last 20 years , however , evolutionary approaches to psychopathology have emerged that focus on evolved strategies and their complex regulators . this directs attention to the evolved functions of motives and emotions , and , importantly for this paper , how they can suppress and coregulate each other . the evolutionary model starts with the fact that a range of emotional and motivational systems evolved because they helped meet the challenges of survival and gene replication in competitive environments . brains and minds therefore monitor their social contexts and change their relationship to them with regard to risks , opportunities , and potential supports . it is now recognized that one of the most important strategic adaptations for primates , and especially humans , is sociality ; we are basically wired to seek helpful connections with others . it is our human motivation for connecting , relating , and communicating that has driven social intelligence , from toolmaking to science , and more recently , the proliferation of communication technologies , social media on a global scale , and emotion regulation . within this drive for connection and relating , humans play out a complex variety of motivations and interacting styles with each other ( eg , they can be supportive , caring , and desirous , or indifferent , exploitative , or hostile ) . what has become clear over the last 20 years is that certain motivational and emotional processes , which are linked to phenotypic and strategic orientations , are more strongly linked to physical and mental health than others . in brief , threat focus and competitive self - focus may be less conducive to wellbeing than a prosocial focus on self and others . in fact , prosocial relationships have major impacts on a range of physiological systems , including genetic expression . fredrickson al found that eudemonic well - being ( positive emotion associated with meaning and helping others ) , in contrast to self - focused pleasure and hedonic well - being , was linked to better physiological profiles involving proinflammatory genes . hence , facilitating affiliative and prosocial processing , rather than ( just ) reducing threat processing or enhancing self - focused competitiveness , becomes the target for therapeutic and preventative interventions . the sections below explore the ways that prosocial behavior is linked to motives and emotions that alleviate and help prevent mental health difficulties . obvious nonsocial ones are harm avoidance , food , and shelter - seeking . in the social domain , for example , sexual behavior involves a ( courting ) dance between two partners , and at any point , miscommunication can result in one partner attacking or taking flight from the other . social motives that require specialist competencies for processing potentially rapidly changing , dynamic , and reciprocal interactional sequences , have been referred to as social mentalities . central to this paper is the issue that different motives and social mentalities organize the brain in very different ways . given that many of our evolved motivational systems are competing for expression and are sensitive to social contexts , this raises the issue of how different motivational systems are related to health , and to vulnerability to psychopathology . to explore this we would ideally have a nosology of motives , but there are no agreed evolution - derived nosologies for social motives and mentalities for example , among the most common social ones are : competition , cooperation , and alliance building ; care providing , care seeking , and sexual . various blends of these would include desires for power , achievement , connection , belonging , socializing , sex , and so on . we can note how different social mentalities create different patterns in a range of biopsychosocial processes , by contrasting two of them such as competitive and caring . individuals who are orientated through competitive motives are highly focused on rank and power issues , shame / pride , are very sensitive to social comparison , vulnerable to envy , and are self - focused and self - monitoring . in contrast , caring motivations do not utilize these social processing competencies , but instead are highly sensitive to signals of distress and needs , and recruit sympathetic and caring competencies . individuals engaged in competitive interactions will be ( neuro)physiologically and psychologically organized in different ways , compared with caring and supportive interactions . they found that : compassion induction was associated with activation in the midbrain periaqueductal gray ( pag ) , a region that is activated during pain and the perception of others ' pain , and that has been implicated in parental nurturance behaviors . pride induction engaged the posterior medial cortex , a region that has been associated with self - referent processing , ( p. 635 ) such studies seek to identify different neurological patterns associated with different motivational systems . the bottom line of this is that prosocial motives that are associated with taking an interest in caring for others , in contrast to self - focused ( competitive ) motives to get ahead or avoid being inferior , shamed , and rejected , are associated with improved well - being and reduce vulnerabilities to psychopathology . derived from social mentality theory , mcewan et al showed that in students and a depressed population , depression was linked to thwarted competitive motives ( feeling inferior , unable to compete in the world , feeling like a loser ) but was not related to caring motives ( being kind , helpful , and trustworthy ) ; depressed patients do not feel inferior in caring domains . in addition , depressed people can be fearful of being open to receiving compassion from others , as well as blocking self - compassion . in regard to motivational systems underpinning prosocial motivation and emotion , there appear to be two main forms . the other relates to alliance - building , cooperation , and friendship - network formations . the simplest form of relating arises in dyads where two individuals come together for various reasons such as caring , helping , sharing , or sexual engagement . working together , with a number of others , and feeling part of a group , from me - ness to we - ness , expands out into issues of group identity and group belonging . it is well - known that kin - like , intimate and close , prosocial , loving , and caring early attachment relationships provide a wealth of resources which shape physiological systems and set phenotypes for increased health and well - being , whereas neglect and/or abuse do the opposite . in addition , there is now considerable evidence that alliances and friendships also play vital roles in physical and mental health , while , in contrast , loneliness and a lack of cooperating alliances ( friendships ) are highly detrimental to well-being.indeed , studies of loneliness show it to be associated with a range of physiological problems , including adverse effects on telomere length . in many forms of psychological difficulty feeling alone , different , and separate(d ) from others , is a very common experience . in addition , shame and self - criticism not only constitute negative self - experiences , in contrast to prosocial , liking , and helpful relationships with oneself , they also interfere with people 's abilities and confidence to develop supportive , affiliative relationships with others . people can be fearful of others being compassionate towards them because of what ( they fear ) might be discovered about them if other get too close , and the risk of rejection . such fears disrupt the potential benefits of prosocial relationships and are associated with depression and anxiety . researchers have long sought to derive an evolution - based , functional classification of emotions and identify their universal regulators , and roles in clinical problems . these include : ( i ) emotions for seeking rewards / resources ; ( ii ) emotions linked to lust which are particularly ( but not only ) focused on sexual stimuli ; ( iii ) emotions linked to caring and affection ; ( iv ) emotions linked to loss and feelings of grief ; ( v ) threat emotions of rage ; and ( vi ) fear ; ( vii ) emotions that are linked to play and give a sense of joyfulness in activities . derived from the work of depue and morrone - strupinsky , ledoux , panksepp , and others , based on clinical observation , and focusing on more general rather than specific functions , gilbert highlighted three core evolved functions of emotions , loosely identified as : emotions that serve the functions of threat detection and generating defensive and safety strategies emotions that serve the functions of detecting , energizing and seeking / acquiring resources for survival and reproduction emotions that serve the function of contentment , satisfaction , calming , settling , and allowing rest and digest . systems , it is more accurate to see them as rooted in patterns of ( neuro ) physiological activation that are constantly blending and coregulating . for example , a threat to people we love raises anxiety , or anger to those who are threatening ; spending time with friends and loved ones can be activating , enjoyable , and exciting ; being in the presence of caring others helps us feel safe and content , and when we are distressed , can be soothing and calming . the reasons why social relationships that signal either threat or positive support / help , have such powerful psychophysiologically regulating impacts , are related to our evolution as socially interacting mammals ; indeed there is general agreement that it was our sociality that drove human intelligence . gilbert bowlby , and mikulincer and shaver highlighted the fact that early attachments provide the young with proximity maintenance , a secure base and safe haven that are fundamental for development . a secure base provides the encouragement and confidence to go out and explore the world ; a safe haven provides a source to return to for protection , soothing , and calming should the infant become distressed . importantly , signals indicating a presence or absence of a secure base and safe haven are linked to all three systems . the value of this way of thinking can be seen when working with trauma , particularly in veterans . during their training , and while on missions , military personnel will experience elevated threat . in essence , the secure base and safe haven become ( re)wired from family and home to signals of the presence of buddies . when they return home with these rewired systems , a sense of connectedness and soothing may ( for some ) no longer be stimulated by wife and children , or even old friends . the removal of a sense of buddy - connectedness and safeness signals can activate the threat - vigilance systems ; typical of major loss / separation . so veterans can experience high levels of threat from the sudden removal of important safeness signals , with a yearning to return to be with their ( safe haven ) buddies . some believe they ought to feel safe and secure ( or excited ) back with their families and are not aware that all three systems have been ( re)wired and so it will take time for them to be wired back into civilian , social contexts . explaining the three - circle model and the possible process of rewiring according to context , to them and their families , can be very helpful and deshaming . trying to understand trauma only through focusing on threat processing will be limited without also discussing the notions of secure base , safe haven , and how the parasympathetic system is linked to the functioning of the soothing system . indeed , understanding the importance of the buddy system has stimulated work on the value of recruiting buddies and fellow veterans in the treatment of trauma . there are a number of key physiology adaptations that have facilitated enhanced affiliative and care - focused relating . amongst evolved challenges for mammalian sociality are : ( i ) for close proximity not to trigger fear / flight or anger / fight ; and ( ii ) provide advantages that support survival . in addition , play became a key source for mammalian interaction , especially where offspring are living close together . porges suggests that the evolution of the myelinated vagus nerve was one part of the solution to such challenges . he has been at the forefront of suggesting that the myelinated parasympathetic system provides the physiological bases to put a brake on predatory , fight - and - flight behavior when individuals are in close proximity , and instead facilitate soothing and calming in the context of close relationships . this provides physiological infrastructures for the development of caring and cooperation ; to enable individuals to help each other . although some of the details of polyvagal theory have been questioned , there is good evidence now that this branch of the parasympathetic system is associated with prosocial behavior and well - being ( see kogan et al for a major review ) . a common way to explore the sympathetic - parasympathetic balance is with measures of heart rate variability there is now good evidence that heart rate variability is an indicator for a range of physical or mental health difficulties and is strongly linked to the quality of social relationships . another key evolved physiological factor in prosociality is the hormone oxytocin , a nine - amino - acid neuropeptide hormone , produced in the hypothalamus . oxytocin plays a fundamental role in a range of physiological processes throughout the body and is a key hormone in prosocial and affiliative behaviors . oxytocin was central to the evolution of the mammalian caring / attachment behavior and now supports conspecific recognition , monogamous bonding , kin - attachment and bonding , increases trust , improves competencies in mind reading tasks , increases feelings of liking others , and reduces activation in the amygdala to threat . importantly , it plays a significant role in threat regulation in general , there being oxytocin receptors in the amygdala . from an evolutionary point of view , oxytocin guides people to be selective in the choice of targets on which they focus prosocial behavior . a central argument of this section is that understanding how affiliative and prosocial systems work is key to understanding threat processing and psychopathology ; one can not understand psychopathology by analyzing threat or symptoms alone . it 's important then to recognize that the ( neuro)physiological mechanisms that support prosocial and affiliative behavior have been identified as problematic in many people with psychological difficulties . given parasympathetic functioning , and in particular heart rate variability , is linked to prosociality and mental well - being , kemp and quintana have provided a major overview of the link between poor heart rate variability and a range of psychological and physical difficulties . low resting heart rate variability has knock - on effects to cardiovascular and immune systems , as well as mood regulation . not only is there a direct connection between feeling socially connected and heart rate variability ; gillie and thayer review the evidence that parasympathetic tone is very important for executive control and the integration of frontal cortical systems with deeper brain systems . they describe how executive control is compromised in people with post - traumatic stress disorder , and identify difficulties in parasympathetic regulation as a potential source . austin et al found that people with borderline difficulties ( reflected in difficulties in regulating emotions , a fragile sense of self , and problems in interpersonal relationships ) do not differ in terms of sympathetic activation to threat compared with controls , but differ significantly in the parasympathetic responses to potential helpfulness actually showing a more fight - flight profile in situations of helpfulness . in a study looking at people 's response to compassion , which involved imagining receiving kindness and compassion from others , rockliff et al found that , when trying to imagine a compassionate other , low self - criticism and secure attachment were associated with improved / increased heart rate variability , but high self - criticism and insecure attachment were associated with a worsening of heart rate variability . for low self - critics , self - reassurance was associated with brain areas for calming . however , for those with higher self - criticism , efforts to be self - reassuring were associated with threat areas , such as the amygdala . hence , for some individuals , efforts to be compassionate , reassuring , and kind to oneself activate threat systems . so , there are many studies suggesting that some of the core physiological systems for prosocial emotion , thinking , and behavior are compromised in people with mental health problems . people are not able to use the parasympathetic - based soothing system for affect regulation and executive control . looking at another major physiological system for prosociality , oxytocin , yuen this may account for some of the depressed person 's feelings of social disconnection , being cut off and different from others , separated , isolated , and lonely . stanley and siever suggested that a number of the interpersonal and emotion regulation difficulties associated with borderline personality disorder are indications of problems in oxytocin . rockliff et al explored the impact of nasal oxytocin and placebo on the ability to generate and feel reassured by compassionate images . given that compassionate and prosocial behavior have such powerful influences on a range of physiological , psychological , and social processes , it follows that training people to cultivate compassion motives and emotions may be therapeutic . this raises the question about the focus for compassion , because compassion can be explored in relation to compassion we have for others , the way we respond to the compassion from others and self - compassion . hutcherson et al found that a brief loving - kindness meditation increases feelings of social connectedness and affiliation towards strangers . fredrickson et al found that six 60-minute weekly group sessions with home practice based on a cd of loving kindness meditation ( compassion directed to self , then others , then strangers ) increases positive emotions , mindfulness , feelings of purpose in life , and social support , and decreases illness symptoms compared with a control group . the beneficial effects of compassion cultivation are not just linked to meditations but to values , and to the ways we live our lives . psychotherapy has also begun to focus on compassion and prosocial cultivation as therapeutic targets in their own right . focusing on the experience and development of compassion has been found to reduce depression , anxiety , and self - criticism in people presenting to a community mental health team , in people with long - term mental health problems , and people in a high - security psychiatric setting . compassion - focused therapy ( cft ) has been shown to be helpful for people with psychosis ; and for people with emotional difficulties and personality disorders . ashworth et al found cft to be a valuable addition in helping people with acquired brain injury . some researchers have focused on a particular and specific kind of self - compassion that involves cultivation of mindfulness ( rather than attentional absorption in difficulties ) , a sense of common humanity ( rather than a sense of shame and isolation ) , and non - judgement ( rather than self - criticalness ) . recent trials in nonclinical populations have shown this to be beneficial to well - being . for depressed people , kuyken et al found that in a mindfulness trial , self - compassion was the significant mediator between mindfulness , change , and recovery from depression . although compassion cultivation training can be helpful , therapists need to be aware that they can run into serious obstacles along the way . as noted above , there are a range of physiological systems that may be compromised and make compassion processing difficult . a series of studies suggest that some patients have negative beliefs about compassion and generally being kind and supportive to oneself . in addition , when some individuals , especially those from a neglectful and abusive background , start to experience compassion , this can ignite a powerful grief process . we should not be surprised by the power of prosocial motives and emotions to create contexts for health , because supportive , affiliative , and helpful relationships provide major benefits for survival and reproduction . the mammalian and human brain and body are highly adapted to be regulated through social relationships . moreover , it has been known for a long time that secure attachment and ongoing support throughout life provide major buffers against stress and vulnerability . despite this , it is comparatively recently that researchers have begun to look at people 's psychological and physiological capacities and competencies for prosocial processing as a way of helping to stabilize affect regulation and sense of self . most pharmacological models ignore these complexities and focus mostly only on threat the physiologies of processing , eg , of anxiety or depression , rather than their regulators , such as social contexts , sense of self - identity , shame , trauma memories , or prosocial competencies and motives . as we understand more about the regulating processes underpinning prosocial relating , we will be better able to develop therapies for cultivating them , and not simply rely on trying to tone down the threat system , be it with drugs , various forms of exposure , or cognitive reappraisals .

traumatic brain injury ( tbi ) is considered the signature injury of soldiers fighting in iraq and afghanistan . between january 2003 and september 2009 , 63,856 service men and women received a diagnosis of tbi . tbi contributes to cognitive impairment , including deficits in planning , problem solving skills , memory , attention , and communication . for some veterans , underlying post - traumatic stress disorder ( ptsd ) and depressive symptoms also complicate the diagnosis and treatment of tbi and may impede recovery . thus , rehabilitation following tbi should promote understanding of and therapy for co - morbid conditions . although the 3 diagnoses ( tbi , ptsd , and depression ) may occur independently in any one patient , the incidence of overlapping diagnoses makes it sensible to combine information about all 3 into 1 intervention . veterans who need intervention also face special challenges in that many are returning to pre - existing jobs and families , and are unable to take time off from work and time out of family functions to receive treatment in a hospital or clinic . the development of a therapeutic intervention that can be achieved at home or work remains paramount given the high incidence of feelings of isolation , loneliness , and anger following combat . the veteran s health administration ( vha ) began using videoconference phones in 2007 to connect directly into the patient s home . as reported by darkins , the vha telemedicine programs have reduced hospitalization , length of stay , and emergency room visits while improving the quality of life for veterans . evaluation of novel telemedicine practices follow a multi - phase process that involves : ( 1 ) identification of a need that warrants treatment provided remotely , ( 2 ) comparison of efficacy between the telehealth treatment and clinic - based treatment [ 47 ] , and ( 3 ) evaluation of participant satisfaction . to date , the majority of studies have found that improvements following intervention via telepractice are approximately equal to those following clinic - based intervention when the results are based on objective measures of progress [ 47 ] . in some cases , measures of patient satisfaction this is presumably due to practical factors : patients can participate in therapy on a schedule that fits their life situation . an online treatment , the military on - line problem solving videophone intervention ( mops - vi ) was developed to address cognitive symptoms of mild tbi in veterans . the mops - vi design adhered to the tenets of traditional cognitive therapy , including duration of treatment , but utilized the internet and videoconference phones to increase treatment adherence . mops - vi taught participants how to compensate for their deficits through utilizing strategies to improve quality of life . participants were provided with functional strategies to improve attention and memory such as writing things down or taking frequent breaks during a time - consuming task . videophones , approved by the department of veterans affairs for use with veterans , were used to provide weekly live support with a speech - language pathologist experienced in treating tbi . the purpose of the present study was 2-fold : 1 ) to determine if treatment adherence improved with a telemedicine approach , and 2 ) to test the efficacy of an online cognitive therapy and education program ( mops - vi ) in improving cognitive functioning in veterans with mild tbi . veterans completed the mops - vi treatment in the convenience of their home or workplace . potential subjects for this phase i study reviewed and signed the informed consent approved by the institutional review board approved by the cincinnati veterans administration medical center and the university of cincinnati before beginning the study . the study was a matched - subject pre / post design , which compared test data collected from 6 veterans who participated in a specialized mops - vi intervention for tbi ( pre- and post - treatment ) vs. test data from 6 veterans who participated in standard clinic - based treatment for tbi ( pre- and post - treatment ) . mops - vi participants were enrolled from 3 cincinnati va tbi clinics and were referred for speech therapy in the clinic , and either : ( 1 ) never came to the initial speech therapy session for a baseline interview , ( 2 ) came for an initial speech therapy session and did not return for follow - up , or ( 3 ) came for less than 2 sessions and did not continue in treatment . initial contact with potential treatment group participants was made by a letter offering participation in the mops - vi program . a postage - paid card was sent with a letter that contained a response box for the participant to indicate interest in participating in the mops - vi study . a member of the research team made a follow - up phone call to potential participants who had not yet responded . inclusion criteria for treatment group ( mops - vi group ) : english as the primary language spoken in the home ; age 20 to 43 years at the time of treatment ; complaints of memory , attention and or concentration deficits on a subjective self - report section of the neurobehavioral symptom inventory ( nsi ) , defined as self - selecting scores in the range of 24 ( indicating moderate to severe impairment per participant report ) on items 15 m , for example , on item15 m the patient is asked to rate the degree to which poor concentration , can not pay attention applies along a continuum between 0 ( indicating the symptom is rarely if ever present ) and 4 ( very severe , the symptom is almost always present and the person has been unable to perform at work , school , or home due to this problem ) . each participant in the mops - vi group was matched to a control participant based on the following factors : age , gender , marital status , and baseline composite scores on the tomal-2 test . control participants were obtained by medical chart review from august 2007 through october 2011 of operation enduring freedom and operation iraqi freedom ( oef / oif ) veterans who were previously treated and discharged from the speech tbi clinic following completion of clinic - based cognitive rehabilitation . following baseline testing , all control participants received education about tbi , memory and attention processes , and compensatory strategies to improve quality of life . this format loosely mirrors the content provided in the mops - vi intervention , with the modification of delivery mode . criteria for matching treatment and control group participants : tomal-2 edition pretreatment scores participants were matched according to their scores on the composite memory index ( 10 out of a possible 100 points ) at the start of therapy . the composite memory index consists of 8 core subtests : ( 1 ) memory for stories , ( 2 ) facial memory , ( 3 ) word - selective reminding , ( 4 ) abstract visual memory , ( 5 ) object recall , ( 6 ) visual sequential memory , ( 7 ) paired recall , and ( 8) memory for location . participants were also matched in pairs on age ( 3 years ) , marital status , ( married / partnered ) , and years of education ( 2 years ) . exclusion criteria for both groups : active treatment for substance abuse , including but not limited to , drugs and alcohol . mops - vi participants came to the cincinnati veterans medical center ( cvamc ) speech - language pathology department for preliminary testing . the peabody picture vocabulary test ( ppvt ) was administered pre - intervention to obtain verbal intelligence scores because pre - injury iq scores were not available . the tomal-2 was selected as a primary outcome given the ease of use , utilization across control subjects , and endorsement by the american veterans association speech - language pathology network . the tomal-2 was administered pre- and post - intervention as an assessment of treatment effects on cognitive functioning . the measures are widely used and have acceptable reliability and validity . in addition , the participants were interviewed to determine highest level of education , number of combat tours , and marital or relationship status . common cognitive complaints among the control and mops - vi participants include deficits in attention , concentration , and memory . participants specifically noted difficulty with medication management , impaired geographical orientation , inability to establish and adhere to routines , and problems paying attention in a classroom or work environment . each mops - vi participant was provided a laptop computer , wireless internet , and a videoconference phone for the duration of the intervention . behavioral issues targeted were related to pragmatics or social communication and were within the scope and practice for licensed speech - language pathologists . for both mops - vi and the control group , intervention followed a conventional approach in that it had dual foci : ( 1 ) education about tbi and ptsd with the aim of improving knowledge about the conditions , and ( 2 ) a therapeutic cognitive intervention focused on memory and attention . the mops - vi portion of the study was designed to be as similar as possible to the control treatment , with the difference that the control treatment was conducted face - to - face in a clinic setting , but the mops - vi was designed to deliver treatment online . the mops - vi incorporated military themes and didactic video clips of veterans discussing how their everyday life had changed since their injury , and how particular strategies helped them . module number 2 , staying positive , highlighted common concerns and problems that veterans may face following a brain injury and provided strategies to improve attitude and communication with others . , focused on organizational strategies to reduce extraneous stress and minimize problems at work , home , or school . the content focused on planning for success using a self - monitoring mnemonic , smart ( stop monitor appraise reflect try ) . module number 5 , controlling your behavior , placed emphasis on participants monitoring and changing their behavior . mops - vi participants were issued a televyou 500sp videophone ( figure 1 ) for use during simulated face - to - face therapy and were required to sign a waiver agreeing to return the videophone upon completion of the study . a more commonly used communication method such as skype would have been the preferred method of simulated face - to - face therapy ; however , va regulations prohibited the use of skype or anything similar . the authors acknowledged the videophones as an inferior technology to what was commercially available at the time . the televyou 500sp utilizes pots ( plain old telephone service ) and is va approved for encryption purposes . all treatment group participants were trained in how to plug the standard telephone wire into the videophone , in addition to making simulated videophone calls while in the presence of the pi to ensure sufficient participation usability . all videophone contact with treatment participants was therapist - generated at a pre - determined appointment time . during the first videophone conference , the pi contacted each participant weekly via videophone to review the session materials and provided a reminder follow - up call to complete the modules if indicated . these videophone sessions lasted approximately 60 minutes , for a total treatment time of 60 minutes per week . additional time was allotted to complete the intervention to take into account unanticipated events such as family illness / death or vacations . thus , the time to complete the intervention varied between participants ( range , 35 months ; average , 3.8 months ) . following completion of all modules , participants were asked to return to the vamc speech - language pathology department , at which time the tomal-2 was re - administered . descriptive data were obtained for the treatment and the control groups ( means and standard deviations or frequencies and percentages , as appropriate . ) in addition , t - tests were used to ensure that the treatment and the control groups were equivalent in age , education , and vocabulary scores as an estimate of iq ( i.e. , ppvt ) . next , the pre - post tomal 2 scores were compared for both groups at 2 points in time ( pre - post test results ) using analysis of variance ( anova ) and the wilcoxon signed ranks test . all data management and analyses potential subjects for this phase i study reviewed and signed the informed consent approved by the institutional review board approved by the cincinnati veterans administration medical center and the university of cincinnati before beginning the study . the study was a matched - subject pre / post design , which compared test data collected from 6 veterans who participated in a specialized mops - vi intervention for tbi ( pre- and post - treatment ) vs. test data from 6 veterans who participated in standard clinic - based treatment for tbi ( pre- and post - treatment ) . mops - vi participants were enrolled from 3 cincinnati va tbi clinics and were referred for speech therapy in the clinic , and either : ( 1 ) never came to the initial speech therapy session for a baseline interview , ( 2 ) came for an initial speech therapy session and did not return for follow - up , or ( 3 ) came for less than 2 sessions and did not continue in treatment . initial contact with potential treatment group participants was made by a letter offering participation in the mops - vi program . a postage - paid card was sent with a letter that contained a response box for the participant to indicate interest in participating in the mops - vi study . a member of the research team made a follow - up phone call to potential participants who had not yet responded . inclusion criteria for treatment group ( mops - vi group ) : english as the primary language spoken in the home ; age 20 to 43 years at the time of treatment ; complaints of memory , attention and or concentration deficits on a subjective self - report section of the neurobehavioral symptom inventory ( nsi ) , defined as self - selecting scores in the range of 24 ( indicating moderate to severe impairment per participant report ) on items 15 m , n , o , and p on the nsi . for example , on item15 m the patient is asked to rate the degree to which poor concentration , can not pay attention applies along a continuum between 0 ( indicating the symptom is rarely if ever present ) and 4 ( very severe , the symptom is almost always present and the person has been unable to perform at work , school , or home due to this problem ) . each participant in the mops - vi group was matched to a control participant based on the following factors : age , gender , marital status , and baseline composite scores on the tomal-2 test . control participants were obtained by medical chart review from august 2007 through october 2011 of operation enduring freedom and operation iraqi freedom ( oef / oif ) veterans who were previously treated and discharged from the speech tbi clinic following completion of clinic - based cognitive rehabilitation . following baseline testing , all control participants received education about tbi , memory and attention processes , and compensatory strategies to improve quality of life . this format loosely mirrors the content provided in the mops - vi intervention , with the modification of delivery mode . criteria for matching treatment and control group participants : tomal-2 edition pretreatment scores participants were matched according to their scores on the composite memory index ( 10 out of a possible 100 points ) at the start of therapy . the composite memory index consists of 8 core subtests : ( 1 ) memory for stories , ( 2 ) facial memory , ( 3 ) word - selective reminding , ( 4 ) abstract visual memory , ( 5 ) object recall , ( 6 ) visual sequential memory , ( 7 ) paired recall , and ( 8) memory for location . participants were also matched in pairs on age ( 3 years ) , marital status , ( married / partnered ) , and years of education ( 2 years ) . exclusion criteria for both groups : active treatment for substance abuse , including but not limited to , drugs and alcohol . mops - vi participants came to the cincinnati veterans medical center ( cvamc ) speech - language pathology department for preliminary testing . the peabody picture vocabulary test ( ppvt ) was administered pre - intervention to obtain verbal intelligence scores because pre - injury iq scores were not available . the tomal-2 was selected as a primary outcome given the ease of use , utilization across control subjects , and endorsement by the american veterans association speech - language pathology network . the tomal-2 was administered pre- and post - intervention as an assessment of treatment effects on cognitive functioning . in addition , the participants were interviewed to determine highest level of education , number of combat tours , and marital or relationship status . common cognitive complaints among the control and mops - vi participants include deficits in attention , concentration , and memory . participants specifically noted difficulty with medication management , impaired geographical orientation , inability to establish and adhere to routines , and problems paying attention in a classroom or work environment . each mops - vi participant was provided a laptop computer , wireless internet , and a videoconference phone for the duration of the intervention . behavioral issues targeted were related to pragmatics or social communication and were within the scope and practice for licensed speech - language pathologists . for both mops - vi and the control group , intervention followed a conventional approach in that it had dual foci : ( 1 ) education about tbi and ptsd with the aim of improving knowledge about the conditions , and ( 2 ) a therapeutic cognitive intervention focused on memory and attention . the mops - vi portion of the study was designed to be as similar as possible to the control treatment , with the difference that the control treatment was conducted face - to - face in a clinic setting , but the mops - vi was designed to deliver treatment online . the mops - vi incorporated military themes and didactic video clips of veterans discussing how their everyday life had changed since their injury , and how particular strategies helped them . module number 2 , staying positive , highlighted common concerns and problems that veterans may face following a brain injury and provided strategies to improve attitude and communication with others . module number 3 , titled getting organized , focused on organizational strategies to reduce extraneous stress and minimize problems at work , home , or school . the content focused on planning for success using a self - monitoring mnemonic , smart ( stop monitor appraise reflect try ) . module number 5 , controlling your behavior , placed emphasis on participants monitoring and changing their behavior . mops - vi participants were issued a televyou 500sp videophone ( figure 1 ) for use during simulated face - to - face therapy and were required to sign a waiver agreeing to return the videophone upon completion of the study . a more commonly used communication method such as skype would have been the preferred method of simulated face - to - face therapy ; however , va regulations prohibited the use of skype or anything similar . the authors acknowledged the videophones as an inferior technology to what was commercially available at the time . the televyou 500sp utilizes pots ( plain old telephone service ) and is va approved for encryption purposes . all treatment group participants were trained in how to plug the standard telephone wire into the videophone , in addition to making simulated videophone calls while in the presence of the pi to ensure sufficient participation usability . all videophone contact with treatment participants was therapist - generated at a pre - determined appointment time . during the first videophone conference , the pi contacted each participant weekly via videophone to review the session materials and provided a reminder follow - up call to complete the modules if indicated . these videophone sessions lasted approximately 60 minutes , for a total treatment time of 60 minutes per week . additional time was allotted to complete the intervention to take into account unanticipated events such as family illness / death or vacations . thus , the time to complete the intervention varied between participants ( range , 35 months ; average , 3.8 months ) . following completion of all modules , participants were asked to return to the vamc speech - language pathology department , at which time the tomal-2 was re - administered . descriptive data were obtained for the treatment and the control groups ( means and standard deviations or frequencies and percentages , as appropriate . ) in addition , t - tests were used to ensure that the treatment and the control groups were equivalent in age , education , and vocabulary scores as an estimate of iq ( i.e. , ppvt ) . next , the pre - post tomal 2 scores were compared for both groups at 2 points in time ( pre - post test results ) using analysis of variance ( anova ) and the wilcoxon signed ranks test . one of the aims of this study was to test the effect of mops - vi on patient compliance . participants were selected from a group of veterans initially non - adherent with clinic - based cognitive rehabilitative services . non - adherence was defined by the following criteria : 1 ) failure to schedule a follow - up appointment after referral for cognitive rehabilitation therapy ; or , 2 ) failure to attend more than 2 therapy sessions . it was hypothesized that the mops - vi therapeutic approach would increase adherence because of decreased travel time and increased scheduling convenience . among the veterans identified as non - adherent with speech therapy for cognitive rehabilitation services using these criteria , 9 signed the informed consent , 8 participated in pre - intervention testing , and 6 ( 67% ) completed the intervention . the 3 who did not complete the intervention included 1 participant who signed the informed consent and was unable to be contacted for baseline testing , a second participant who dropped out after being diagnosed with cancer , and a third participant who was unable to complete the intervention due to redeployment shortly after completing baseline testing . six veterans who initially did not follow through with clinic - based cognitive treatment completed the mops - vi telemedicine treatment . these 6 mops - vi participants ranged in age from 23 to 38 years , with a mean age of 30.17 years . the control group consisted of participants seen face - to - face ( clinic - based ) who were selected according to inclusion / exclusion data and were similar to the treatment group participants . of the 6 participants who completed the intervention , most of the sessions were completed sequentially and according to schedule . the few exceptions included 1 participant who re - scheduled his initial videophone session 2 times because he had not yet completed the self - guided module preceding the session and required 2 sessions for post - intervention testing . an additional participant called to re - schedule his third videophone session because of traffic . detailed compliance data was not available for control group participants due to the study design . there were no significant differences between the groups in mean age , marital status , years of education , and tomal-2nd pre - test composite score , indicating that the matching procedure was successful . results of a 2-way ( pre- vs. post- assessments ) ( standard vs. mops - vi ) analysis of variance ( anova ) revealed a significant pre - post assessment effect , f(1,10)=50.38 , p<0.001 , indicating that participants memory improved after treatment for both mops - vi and standard treatment groups ( table 2 ) . there was no significant interaction between treatment groups and pre - post assessment , f(1,10)=3.43 , p=0.09 . there was no significant difference between face - to - face therapy and mops - vi therapy ( f(1 , 10)=0.39 , p=0.55 ) suggesting that mops - vi therapy is as effective as clinic - based treatment and therefore is a viable alternative . because of the small n of the mops - vi treatment group , the nonparametric test , the wilcoxon signed ranks test was also conducted to compare the composite memory index score pre- and post - intervention . the z score was 2.21 with a p=0.027 . therefore , both the parametric and nonparametric statistical analyses indicated a significant improvement in pre- and post - treatment performance on the tomal-2 . partial eta - squared was used to provide an estimate of the magnitude of the effects of treatment as assessed by changes over time . the change in tomal-2nd scores from the pre- to post - treatment contributed 0.83 , which indicates a strong effect of treatment on composite test scores regardless of whether the treatment was clinic - based or mops - vi ( figure 2 ) . although both therapy groups experienced a positive change in tomal-2 test scores , the mops - vi group spent approximately 6 months less time in treatment ( mean=3.87 ) compared to the control group ( mean=9.33 ) . mops - vi participants were recruited from the 36% of veteran s non - adherent to standard face - to - face treatment . these numbers suggest that of 100 randomly chosen patients , 64 would complete treatment if offered clinic - based therapy , and 36 would drop out , but 27 ( 67% ) would complete treatment if offered mops - vi . this suggests that if both clinic - based therapy and mops - vi were offered as alternative treatments to veterans , a 91% compliance rate could be achieved with no diminution in the quality of results . this would be a major advance in the effort to maximize these veterans ability to transition to normal lives . a consensus conference sponsored by the national institutes of health outlined the characteristics thought to define effective cognitive interventions in tbi . their recommendations included the following optimal characteristics of cognitive therapy : structured , systematic , goal - directed , individualized , and involving learning , practice , social contact , and a relevant context . the mops - vi intervention was structured to include modules that were delivered sequentially in a systematic fashion ; participants completed the program at their own pace . a speech - language pathologist called the participants on a weekly basis to provide clarification and support and to encourage use of the strategies in social contexts . these follow - up videophone sessions also facilitated generalization of the content learned in the web - based modules . the cognitive rehabilitation task force ( acrm , bi - isig ) conducted a systematic review of cognitive rehabilitation after tbi , and provided updated evidence - based practice recommendations . the report addressed the full spectrum of tbi severity and recommended the use of attention , problem solving , and memory training during post - acute rehabilitation of individuals with tbi . emphasis is placed on the use in training of metacognitive strategies that increase awareness of anticipated difficulties and help develop online monitoring and self - regulation skills . such skills are thought to be necessary to promote the generalization of newly acquired compensatory strategies to real - world tasks . the mops - vi intervention placed considerable emphasis on metacognitive strategies and generalization and yielded results similar to standard therapy , even though the mops - vi was conducted independently in the veterans homes or workplaces with weekly videophone support over a shorter time period . results of the mops - vi intervention are consistent with the findings of cicerone , who reported that cognitive therapies directed at multiple domains of impairment can significantly improve neuropsychological performance in particular skill areas ( e.g. , attention , memory , and problem solving ) . the mops - vi is an example of a cognitive intervention that addresses the constellation of neurobehavioral deficits commonly encountered in veterans . the results of the current study suggest that veterans recovering from mild tbi might benefit from an online treatment program with videophone support to improve memory and learning . for example , schoenberg et al . compared the effectiveness of a commercially available computer - based cognitive rehabilitative teletherapy program and traditional outpatient speech therapy , finding that the teletherapy group spent significantly more time in therapy compared to the face - to - face group . however , mops - vi participants spent less time in treatment ( 3.87 months ) than the clinic - based control group ( 9.33 months ) , yet both groups had similar outcomes , suggesting that effective telehealth therapies can be briefer than standard office - based treatments . fluctuating numbers of participants from which to recruit hinders optimal structuring of methodology . in the current study , logistical restraints due to limited funding prohibited an extended time for recruitment , contributing to a small sample size . the researchers evaluated whether or not this intervention improved compliance with cognitive therapy for those who initially did not comply . comparisons were made to those who complied with traditional face - to - face cognitive therapy . although matched groups are not ideal given the baseline variability between the populations ( initially compliant vs. not initially compliant ) , the researchers acknowledge these limitations a priori . the purpose of this study was to test the efficacy of cognitive therapy and education using a telemedicine treatment for veterans with mild tbi in the convenience of their homes or workplaces . there was also a practical advantage to mops - vi ; many individuals requiring therapeutic intervention are unable to take time off from work and time out of family functions to receive treatment in a hospital or clinic . because , because all participants had a diagnosis of a mild tbi , findings may not generalize to a more severely injured sample . nevertheless , we were able to demonstrate improvement in treatment compliance and cognitive test scores following participation in an online program with phone support ( mops - vi ) . the mops - vi resulted in better compliance and a similar degree of improvement in cognitive test scores within a shorter time period than standard clinic - based treatment . overall , these results support the feasibility of the mops - vi approach and provide evidence of efficacy , defined as increased compliance in a group of veterans who were not compliant with traditional face - to - face therapy . furthermore , improvement in standardized cognitive test performance was comparable to clinic - based treatment .

backgroundwe report findings from an intervention study using telehealth modalities to determine whether provision of telehealth services can improve access to care and increase adherence to cognitive therapy in veterans with mild traumatic brain injury ( tbi ) while matching traditional care in terms of outcomes.material/methodsveterans who were initially non - adherent to clinic - based cognitive therapy were offered a newly developed treatment . the control participants were selected from patient records of veterans who had completed cognitive treatment and matched to mops - vi participants on the basis of age , marital or relationship status , and composite memory index score . baseline and post - treatment cognitive functioning as assessed by the test of memory and learning 2nd edition ( tomal-2 ) was obtained for all participants . the mops - vi modules were designed to increase understanding of tbi and elicit problem - solving skills for attention and memory impairment.resultssixty-seven percent of veterans ( who were assigned to the mops - vi treatment group because they were initially non - adherent with the clinic - based treatment ) completed the mops - vi telemedicine treatment . results of a two - way analysis of variance ( anova ) comparing baseline and follow - up scores on the tomal-2 in the mops - vi and control groups revealed there was a significant pre - post assessment effect , indicating that participant s memory and learning improved after treatment for both mops - vi and standard treatment groups . there was no significant difference between clinic - based treatment and mops - vi therapy.conclusionspreliminary evidence supports the efficacy of the treatment , defined as increased compliance in completing the treatment program , and improvements in standardized memory and learning test results comparable to those following clinic - based treatment .

Background Material and Methods Subjects Treatment group (MOPS-VI group) Control group Procedures Data analysis Results Discussion Conclusions

traumatic brain injury ( tbi ) is considered the signature injury of soldiers fighting in iraq and afghanistan . tbi contributes to cognitive impairment , including deficits in planning , problem solving skills , memory , attention , and communication . for some veterans , underlying post - traumatic stress disorder ( ptsd ) and depressive symptoms also complicate the diagnosis and treatment of tbi and may impede recovery . veterans who need intervention also face special challenges in that many are returning to pre - existing jobs and families , and are unable to take time off from work and time out of family functions to receive treatment in a hospital or clinic . evaluation of novel telemedicine practices follow a multi - phase process that involves : ( 1 ) identification of a need that warrants treatment provided remotely , ( 2 ) comparison of efficacy between the telehealth treatment and clinic - based treatment [ 47 ] , and ( 3 ) evaluation of participant satisfaction . to date , the majority of studies have found that improvements following intervention via telepractice are approximately equal to those following clinic - based intervention when the results are based on objective measures of progress [ 47 ] . an online treatment , the military on - line problem solving videophone intervention ( mops - vi ) was developed to address cognitive symptoms of mild tbi in veterans . the mops - vi design adhered to the tenets of traditional cognitive therapy , including duration of treatment , but utilized the internet and videoconference phones to increase treatment adherence . participants were provided with functional strategies to improve attention and memory such as writing things down or taking frequent breaks during a time - consuming task . the purpose of the present study was 2-fold : 1 ) to determine if treatment adherence improved with a telemedicine approach , and 2 ) to test the efficacy of an online cognitive therapy and education program ( mops - vi ) in improving cognitive functioning in veterans with mild tbi . veterans completed the mops - vi treatment in the convenience of their home or workplace . the study was a matched - subject pre / post design , which compared test data collected from 6 veterans who participated in a specialized mops - vi intervention for tbi ( pre- and post - treatment ) vs. test data from 6 veterans who participated in standard clinic - based treatment for tbi ( pre- and post - treatment ) . mops - vi participants were enrolled from 3 cincinnati va tbi clinics and were referred for speech therapy in the clinic , and either : ( 1 ) never came to the initial speech therapy session for a baseline interview , ( 2 ) came for an initial speech therapy session and did not return for follow - up , or ( 3 ) came for less than 2 sessions and did not continue in treatment . initial contact with potential treatment group participants was made by a letter offering participation in the mops - vi program . a postage - paid card was sent with a letter that contained a response box for the participant to indicate interest in participating in the mops - vi study . a member of the research team made a follow - up phone call to potential participants who had not yet responded . inclusion criteria for treatment group ( mops - vi group ) : english as the primary language spoken in the home ; age 20 to 43 years at the time of treatment ; complaints of memory , attention and or concentration deficits on a subjective self - report section of the neurobehavioral symptom inventory ( nsi ) , defined as self - selecting scores in the range of 24 ( indicating moderate to severe impairment per participant report ) on items 15 m , for example , on item15 m the patient is asked to rate the degree to which poor concentration , can not pay attention applies along a continuum between 0 ( indicating the symptom is rarely if ever present ) and 4 ( very severe , the symptom is almost always present and the person has been unable to perform at work , school , or home due to this problem ) . each participant in the mops - vi group was matched to a control participant based on the following factors : age , gender , marital status , and baseline composite scores on the tomal-2 test . control participants were obtained by medical chart review from august 2007 through october 2011 of operation enduring freedom and operation iraqi freedom ( oef / oif ) veterans who were previously treated and discharged from the speech tbi clinic following completion of clinic - based cognitive rehabilitation . following baseline testing , all control participants received education about tbi , memory and attention processes , and compensatory strategies to improve quality of life . this format loosely mirrors the content provided in the mops - vi intervention , with the modification of delivery mode . criteria for matching treatment and control group participants : tomal-2 edition pretreatment scores participants were matched according to their scores on the composite memory index ( 10 out of a possible 100 points ) at the start of therapy . the composite memory index consists of 8 core subtests : ( 1 ) memory for stories , ( 2 ) facial memory , ( 3 ) word - selective reminding , ( 4 ) abstract visual memory , ( 5 ) object recall , ( 6 ) visual sequential memory , ( 7 ) paired recall , and ( 8) memory for location . participants were also matched in pairs on age ( 3 years ) , marital status , ( married / partnered ) , and years of education ( 2 years ) . mops - vi participants came to the cincinnati veterans medical center ( cvamc ) speech - language pathology department for preliminary testing . the tomal-2 was selected as a primary outcome given the ease of use , utilization across control subjects , and endorsement by the american veterans association speech - language pathology network . the tomal-2 was administered pre- and post - intervention as an assessment of treatment effects on cognitive functioning . in addition , the participants were interviewed to determine highest level of education , number of combat tours , and marital or relationship status . common cognitive complaints among the control and mops - vi participants include deficits in attention , concentration , and memory . each mops - vi participant was provided a laptop computer , wireless internet , and a videoconference phone for the duration of the intervention . for both mops - vi and the control group , intervention followed a conventional approach in that it had dual foci : ( 1 ) education about tbi and ptsd with the aim of improving knowledge about the conditions , and ( 2 ) a therapeutic cognitive intervention focused on memory and attention . the mops - vi portion of the study was designed to be as similar as possible to the control treatment , with the difference that the control treatment was conducted face - to - face in a clinic setting , but the mops - vi was designed to deliver treatment online . the mops - vi incorporated military themes and didactic video clips of veterans discussing how their everyday life had changed since their injury , and how particular strategies helped them . mops - vi participants were issued a televyou 500sp videophone ( figure 1 ) for use during simulated face - to - face therapy and were required to sign a waiver agreeing to return the videophone upon completion of the study . all treatment group participants were trained in how to plug the standard telephone wire into the videophone , in addition to making simulated videophone calls while in the presence of the pi to ensure sufficient participation usability . following completion of all modules , participants were asked to return to the vamc speech - language pathology department , at which time the tomal-2 was re - administered . descriptive data were obtained for the treatment and the control groups ( means and standard deviations or frequencies and percentages , as appropriate . ) in addition , t - tests were used to ensure that the treatment and the control groups were equivalent in age , education , and vocabulary scores as an estimate of iq ( i.e. next , the pre - post tomal 2 scores were compared for both groups at 2 points in time ( pre - post test results ) using analysis of variance ( anova ) and the wilcoxon signed ranks test . the study was a matched - subject pre / post design , which compared test data collected from 6 veterans who participated in a specialized mops - vi intervention for tbi ( pre- and post - treatment ) vs. test data from 6 veterans who participated in standard clinic - based treatment for tbi ( pre- and post - treatment ) . mops - vi participants were enrolled from 3 cincinnati va tbi clinics and were referred for speech therapy in the clinic , and either : ( 1 ) never came to the initial speech therapy session for a baseline interview , ( 2 ) came for an initial speech therapy session and did not return for follow - up , or ( 3 ) came for less than 2 sessions and did not continue in treatment . initial contact with potential treatment group participants was made by a letter offering participation in the mops - vi program . a postage - paid card was sent with a letter that contained a response box for the participant to indicate interest in participating in the mops - vi study . a member of the research team made a follow - up phone call to potential participants who had not yet responded . inclusion criteria for treatment group ( mops - vi group ) : english as the primary language spoken in the home ; age 20 to 43 years at the time of treatment ; complaints of memory , attention and or concentration deficits on a subjective self - report section of the neurobehavioral symptom inventory ( nsi ) , defined as self - selecting scores in the range of 24 ( indicating moderate to severe impairment per participant report ) on items 15 m , n , o , and p on the nsi . each participant in the mops - vi group was matched to a control participant based on the following factors : age , gender , marital status , and baseline composite scores on the tomal-2 test . control participants were obtained by medical chart review from august 2007 through october 2011 of operation enduring freedom and operation iraqi freedom ( oef / oif ) veterans who were previously treated and discharged from the speech tbi clinic following completion of clinic - based cognitive rehabilitation . following baseline testing , all control participants received education about tbi , memory and attention processes , and compensatory strategies to improve quality of life . this format loosely mirrors the content provided in the mops - vi intervention , with the modification of delivery mode . criteria for matching treatment and control group participants : tomal-2 edition pretreatment scores participants were matched according to their scores on the composite memory index ( 10 out of a possible 100 points ) at the start of therapy . the composite memory index consists of 8 core subtests : ( 1 ) memory for stories , ( 2 ) facial memory , ( 3 ) word - selective reminding , ( 4 ) abstract visual memory , ( 5 ) object recall , ( 6 ) visual sequential memory , ( 7 ) paired recall , and ( 8) memory for location . participants were also matched in pairs on age ( 3 years ) , marital status , ( married / partnered ) , and years of education ( 2 years ) . mops - vi participants came to the cincinnati veterans medical center ( cvamc ) speech - language pathology department for preliminary testing . the tomal-2 was selected as a primary outcome given the ease of use , utilization across control subjects , and endorsement by the american veterans association speech - language pathology network . the tomal-2 was administered pre- and post - intervention as an assessment of treatment effects on cognitive functioning . in addition , the participants were interviewed to determine highest level of education , number of combat tours , and marital or relationship status . common cognitive complaints among the control and mops - vi participants include deficits in attention , concentration , and memory . each mops - vi participant was provided a laptop computer , wireless internet , and a videoconference phone for the duration of the intervention . for both mops - vi and the control group , intervention followed a conventional approach in that it had dual foci : ( 1 ) education about tbi and ptsd with the aim of improving knowledge about the conditions , and ( 2 ) a therapeutic cognitive intervention focused on memory and attention . the mops - vi portion of the study was designed to be as similar as possible to the control treatment , with the difference that the control treatment was conducted face - to - face in a clinic setting , but the mops - vi was designed to deliver treatment online . the mops - vi incorporated military themes and didactic video clips of veterans discussing how their everyday life had changed since their injury , and how particular strategies helped them . mops - vi participants were issued a televyou 500sp videophone ( figure 1 ) for use during simulated face - to - face therapy and were required to sign a waiver agreeing to return the videophone upon completion of the study . all treatment group participants were trained in how to plug the standard telephone wire into the videophone , in addition to making simulated videophone calls while in the presence of the pi to ensure sufficient participation usability . during the first videophone conference , the pi contacted each participant weekly via videophone to review the session materials and provided a reminder follow - up call to complete the modules if indicated . following completion of all modules , participants were asked to return to the vamc speech - language pathology department , at which time the tomal-2 was re - administered . descriptive data were obtained for the treatment and the control groups ( means and standard deviations or frequencies and percentages , as appropriate . ) in addition , t - tests were used to ensure that the treatment and the control groups were equivalent in age , education , and vocabulary scores as an estimate of iq ( i.e. next , the pre - post tomal 2 scores were compared for both groups at 2 points in time ( pre - post test results ) using analysis of variance ( anova ) and the wilcoxon signed ranks test . one of the aims of this study was to test the effect of mops - vi on patient compliance . participants were selected from a group of veterans initially non - adherent with clinic - based cognitive rehabilitative services . non - adherence was defined by the following criteria : 1 ) failure to schedule a follow - up appointment after referral for cognitive rehabilitation therapy ; or , 2 ) failure to attend more than 2 therapy sessions . it was hypothesized that the mops - vi therapeutic approach would increase adherence because of decreased travel time and increased scheduling convenience . among the veterans identified as non - adherent with speech therapy for cognitive rehabilitation services using these criteria , 9 signed the informed consent , 8 participated in pre - intervention testing , and 6 ( 67% ) completed the intervention . six veterans who initially did not follow through with clinic - based cognitive treatment completed the mops - vi telemedicine treatment . these 6 mops - vi participants ranged in age from 23 to 38 years , with a mean age of 30.17 years . the control group consisted of participants seen face - to - face ( clinic - based ) who were selected according to inclusion / exclusion data and were similar to the treatment group participants . the few exceptions included 1 participant who re - scheduled his initial videophone session 2 times because he had not yet completed the self - guided module preceding the session and required 2 sessions for post - intervention testing . there were no significant differences between the groups in mean age , marital status , years of education , and tomal-2nd pre - test composite score , indicating that the matching procedure was successful . results of a 2-way ( pre- vs. post- assessments ) ( standard vs. mops - vi ) analysis of variance ( anova ) revealed a significant pre - post assessment effect , f(1,10)=50.38 , p<0.001 , indicating that participants memory improved after treatment for both mops - vi and standard treatment groups ( table 2 ) . there was no significant interaction between treatment groups and pre - post assessment , f(1,10)=3.43 , p=0.09 . there was no significant difference between face - to - face therapy and mops - vi therapy ( f(1 , 10)=0.39 , p=0.55 ) suggesting that mops - vi therapy is as effective as clinic - based treatment and therefore is a viable alternative . because of the small n of the mops - vi treatment group , the nonparametric test , the wilcoxon signed ranks test was also conducted to compare the composite memory index score pre- and post - intervention . therefore , both the parametric and nonparametric statistical analyses indicated a significant improvement in pre- and post - treatment performance on the tomal-2 . partial eta - squared was used to provide an estimate of the magnitude of the effects of treatment as assessed by changes over time . the change in tomal-2nd scores from the pre- to post - treatment contributed 0.83 , which indicates a strong effect of treatment on composite test scores regardless of whether the treatment was clinic - based or mops - vi ( figure 2 ) . although both therapy groups experienced a positive change in tomal-2 test scores , the mops - vi group spent approximately 6 months less time in treatment ( mean=3.87 ) compared to the control group ( mean=9.33 ) . mops - vi participants were recruited from the 36% of veteran s non - adherent to standard face - to - face treatment . these numbers suggest that of 100 randomly chosen patients , 64 would complete treatment if offered clinic - based therapy , and 36 would drop out , but 27 ( 67% ) would complete treatment if offered mops - vi . this suggests that if both clinic - based therapy and mops - vi were offered as alternative treatments to veterans , a 91% compliance rate could be achieved with no diminution in the quality of results . the mops - vi intervention was structured to include modules that were delivered sequentially in a systematic fashion ; participants completed the program at their own pace . these follow - up videophone sessions also facilitated generalization of the content learned in the web - based modules . the report addressed the full spectrum of tbi severity and recommended the use of attention , problem solving , and memory training during post - acute rehabilitation of individuals with tbi . the mops - vi intervention placed considerable emphasis on metacognitive strategies and generalization and yielded results similar to standard therapy , even though the mops - vi was conducted independently in the veterans homes or workplaces with weekly videophone support over a shorter time period . results of the mops - vi intervention are consistent with the findings of cicerone , who reported that cognitive therapies directed at multiple domains of impairment can significantly improve neuropsychological performance in particular skill areas ( e.g. the mops - vi is an example of a cognitive intervention that addresses the constellation of neurobehavioral deficits commonly encountered in veterans . the results of the current study suggest that veterans recovering from mild tbi might benefit from an online treatment program with videophone support to improve memory and learning . compared the effectiveness of a commercially available computer - based cognitive rehabilitative teletherapy program and traditional outpatient speech therapy , finding that the teletherapy group spent significantly more time in therapy compared to the face - to - face group . however , mops - vi participants spent less time in treatment ( 3.87 months ) than the clinic - based control group ( 9.33 months ) , yet both groups had similar outcomes , suggesting that effective telehealth therapies can be briefer than standard office - based treatments . the purpose of this study was to test the efficacy of cognitive therapy and education using a telemedicine treatment for veterans with mild tbi in the convenience of their homes or workplaces . there was also a practical advantage to mops - vi ; many individuals requiring therapeutic intervention are unable to take time off from work and time out of family functions to receive treatment in a hospital or clinic . the mops - vi resulted in better compliance and a similar degree of improvement in cognitive test scores within a shorter time period than standard clinic - based treatment . overall , these results support the feasibility of the mops - vi approach and provide evidence of efficacy , defined as increased compliance in a group of veterans who were not compliant with traditional face - to - face therapy . furthermore , improvement in standardized cognitive test performance was comparable to clinic - based treatment .

clinical teaching is a critical and indispensable section in medical education ( 1 ) that results in the evolution of knowledge , attitude and skills ( 2 ) ; i.e. , the clinical competency of the students ( 3 ) . clinical teaching consists of various fields , one of which is clinical rounds ( 4 ) . bedside teaching is a patient - based teaching method in medical education that facilitates acquiring real knowledge , practical skills and professional attitudes ( 5 ) . this field of learning includes every situation where learning takes place in the presence of the patient , regardless of the environment in which such education is presented ( 6 ) . bedside teaching engages the training physicians with interaction with the patient beside their bed to extract a patient s records , represent the key features of clinical examination and discuss the best approach to diagnosing the disease and treating the patient ( 7 ) . bedside teaching provides active learning in real situations , observing students skills , increasing their motivation , professional thinking , clinical integration , communicative skills , problem - solving skills , decision - making and moral skills along with improving the understanding of patients ( 1 , 8) . in addition , it transfers the human aspects of patient care to the medical students ( 5 , 9 ) and helps the physician to view the patient as a real person and not merely a summary of the disease ( 4 ) . this is why these qualities can not be developed effectively in classrooms ( 1 , 10 ) . in clinical medicine , 56% of the patients problems could be well recognized after a complete history taking , and this increases to 72% after physical examination . in some cases , in other words , performing a comprehensive physical examination could assist the individual physician to reach a diagnosis faster ( 1 , 8 , 11 , and 12 ) . previous studies indicate that real clinical teaching emphasizing the history and physical diagnosis over the last 17 years has decreased from 75 to 16% , and it is even less today ( 4 , 10 , and 13 ) . in addition , clinical teaching has withdrawn from the bedside to hospital corridors , nursing stations and finally to the conference hall ( 5 , 11 , and 14 ) . claim that only 48% of the trainees have stated that they have had bedside teaching during their study . these individuals are 100% sure that bedside learning is the most effective method for learning clinical skills ( 15 ) . since the aim of clinical teaching is to provide opportunities for students to link theoretical information with practical realities , promoting its quality could result in training qualified students in different clinical domains ( 16 , 17 ) . unfortunately , no experimental evidence indicates that bedside teaching is the most effective strategy ( 6 ) . since no official evaluation has been conducted on this important educational field in medical universities , no reliable information shows that bedside teaching objectives , including the increase of concept understanding and personal skills , have been achieved through this strategy . this study was planned and conducted with the aim of determining the quality of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad . the present study has been conducted with the general objective of determining bedside teaching quality in internal wards of qaem and imam reza educational hospitals in mashhad in 2014 . the specific objectives of this study include : determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 clinical teaching is a critical and indispensable section in medical education ( 1 ) that results in the evolution of knowledge , attitude and skills ( 2 ) ; i.e. , the clinical competency of the students ( 3 ) . clinical teaching consists of various fields , one of which is clinical rounds ( 4 ) . bedside teaching is a patient - based teaching method in medical education that facilitates acquiring real knowledge , practical skills and professional attitudes ( 5 ) . this field of learning includes every situation where learning takes place in the presence of the patient , regardless of the environment in which such education is presented ( 6 ) . bedside teaching engages the training physicians with interaction with the patient beside their bed to extract a patient s records , represent the key features of clinical examination and discuss the best approach to diagnosing the disease and treating the patient ( 7 ) . bedside teaching provides active learning in real situations , observing students skills , increasing their motivation , professional thinking , clinical integration , communicative skills , problem - solving skills , decision - making and moral skills along with improving the understanding of patients ( 1 , 8) . in addition , it transfers the human aspects of patient care to the medical students ( 5 , 9 ) and helps the physician to view the patient as a real person and not merely a summary of the disease ( 4 ) . this is why these qualities can not be developed effectively in classrooms ( 1 , 10 ) . in clinical medicine , 56% of the patients problems could be well recognized after a complete history taking , and this increases to 72% after physical examination . in some cases , in other words , performing a comprehensive physical examination could assist the individual physician to reach a diagnosis faster ( 1 , 8 , 11 , and 12 ) . previous studies indicate that real clinical teaching emphasizing the history and physical diagnosis over the last 17 years has decreased from 75 to 16% , and it is even less today ( 4 , 10 , and 13 ) . in addition , clinical teaching has withdrawn from the bedside to hospital corridors , nursing stations and finally to the conference hall ( 5 , 11 , and 14 ) . claim that only 48% of the trainees have stated that they have had bedside teaching during their study . these individuals are 100% sure that bedside learning is the most effective method for learning clinical skills ( 15 ) . since the aim of clinical teaching is to provide opportunities for students to link theoretical information with practical realities , promoting its quality could result in training qualified students in different clinical domains ( 16 , 17 ) . unfortunately , no experimental evidence indicates that bedside teaching is the most effective strategy ( 6 ) . since no official evaluation has been conducted on this important educational field in medical universities , no reliable information shows that bedside teaching objectives , including the increase of concept understanding and personal skills , have been achieved through this strategy . this study was planned and conducted with the aim of determining the quality of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad . the present study has been conducted with the general objective of determining bedside teaching quality in internal wards of qaem and imam reza educational hospitals in mashhad in 2014 . the specific objectives of this study include : determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 the method employed in this research was a qualitative study to design tools and apply them to educational clinical rounds held in the internal wards of qaem and imam reza educational hospitals in mashhad in october 2014 . these two hospitals were selected since they are currently regarded as general hospitals and among the largest centers of medical education and health in northeastern iran . training of general practitioners as well as specialty and subspecialty levels is mostly done in these two hospitals . sample size was calculated as at least 96 rounds using the cochran formula ; in total we investigated 131 educational rounds in both hospitals . 59 and 54 educational rounds were held in imam reza and qaem hospitals respectively . to supply the calculated sample size , then they were selected randomly and evaluated proportionally to the rank of the holding members of the faculty . to collect data , to do so , persian and english keywords of medical education , quality of education , clinical teaching , challenges and strategies in bedside teaching , along with new models in bedside and patient - based teaching . the above - mentioned keywords , in combination and separately , were then used as search terms in pubmed , google , scientific information database ( sid ) , eric , web of knowledge , medline , science direct , scopus , magiran , google scholar , proquest , elsevier , and the iranian journal of medical education without considering the time limit . in total , the search yielded 138 articles published from 2003 to 2014 . the author investigated the obtained texts based on the titles and abstracts . in this step , then the sections related to quality based on variables mentioned in these studies were collected . in the next step , the resulting items were investigated and discussed in numerous meetings with experts and professors , and the overlapping cases were cancelled or merged together . finally , the contents were classified into three domains : patient comfort , targeted - teaching and group dynamics , in accordance with the three - domain model of janicik and fletcher . the resulting checklist was presented to a number of medical education and clinical medicine specialists for content validity . the reliability of the checklist was verified using cronbach s alpha coefficient ( 0.74 ) . the first part of the author s checklist consisted of demographic information including the name of the hospital , type of internal ward and the rank of the faculty member conducting the educational rounds . the second part of the checklist included 30 questions involving the factors of effective bedside teaching , including the domains of patient comfort ( 8 questions ) , targeted teaching ( 14 questions ) and group dynamics ( 8 questions ) ( table 1 ) . the students were asked to score the checklist questions from 0 to 10 ( 0= not done , 10= done perfectly ) . first , the researcher obtained permission from the department of higher education and the vice - chancellor for medical school . the subject of the study was presented to the heads of the internal wards of the mentioned hospitals , and permission was then obtained from the hospitals . in the next step , a list of the medical students in the clinical teaching course was provided ; those who were passing the internal ward course and the professors rounds were selected randomly . then the content of the checklist was given to the students to familiarize them with its content and data collection method . finally , the students filled out the checklists after attending the selected educational rounds in internal wards , maintaining the confidentiality of their answers . this study was conducted under the license of the research ethics committee of mashhad university of medical sciences . participation in this study was voluntary , and the names of the members of the faculty who held educational rounds were not entered on the data collection forms to maintain privacy and confidentiality of information . data description was conducted using descriptive statistical indices as frequency and mean along with standard deviation . to ensure the normal distribution of data , the kolmogorov - smirnov test ( k s test or ks test ) was first conducted . then , independent t - test , one - way anova and variance analysis were used , in which ( p<0.05 ) was regarded as statistically significant . the method employed in this research was a qualitative study to design tools and apply them to educational clinical rounds held in the internal wards of qaem and imam reza educational hospitals in mashhad in october 2014 . these two hospitals were selected since they are currently regarded as general hospitals and among the largest centers of medical education and health in northeastern iran . training of general practitioners as well as specialty and subspecialty levels is mostly done in these two hospitals . sample size was calculated as at least 96 rounds using the cochran formula ; in total we investigated 131 educational rounds in both hospitals . 59 and 54 educational rounds were held in imam reza and qaem hospitals respectively . to supply the calculated sample size , then they were selected randomly and evaluated proportionally to the rank of the holding members of the faculty . to collect data , first a checklist was designed . to do so , persian and english keywords of medical education , quality of education , clinical teaching , challenges and strategies in bedside teaching , along with new models in bedside and patient - based teaching . the above - mentioned keywords , in combination and separately , were then used as search terms in pubmed , google , scientific information database ( sid ) , eric , web of knowledge , medline , science direct , scopus , magiran , google scholar , proquest , elsevier , and the iranian journal of medical education without considering the time limit . in total , the search yielded 138 articles published from 2003 to 2014 . the author investigated the obtained texts based on the titles and abstracts . in this step , then the sections related to quality based on variables mentioned in these studies were collected . in the next step , the resulting items were investigated and discussed in numerous meetings with experts and professors , and the overlapping cases were cancelled or merged together . finally , the contents were classified into three domains : patient comfort , targeted - teaching and group dynamics , in accordance with the three - domain model of janicik and fletcher . the resulting checklist was presented to a number of medical education and clinical medicine specialists for content validity . the reliability of the checklist was verified using cronbach s alpha coefficient ( 0.74 ) . the first part of the author s checklist consisted of demographic information including the name of the hospital , type of internal ward and the rank of the faculty member conducting the educational rounds . the second part of the checklist included 30 questions involving the factors of effective bedside teaching , including the domains of patient comfort ( 8 questions ) , targeted teaching ( 14 questions ) and group dynamics ( 8 questions ) ( table 1 ) . the students were asked to score the checklist questions from 0 to 10 ( 0= not done , 10= done perfectly ) . first , the researcher obtained permission from the department of higher education and the vice - chancellor for medical school . the subject of the study was presented to the heads of the internal wards of the mentioned hospitals , and permission was then obtained from the hospitals . in the next step , a list of the medical students in the clinical teaching course was provided ; those who were passing the internal ward course and the professors rounds were selected randomly . then the content of the checklist was given to the students to familiarize them with its content and data collection method . finally , the students filled out the checklists after attending the selected educational rounds in internal wards , maintaining the confidentiality of their answers . this study was conducted under the license of the research ethics committee of mashhad university of medical sciences . participation in this study was voluntary , and the names of the members of the faculty who held educational rounds were not entered on the data collection forms to maintain privacy and confidentiality of information . data description was conducted using descriptive statistical indices as frequency and mean along with standard deviation . to ensure the normal distribution of data , the kolmogorov - smirnov test ( k s test or ks test ) was first conducted . then , independent t - test , one - way anova and variance analysis were used , in which ( p<0.05 ) was regarded as statistically significant . in this study , 113 educational rounds in total were investigated ( table 2 ) . among them , 59 ( 52.2% ) and 54 ( 47.8% ) of educational rounds were held in imam reza and qaem hospitals , respectively . among all educational rounds investigated , educational rounds held by the members of the faculty with the rank of professor was 19 ( 16.8% ) , assistant professor 63 ( 55.8% ) and associate professor 31 ; this conformed to the real ratio of the members of the faculty . in general , the average total quality score of bedside teaching in the internal wards of imam reza and qaem hospitals was 180.8 out of the total score of 300 . the lowest score related to the quality of bedside teaching was 0 and the highest score was 300 . in the domains of considering patient comfort and group dynamics , the lowest and highest scores were 0 and 80 , and in the case of targeted teaching the lowest and highest scores were 0 and 140 , respectively . the average quality score of bedside teaching in the internal wards of imam reza and qaem hospitals was 163.7 ( 46.4 ) and 197.9 ( 66.4 ) , respectively . the average score of bedside teaching was compared for both groups using independent t - test ( table 3 ) , the difference of which was not significant ( p>0.05 ) . the average quality score of patient comfort in the internal wards of imam reza and qaem hospitals was 26.5 ( 15.4 ) and 31.1 ( 20.4 ) out of 80 , respectively ( table 3 ) . the average score related to patient comfort in both groups was compared using independent t - test , and the difference was not significant ( p>0.05 ) . the average quality score of targeted teaching in bedside teaching in the internal wards of imam reza and qaem hospitals was 101.3 ( 25.7 ) and 110.7 ( 33.3 ) out of 140 , respectively ( table 3 ) . the average score of targeted teaching in bedside teaching in both groups was compared using independent t - test , and the difference was not significant ( p>0.05 ) . the average quality score of group dynamics in bedside teaching in internal ward of imam reza and qaem hospitals was 35.7 ( 15.1 ) and 56.0 ( 21.6 ) out of 80 ( table 3 ) . the average score of group dynamics was compared in both groups using independent t - test , and the difference was significant ( p<0.05 ) . the average total score of bedside teaching quality in terms of patient comfort , targeted teaching and group dynamics was compared in the two groups using independent t - test . by supposing equal variances , the difference was not significant in terms of patient comfort and targeted teaching ( p>0.05 ) . however , it was significant in terms of group dynamics ( p<0.05 ) . nevertheless , the quality of bedside teaching was not significantly different in the two hospitals ( p>0.05 ) . one - way anova was employed to compare the quality of bedside teaching in terms of the rank of faculty members . no significant difference was observed in terms of patient comfort and targeted teaching ( p=0.235 and p=0.121 , respectively ) . however , the quality was significantly different in terms of group dynamics considering the rank of faculty members ( p=0.027 ) . in sum , bedside teaching quality was not significantly different in the two hospitals in terms of the rank of faculty members ( p=0.129 ) . the quality of bedside teaching was significantly different only in terms of group dynamics among assistant to associate ranks of faculty members ( p=0.049 ) in such a way that the average score obtained by assistant professors was higher for 10.7 . in general , the results of the study showed that the quality of bedside teaching in internal wards of qaem and imam reza educational hospitals is not acceptable according to the indicators of patient comfort , targeted teaching and group dynamics . in the study by ziaee , the percentage of students consent from clinical teaching was 38.8% , which conformed to the results of this study . in the previous study , only a few items including the educational objectives being specified , the number of participating students and having a course plan were investigated ( 18 ) . the results of the present study conform to the results of khorasani study in 2007 , which had reported students attitudes toward the current situation of clinical teaching as negative . however , in that study , only limited aspects of clinical teaching were addressed , including the educational objectives being specified , the possibility of independent visits by the trainee , the possibility of prescribing medicine independently and criticizing and correcting the trainee s history ( 19 ) . on the other hand , another study conducted in the iran university of medical sciences in 2004 showed that the quality of clinical teaching was regarded as relatively favorable , according to medical students . in that study , domains of scientific mastery of the professor , educational management and the quality of communication and consulting were considered . that study did not consider patient comfort as an independent item , and it conformed to our study only in the domains of targeted teaching and group dynamics ( 20 ) . in his study in 2012 , bagheriyan represented that the situation of clinical teaching is regarded as desirable , according to the students of anesthesiology and operating room in tabriz . in this study , the trainer , clarity of educational objectives , and the educational environment were shown to have the highest effect in the quality of clinical teaching ( 21 ) . in the study by mardani in 2010 , the situation of clinical teaching was reported from nursing students perspective . in this study , the evaluation method and proper manners of the trainer were regarded as strong points , and the lack of coordination of clinical teachings with theory was regarded as the weak point ( 22 ) . it appears that the current study has included aspects that play more important roles in clinical teaching . possibly the strong point of the present study in comparison to others is its special attention to patient preferences as one of the factors affecting quality . in a study by lubetkin at cornell university , the quality of clinical teaching was regarded as desirable according to the professors and students , although the professors were more satisfied than the student ( 23 ) . the results of that study did not conform to the results of our study . in the above - mentioned studies , items including educational equipment , mastery of professors of the subjects and the physical environment were considered . the results of the present study are devastating in terms of the quality related to patient comfort in bedside teaching , and these conform to the results of the study by dehghani et al . this category has been emphasized in numerous studies , and it is regarded as a high - quality component of clinical teaching ( 6 , 2527 ) . a study conducted in isfahan in 2006 showed that talking about mental and social issues satisfies only 40% of the patients , and nearly 60% of the patients were satisfied in terms of having emotional connection with their physicians . the patient s participation in the treatment process was not satisfying for 50% of the patients in terms of having treatment methods clearly explained , being included in understandable discussions , and feeling that their feedback was valued in terms of performing or not performing a treatment . 40% of the patients felt unsafe about scattered talks during clinical teaching and stated that they felt themselves to be regarded as educational tools ( 4 ) . in lehmann s study of educational hospitals of the usa , it appears that not allowing the patient to discuss their emotional issues and social conditions , lack of participation and getting feedback from them in discussions , along with medical decisions and low emotional relations of the physician with the patient , have resulted in ignoring human aspects . however , in related studies , indicators such as good relations with the patient , patients participation in decisions , considering patients concerns , accelerating the solution of patients problems and answering their questions are considered to be measures of humanitarian behavior of the physician ( 13 , 29 ) . in 2012 , a study by adibi in isfahan revealed that repeated examinations and visits , along with crowded and long clinical rounds in which information is presented to the patient in an unclear way , have resulted in patient dissatisfaction . in effect , patients feel that they are employed as educational tools ( 13 ) . factors such as a high number of questions and examining individuals in clinical rounds and the main physician not being recognized , scattered and contradictory discussions in the patient s presence , along with the lack of presenting sufficient and understandable explanations of the disease and treatment measures , results in increasing the patient s sense of insecurity . in addition , it results in greater uncertainty about being treated by an individual who is not the main physician . the necessity of introducing the people present in clinical rounds and identifying the person in charge , along with explaining treatments and procedures clearly , are given serious emphasis ( 4 , 30 ) . these results conformed to the results of the present study . in our study , the following cases in the domain of patient comfort have been considered : coordination with the patient , stating educational objectives , introducing people , considering the patients concerns , minimizing possible patient misunderstandings at the end of teaching and the importance of patients preferences . in the section related to the quality of group dynamics in clinical teaching , the results of our study showed that this quality has been lower than average in the internal ward of imam reza hospital , and it was average in that of qaem hospital . the results of our study were similar to the results of bazzazi s study ( 31 ) . it appears that to achieve desirable conditions , new ways of increasing students participation must be employed . in this regard , a more accurate evaluation of the current situation appears essential along with discovering weak and strong points . possibly one of the obstacles to achieving this goal is providing students an opportunity to judge professors capabilities . in their discussion of learning environments , kroenke and omori ( 2010 ) stated that during educational rounds , physicians are maybe anxious about their abilities in knowledge transmission . they claim that this fear may be more prevalent in young lecturers who do not have much clinical teaching experience . therefore , having friendly relations with students at the beginning of teaching would result in a more positive learning environment ( 10 ) . in this regard , ramani in his 2013 study states that the professor should challenge the students minds without humiliating them , and he suggests that the professor should involve all learners in teaching process . this could be achieved by assuring that all students have an opportunity to answer questions ( 32 ) . recommended that before visiting the first patient , it is preferable to set a time for discussion with students . by introducing all the team members from the professors to the residents and the medical students , they will have the opportunity to become familiar with each other . the professor should state his objectives and expectations for the students at the beginning as well ( 27 ) . in our study , in the domain of quality related to the group dynamics for which an average score was obtained for both hospitals , the following issues were considered : team cooperation , freedom in presenting comments , creating an intimate relationship at the beginning of teaching , giving responsibility to students and creating a safe atmosphere for free discussion . in the section comparing clinical teaching quality in the internal wards of imam reza and qaem hospitals , considering the indicators related to patient comfort , targeted teaching and group dynamics , the results of our study showed that generally the quality of clinical teaching in both hospitals does not differ significantly . however , the score related to the quality of group dynamics in qaem hospital was better compared to that of imam reza hospital . the quality of clinical teaching according to the rank of the member of the faculty was not different for both hospitals . more dynamics were observed in teachers with the rank of professor compared to associate professors . to answer the present ambiguities in this regard , more research is required . in this study , we investigated the quality according to the three - domain model of janicik and fletcher , which includes patient comfort , targeted teaching and group dynamics . other studies conducted from the perspectives of nursing students and clinical professors have investigated the factors affecting clinical teaching from different points of view ( 22 , 33 , and 34 ) . another study in gonabad revealed that the most effective factor in the quality of clinical teaching , according to both students and lecturers , has been the performance of instructors . collaborative working environment , educational equipment in clinical environment , amenities in the clinical setting and the ward setting being proportional to the number of students are domains considered in the mentioned study ( 33 ) . in that study , collaborative working environment is regarded as the weakest factor . this finding conforms to the results of our study in terms of low collaborative working environment as one of the components of group dynamics . a 2010 study by mardani in ahwaz revealed that the interns and trainees views of clinical teaching is average and better than average . in this study , the strongest point of clinical teaching students awareness from clinical evaluation method at the beginning of practical course and proper behavioral treatment by the clinical instructor . in addition , the weakest points were related to the lack of cooperation of the ward with students and the lack of harmony between theoretical learning and clinical activities ( 22 ) . in summary , the results of this study showed that the quality of clinical teaching in the internal wards of qaem and imam reza educational hospitals is weak in the domains related to patient comfort and group dynamics . improving and promoting the quality of bedside teaching requires continuous assessment of the current situation , recognizing strong points and correcting weak points , a process in which the comments and ideas of both professors and students could pave the way for later plans . it is suggested that mashhad university of medical sciences take some steps to improve the quality of clinical teaching , especially in domains related to the patient s comfort and trainee s participation through holding educational workshops and presenting new teaching methods , along with investigating possible problems .

background : bedside teaching is a patient - based teaching method in medical education . the present study has been conducted with the aim of investigating the quality of bedside teaching in the internal wards of qaem and imam reza educational hospitals.methods:this study follows a mixed qualitative - quantitative approach using checklists on educational clinical rounds in imam reza and qaem hospitals in mashhad . in the first stage consisting of qualitative study , the parts related to the quality of bedside teaching were recognized and a checklist was designed in three domains of patient comfort ( 8 questions ) , targeted teaching ( 14 questions ) and group dynamics ( 8 questions ) , and its reliability and validity were verified . in the next step , data were collected and then analyzed using spss 16 software through statistical techniques of independent t - test , one - way anova and variance analysis.results:in total , 113 educational rounds were investigated in this study . among them , 59 ( 52.2% ) and 54 ( 47.8% ) educational rounds have been investigated in imam reza and qaem hospitals , respectively . the average total score of bedside teaching was 180.8 out of 300 in the internal wards of both imam reza and qaem hospitals.conclusion:the results of this study showed that generally the quality of bedside teaching in imam reza and qaem hospitals of mashhad is low according to the qualitative standards considered in this study . holding educational workshops along with more familiarity of the professors with effective bedside teaching strategies could be effective in improving the quality of educational rounds .

Introduction Background Statement of the problem Objectives Material and Methods Research Design and Setting Sampling Measurement Tool Data Collection Ethical Consideration Statistical Analysis Results Discussion Conclusions

clinical teaching is a critical and indispensable section in medical education ( 1 ) that results in the evolution of knowledge , attitude and skills ( 2 ) ; i.e. bedside teaching is a patient - based teaching method in medical education that facilitates acquiring real knowledge , practical skills and professional attitudes ( 5 ) . bedside teaching engages the training physicians with interaction with the patient beside their bed to extract a patient s records , represent the key features of clinical examination and discuss the best approach to diagnosing the disease and treating the patient ( 7 ) . bedside teaching provides active learning in real situations , observing students skills , increasing their motivation , professional thinking , clinical integration , communicative skills , problem - solving skills , decision - making and moral skills along with improving the understanding of patients ( 1 , 8) . in addition , it transfers the human aspects of patient care to the medical students ( 5 , 9 ) and helps the physician to view the patient as a real person and not merely a summary of the disease ( 4 ) . since the aim of clinical teaching is to provide opportunities for students to link theoretical information with practical realities , promoting its quality could result in training qualified students in different clinical domains ( 16 , 17 ) . since no official evaluation has been conducted on this important educational field in medical universities , no reliable information shows that bedside teaching objectives , including the increase of concept understanding and personal skills , have been achieved through this strategy . this study was planned and conducted with the aim of determining the quality of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad . the present study has been conducted with the general objective of determining bedside teaching quality in internal wards of qaem and imam reza educational hospitals in mashhad in 2014 . the specific objectives of this study include : determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 clinical teaching is a critical and indispensable section in medical education ( 1 ) that results in the evolution of knowledge , attitude and skills ( 2 ) ; i.e. bedside teaching is a patient - based teaching method in medical education that facilitates acquiring real knowledge , practical skills and professional attitudes ( 5 ) . bedside teaching engages the training physicians with interaction with the patient beside their bed to extract a patient s records , represent the key features of clinical examination and discuss the best approach to diagnosing the disease and treating the patient ( 7 ) . in addition , it transfers the human aspects of patient care to the medical students ( 5 , 9 ) and helps the physician to view the patient as a real person and not merely a summary of the disease ( 4 ) . since no official evaluation has been conducted on this important educational field in medical universities , no reliable information shows that bedside teaching objectives , including the increase of concept understanding and personal skills , have been achieved through this strategy . this study was planned and conducted with the aim of determining the quality of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad . the present study has been conducted with the general objective of determining bedside teaching quality in internal wards of qaem and imam reza educational hospitals in mashhad in 2014 . the specific objectives of this study include : determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 the method employed in this research was a qualitative study to design tools and apply them to educational clinical rounds held in the internal wards of qaem and imam reza educational hospitals in mashhad in october 2014 . 59 and 54 educational rounds were held in imam reza and qaem hospitals respectively . to collect data , to do so , persian and english keywords of medical education , quality of education , clinical teaching , challenges and strategies in bedside teaching , along with new models in bedside and patient - based teaching . the above - mentioned keywords , in combination and separately , were then used as search terms in pubmed , google , scientific information database ( sid ) , eric , web of knowledge , medline , science direct , scopus , magiran , google scholar , proquest , elsevier , and the iranian journal of medical education without considering the time limit . in this step , then the sections related to quality based on variables mentioned in these studies were collected . in the next step , the resulting items were investigated and discussed in numerous meetings with experts and professors , and the overlapping cases were cancelled or merged together . finally , the contents were classified into three domains : patient comfort , targeted - teaching and group dynamics , in accordance with the three - domain model of janicik and fletcher . the second part of the checklist included 30 questions involving the factors of effective bedside teaching , including the domains of patient comfort ( 8 questions ) , targeted teaching ( 14 questions ) and group dynamics ( 8 questions ) ( table 1 ) . the subject of the study was presented to the heads of the internal wards of the mentioned hospitals , and permission was then obtained from the hospitals . in the next step , a list of the medical students in the clinical teaching course was provided ; those who were passing the internal ward course and the professors rounds were selected randomly . then the content of the checklist was given to the students to familiarize them with its content and data collection method . finally , the students filled out the checklists after attending the selected educational rounds in internal wards , maintaining the confidentiality of their answers . participation in this study was voluntary , and the names of the members of the faculty who held educational rounds were not entered on the data collection forms to maintain privacy and confidentiality of information . then , independent t - test , one - way anova and variance analysis were used , in which ( p<0.05 ) was regarded as statistically significant . the method employed in this research was a qualitative study to design tools and apply them to educational clinical rounds held in the internal wards of qaem and imam reza educational hospitals in mashhad in october 2014 . 59 and 54 educational rounds were held in imam reza and qaem hospitals respectively . to do so , persian and english keywords of medical education , quality of education , clinical teaching , challenges and strategies in bedside teaching , along with new models in bedside and patient - based teaching . the above - mentioned keywords , in combination and separately , were then used as search terms in pubmed , google , scientific information database ( sid ) , eric , web of knowledge , medline , science direct , scopus , magiran , google scholar , proquest , elsevier , and the iranian journal of medical education without considering the time limit . in this step , then the sections related to quality based on variables mentioned in these studies were collected . in the next step , the resulting items were investigated and discussed in numerous meetings with experts and professors , and the overlapping cases were cancelled or merged together . finally , the contents were classified into three domains : patient comfort , targeted - teaching and group dynamics , in accordance with the three - domain model of janicik and fletcher . the first part of the author s checklist consisted of demographic information including the name of the hospital , type of internal ward and the rank of the faculty member conducting the educational rounds . the second part of the checklist included 30 questions involving the factors of effective bedside teaching , including the domains of patient comfort ( 8 questions ) , targeted teaching ( 14 questions ) and group dynamics ( 8 questions ) ( table 1 ) . the subject of the study was presented to the heads of the internal wards of the mentioned hospitals , and permission was then obtained from the hospitals . in the next step , a list of the medical students in the clinical teaching course was provided ; those who were passing the internal ward course and the professors rounds were selected randomly . then the content of the checklist was given to the students to familiarize them with its content and data collection method . finally , the students filled out the checklists after attending the selected educational rounds in internal wards , maintaining the confidentiality of their answers . this study was conducted under the license of the research ethics committee of mashhad university of medical sciences . participation in this study was voluntary , and the names of the members of the faculty who held educational rounds were not entered on the data collection forms to maintain privacy and confidentiality of information . then , independent t - test , one - way anova and variance analysis were used , in which ( p<0.05 ) was regarded as statistically significant . in this study , 113 educational rounds in total were investigated ( table 2 ) . among them , 59 ( 52.2% ) and 54 ( 47.8% ) of educational rounds were held in imam reza and qaem hospitals , respectively . among all educational rounds investigated , educational rounds held by the members of the faculty with the rank of professor was 19 ( 16.8% ) , assistant professor 63 ( 55.8% ) and associate professor 31 ; this conformed to the real ratio of the members of the faculty . in general , the average total quality score of bedside teaching in the internal wards of imam reza and qaem hospitals was 180.8 out of the total score of 300 . the lowest score related to the quality of bedside teaching was 0 and the highest score was 300 . in the domains of considering patient comfort and group dynamics , the lowest and highest scores were 0 and 80 , and in the case of targeted teaching the lowest and highest scores were 0 and 140 , respectively . the average quality score of bedside teaching in the internal wards of imam reza and qaem hospitals was 163.7 ( 46.4 ) and 197.9 ( 66.4 ) , respectively . the average score of bedside teaching was compared for both groups using independent t - test ( table 3 ) , the difference of which was not significant ( p>0.05 ) . the average quality score of patient comfort in the internal wards of imam reza and qaem hospitals was 26.5 ( 15.4 ) and 31.1 ( 20.4 ) out of 80 , respectively ( table 3 ) . the average score related to patient comfort in both groups was compared using independent t - test , and the difference was not significant ( p>0.05 ) . the average quality score of targeted teaching in bedside teaching in the internal wards of imam reza and qaem hospitals was 101.3 ( 25.7 ) and 110.7 ( 33.3 ) out of 140 , respectively ( table 3 ) . the average score of targeted teaching in bedside teaching in both groups was compared using independent t - test , and the difference was not significant ( p>0.05 ) . the average quality score of group dynamics in bedside teaching in internal ward of imam reza and qaem hospitals was 35.7 ( 15.1 ) and 56.0 ( 21.6 ) out of 80 ( table 3 ) . the average score of group dynamics was compared in both groups using independent t - test , and the difference was significant ( p<0.05 ) . the average total score of bedside teaching quality in terms of patient comfort , targeted teaching and group dynamics was compared in the two groups using independent t - test . by supposing equal variances , the difference was not significant in terms of patient comfort and targeted teaching ( p>0.05 ) . nevertheless , the quality of bedside teaching was not significantly different in the two hospitals ( p>0.05 ) . one - way anova was employed to compare the quality of bedside teaching in terms of the rank of faculty members . no significant difference was observed in terms of patient comfort and targeted teaching ( p=0.235 and p=0.121 , respectively ) . in sum , bedside teaching quality was not significantly different in the two hospitals in terms of the rank of faculty members ( p=0.129 ) . the quality of bedside teaching was significantly different only in terms of group dynamics among assistant to associate ranks of faculty members ( p=0.049 ) in such a way that the average score obtained by assistant professors was higher for 10.7 . in general , the results of the study showed that the quality of bedside teaching in internal wards of qaem and imam reza educational hospitals is not acceptable according to the indicators of patient comfort , targeted teaching and group dynamics . in the study by ziaee , the percentage of students consent from clinical teaching was 38.8% , which conformed to the results of this study . in the previous study , only a few items including the educational objectives being specified , the number of participating students and having a course plan were investigated ( 18 ) . the results of the present study conform to the results of khorasani study in 2007 , which had reported students attitudes toward the current situation of clinical teaching as negative . however , in that study , only limited aspects of clinical teaching were addressed , including the educational objectives being specified , the possibility of independent visits by the trainee , the possibility of prescribing medicine independently and criticizing and correcting the trainee s history ( 19 ) . on the other hand , another study conducted in the iran university of medical sciences in 2004 showed that the quality of clinical teaching was regarded as relatively favorable , according to medical students . in that study , domains of scientific mastery of the professor , educational management and the quality of communication and consulting were considered . that study did not consider patient comfort as an independent item , and it conformed to our study only in the domains of targeted teaching and group dynamics ( 20 ) . in this study , the trainer , clarity of educational objectives , and the educational environment were shown to have the highest effect in the quality of clinical teaching ( 21 ) . in this study , the evaluation method and proper manners of the trainer were regarded as strong points , and the lack of coordination of clinical teachings with theory was regarded as the weak point ( 22 ) . in a study by lubetkin at cornell university , the quality of clinical teaching was regarded as desirable according to the professors and students , although the professors were more satisfied than the student ( 23 ) . the results of the present study are devastating in terms of the quality related to patient comfort in bedside teaching , and these conform to the results of the study by dehghani et al . a study conducted in isfahan in 2006 showed that talking about mental and social issues satisfies only 40% of the patients , and nearly 60% of the patients were satisfied in terms of having emotional connection with their physicians . in lehmann s study of educational hospitals of the usa , it appears that not allowing the patient to discuss their emotional issues and social conditions , lack of participation and getting feedback from them in discussions , along with medical decisions and low emotional relations of the physician with the patient , have resulted in ignoring human aspects . in 2012 , a study by adibi in isfahan revealed that repeated examinations and visits , along with crowded and long clinical rounds in which information is presented to the patient in an unclear way , have resulted in patient dissatisfaction . factors such as a high number of questions and examining individuals in clinical rounds and the main physician not being recognized , scattered and contradictory discussions in the patient s presence , along with the lack of presenting sufficient and understandable explanations of the disease and treatment measures , results in increasing the patient s sense of insecurity . these results conformed to the results of the present study . in our study , the following cases in the domain of patient comfort have been considered : coordination with the patient , stating educational objectives , introducing people , considering the patients concerns , minimizing possible patient misunderstandings at the end of teaching and the importance of patients preferences . in the section related to the quality of group dynamics in clinical teaching , the results of our study showed that this quality has been lower than average in the internal ward of imam reza hospital , and it was average in that of qaem hospital . in this regard , ramani in his 2013 study states that the professor should challenge the students minds without humiliating them , and he suggests that the professor should involve all learners in teaching process . in our study , in the domain of quality related to the group dynamics for which an average score was obtained for both hospitals , the following issues were considered : team cooperation , freedom in presenting comments , creating an intimate relationship at the beginning of teaching , giving responsibility to students and creating a safe atmosphere for free discussion . in the section comparing clinical teaching quality in the internal wards of imam reza and qaem hospitals , considering the indicators related to patient comfort , targeted teaching and group dynamics , the results of our study showed that generally the quality of clinical teaching in both hospitals does not differ significantly . however , the score related to the quality of group dynamics in qaem hospital was better compared to that of imam reza hospital . the quality of clinical teaching according to the rank of the member of the faculty was not different for both hospitals . in this study , we investigated the quality according to the three - domain model of janicik and fletcher , which includes patient comfort , targeted teaching and group dynamics . another study in gonabad revealed that the most effective factor in the quality of clinical teaching , according to both students and lecturers , has been the performance of instructors . this finding conforms to the results of our study in terms of low collaborative working environment as one of the components of group dynamics . in this study , the strongest point of clinical teaching students awareness from clinical evaluation method at the beginning of practical course and proper behavioral treatment by the clinical instructor . in addition , the weakest points were related to the lack of cooperation of the ward with students and the lack of harmony between theoretical learning and clinical activities ( 22 ) . in summary , the results of this study showed that the quality of clinical teaching in the internal wards of qaem and imam reza educational hospitals is weak in the domains related to patient comfort and group dynamics . improving and promoting the quality of bedside teaching requires continuous assessment of the current situation , recognizing strong points and correcting weak points , a process in which the comments and ideas of both professors and students could pave the way for later plans . it is suggested that mashhad university of medical sciences take some steps to improve the quality of clinical teaching , especially in domains related to the patient s comfort and trainee s participation through holding educational workshops and presenting new teaching methods , along with investigating possible problems .

the cycloset safety trial study protocol has been previously published in abbreviated form ( 11 ) . eligible patients had type 2 diabetes , as defined by the 2004 american diabetes association guidelines ( 18 ) , were between the ages of 30 and 80 years , had a bmi of < 43 kg / m , and an a1c level 10.0% . exclusion criteria were current chronic ( greater than 10 days ) use of prescription sympathomimetic drugs , ergot alkaloid derivatives , or abortive migraine medications , clinically significant comorbid conditions such as uncontrolled hypertension , new york heart classification ( nyhc ) iii - iv congestive heart failure , renal failure , or cancer ( other than nonmelanoma skin or nonmetastatic prostate cancer ) within the past 5 years . patients with nyhc i - ii congestive heart failure were allowed in the trial as were those with significant cardiovascular disease , including a cardiovascular event prior to 6 months before screening . patients were required to be on a stable antidiabetes regimen consisting of either diet , oral hypoglycemic agents ( no more than two ) , or insulin ( alone or with no more than one oral hypoglycemic agent ) for at least 30 days prior to randomization after a two - week lead - in period . patients were randomized in a 2:1 ratio to their usual antidiabetes regimen plus bromocriptine - qr or placebo taken with their morning meal . during the first 6 weeks of the study , the daily dose of the study drug was titrated up by one tablet ( 0.8 mg ) per day on a weekly basis until a maximal dose of up to six tablets ( 4.8 mg ) per day was achieved or until the patient could not tolerate a higher dose . patients were required to continue their usual antidiabetes regimen during the first 3 months of the study but were allowed to alter the dosages of these medications to optimize blood glucose control as deemed appropriate by the site investigator ( 19 ) . after 3 months , alterations to antidiabetes medications ( elimination or addition of an agent ) were allowed when necessary , but if a medication was added the final regimen was limited to no more than two oral antidiabetes agents , or no more than one antidiabetes agent if taking insulin . during the first 6 weeks of the study , patients were called weekly , had office visits at weeks 3 and 6 , and then visits every 3 months until the study end ( week 52 ) or early termination . patients were contacted 30 days after stopping a study drug to record any adverse events that occurred after cessation . physical exams and laboratory assessments ( blood chemistries , hematology , and urine analyses ) were obtained at baseline , week 24 , and week 52 or study termination . the study protocol was approved by each va hospital institutional review board and a central or academic - affiliated institutional review board for non - va centers . there were two main objectives of the cycloset safety trial : 1 ) assessment of overall safety of bromocriptine - qr by measuring the frequency of serious adverse events ( saes ) among patients taking bromocriptine - qr and placebo , and 2 ) cardiovascular safety assessed by determining the frequency of major cardiovascular events , defined as a composite of first myocardial infarction , stroke , coronary revascularization , or hospitalization for angina or congestive heart failure that occurred after randomization . an independent adverse event adjudication committee ( aeac ) consisting of two cardiologists and an endocrinologist , blinded to treatment assignment and medical dictionary for regulatory activities ( meddra ) coding of events by the study team , made the final sae system organ class ( soc ) classifications and assignment of an sae as a cardiovascular end point . additional safety measures included laboratory measures ( blood chemistries , hematology , and urine analyses ) at weeks 0 , 24 , and 52 of the study and evaluation of electrocardiograms ( ecgs ) at weeks 0 , 24 , 52 or early termination . an analysis of noninferiority tested the hypothesis that usual antidiabetes therapy ( uadt ) plus bromocriptine - qr is not inferior to that of uadt plus placebo in terms of the occurrence of all - cause saes using a one - sided = 0.05 level . in order to test the primary hypothesis at a one - sided = 0.05 and with a power of one = 0.90 when the noninferiority margin is 1.5 , the total number of patients with all - cause saes needed during the study was calculated to be 235 . all randomized patients that took at least one dose of study drug were included in the all - cause sae analysis . saes that occurred on a study drug or within 30 days of stopping a study drug were considered as treatment emergent events . statistical testing between the two groups was conducted using the two - sided 91.9% ci for the rate ratio of all - cause saes because this provides the same confidence boundaries as a one - sided 96% ci . the ci was estimated by the corresponding estimate for the hazard ratio ( hr ) with cox regression analysis with consideration for possible center interaction . the analysis of the composite cardiovascular endpoint used the modified intent - to - treat population of patients receiving at least one dose of a study drug . superiority or noninferiority between bromocriptine - qr and placebo for the composite cvd end point was defined as the upper bound of the two - sided 95% confidence limit being < 0 and < 1.5 , respectively . assuming a cvd composite event rate of 3.4% , this sample size provided a power of 75% to demonstrate cvd noninferiority . upon confirming noninferiority , a sequential superiority analysis of cvd end point based on the cox proportional - hazards regression with two - sided p values analysis of the cvd end point was conducted adjusting for baseline covariates including history of stroke and cardiovascular revascularization and center . changes in laboratory measures were assessed using the wilcoxon rank sum test except for a1c , which was based on a general linear model with baseline and treatment as fixed effects . the significant level was set at p < 0.05 . ( toronto , canada ) using sas software version 8.2 ( cary , nc ) . there were two main objectives of the cycloset safety trial : 1 ) assessment of overall safety of bromocriptine - qr by measuring the frequency of serious adverse events ( saes ) among patients taking bromocriptine - qr and placebo , and 2 ) cardiovascular safety assessed by determining the frequency of major cardiovascular events , defined as a composite of first myocardial infarction , stroke , coronary revascularization , or hospitalization for angina or congestive heart failure that occurred after randomization . an independent adverse event adjudication committee ( aeac ) consisting of two cardiologists and an endocrinologist , blinded to treatment assignment and medical dictionary for regulatory activities ( meddra ) coding of events by the study team , made the final sae system organ class ( soc ) classifications and assignment of an sae as a cardiovascular end point . additional safety measures included laboratory measures ( blood chemistries , hematology , and urine analyses ) at weeks 0 , 24 , and 52 of the study and evaluation of electrocardiograms ( ecgs ) at weeks 0 , 24 , 52 or early termination . an analysis of noninferiority tested the hypothesis that usual antidiabetes therapy ( uadt ) plus bromocriptine - qr is not inferior to that of uadt plus placebo in terms of the occurrence of all - cause saes using a one - sided = 0.05 level . in order to test the primary hypothesis at a one - sided = 0.05 and with a power of one = 0.90 when the noninferiority margin is 1.5 , the total number of patients with all - cause saes needed during the study was calculated to be 235 . all randomized patients that took at least one dose of study drug were included in the all - cause sae analysis . saes that occurred on a study drug or within 30 days of stopping a study drug were considered as treatment emergent events . statistical testing between the two groups was conducted using the two - sided 91.9% ci for the rate ratio of all - cause saes because this provides the same confidence boundaries as a one - sided 96% ci . the ci was estimated by the corresponding estimate for the hazard ratio ( hr ) with cox regression analysis with consideration for possible center interaction . the analysis of the composite cardiovascular endpoint used the modified intent - to - treat population of patients receiving at least one dose of a study drug . superiority or noninferiority between bromocriptine - qr and placebo for the composite cvd end point was defined as the upper bound of the two - sided 95% confidence limit being < 0 and < 1.5 , respectively . assuming a cvd composite event rate of 3.4% , this sample size provided a power of 75% to demonstrate cvd noninferiority . upon confirming noninferiority , a sequential superiority analysis of cvd end point based on the cox proportional - hazards regression with two - sided p values analysis of the cvd end point was conducted adjusting for baseline covariates including history of stroke and cardiovascular revascularization and center . changes in laboratory measures were assessed using the wilcoxon rank sum test except for a1c , which was based on a general linear model with baseline and treatment as fixed effects . the significant level was set at p < 0.05 . ( toronto , canada ) using sas software version 8.2 ( cary , nc ) . a total of 4,074 patients were screened and 879 patients were excluded for not meeting eligibility criteria , withdrawal of consent , or other reasons . of the 3,070 patients that received a study drug , 47% stopped treatment on bromocriptine - qr and the on treatment exposure time was 1,643 person - years and 914 person - years for bromocriptine - qr and placebo , respectively , which represents 77 and 86% of the expected person - time , respectively . additionally , 82% of the study patients had final assessments , which accounts for 91% of the expected person time for this trial ( 2,906 of 3,193 possible total person - years ) ( appendix iv , figure a , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-2009/dc1 ) . the study included a diverse population of patients with type 2 diabetes ( table 1 ) with the majority of patients receiving cardioprotective medications . on average for the study population , the baseline a1c , lipid , and blood pressure values indicated reasonably good control of hyperglycemia , dyslipidemia , and hypertension , respectively ; however , the presence of cardiovascular disease was still prevalent . overall , there were no differences in baseline characteristics between the groups that would not be expected by chance . saes occurred among 9.6% of placebo - treated patients versus 8.6% of bromocriptine - qr treated patients . aside from the cardiac soc class , the frequency of all remaining saes grouped by soc was similar between bromocriptine - qr and placebo . it should be noted that the cardiac soc includes a much broader definition comprising thirty - seven preferred terms ( i.e. , arrhythmia , chest pain , syncope ) as opposed to the prespecified cardiovascular end point . saes by soc and all - cause safety and composite cardiovascular end point * soc as determined by meddra coding of preferred term provided by study investigator . patients may appear in more than one system organ category if they experienced multiple aes belonging to different socs . all saes were adjudicated by an independent aeac . * * the cardiac soc includes 37 different preferred terms such as arrhythmia , myocardial infarction , chest pain , and coronary artery disease ; however the composite cardiovascular end point comprised events that met the prespecified end point criteria set forth by an independent aeac that was blinded to treatment assignment . patients may appear in more than one component of the composite cardiovascular end point if they experienced multiple cardiovascular aes . in the analysis of time to first all - cause sae , the and upper bound of the one - sided confidence limit support noninferiority between bromocriptine - qr and placebo , ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . the treatment effect did not change appreciably with the addition of the baseline covariates of age , duration of diabetes , insulin usage , sex and race . the composite cvd end point occurred in 37 bromocriptine - qr treated ( 1.8% ) and 32 placebo - treated ( 3.2% ) patients resulting in a 40% cvd relative risk reduction ( hr 0.60 [ 95% two - sided ci 0.370.96 ] ; table 2 ) . the observed cvd risk reduction did not change appreciably with the addition of the baseline covariates of age , baseline a1c level , sex , race , and prior history of stroke or prior history of coronary revascularization . figure 1 shows the kaplan - meier estimates of the proportion of patients by treatment that experienced an event within the composite end point and demonstrates a clear separation in the curves that begins after 3 months of treatment . kaplan - meier curve of the occurrence of the prespecified composite cardiovascular end point among patients randomized to bromocriptine - qr or placebo . the composite cardiovascular end point consisted of the time to first myocardial infarction , stroke , coronary revascularization , hospitalization for unstable angina , or hospitalization for congestive heart failure that occurred after randomization . the hazard ratio of bromocriptine - qr versus placebo for the occurrence of the composite cardiovascular end point was 0.60 ( 95% ci 0.370.96 ) . the effect of treatment was estimated from the unadjusted cox proportional - hazard model that used all the available data . the frequency of deaths that occurred while on a study drug or within 30 days of stopping a study drug was similar between the treatment groups ( 0.19% bromocriptine - qr , 0.20% placebo ) . five patients randomized to bromocriptine - qr and one randomized to placebo died in excess of 30 days after stopping the study drug . adverse events ( aes ) occurred in 89% of bromocriptine - qr treated and 83% of placebo - treated patients . aes were not commonly reported as severe in either treatment group ( 17% events reported as severe on bromocriptine - qr compared with 14% on placebo ) . more patients discontinued bromocriptine - qr than placebo due to an adverse event ( 24% vs. 11% , respectively ) . the most commonly reported adverse event among patients who discontinued bromocriptine - qr was nausea ( 7.6% bromocriptine - qr vs. 1% placebo ) . the only aes that occurred at a frequency rate of at least 5% and were numerically greater in the bromocriptine - qr arm were nausea , vomiting , dizziness , headache , and diarrhea . nausea was the most common adverse event ( 32.2% bromocriptine - qr vs. 7.6% placebo ) . the majority of the commonly occurring adverse events attributed to bromocriptine - qr occurred during the initial titration phase were dose related and transient in nature ( mean duration of < 14 days ) . after the initial titration phase , commonly occurring adverse events were reported at a frequency similar to that observed in the placebo treated arm . somnolence ( 4.3 bromocriptine - qr vs. 1.3% placebo ) and hypoesthesia ( 1.4 bromocriptine - qr vs. 1.1% placebo ) were the only aes within the nervous system organ class that were reported at a rate of < 5% and 1% and that occurred at a numerically greater frequency among bromocriptine - qr - treated patients . additionally , 15 ( 0.7% ) of patients on bromocriptine - qr and 14 ( 1.4% ) on placebo reported depression or depressed mood , and 13 ( 0.6% ) patients on bromocriptine - qr and 8 ( 0.8% ) on placebo reported anxiety . hypoglycemic aes occurred infrequently , with 6.9% bromocriptine - qr patients reporting an event versus 5.3% in the placebo - treated arm . there were no significant differences between the treatment arms in changes from baseline in hematology or chemistry laboratory values . there were minimal changes from baseline in lipid measures and blood pressure in both groups ( online appendix iv , table b ) . compared with those on placebo , heart rate among patients randomized to bromocriptine - qr decreased 1 bpm from a baseline study population mean heart rate of 68 bpm at week 52 ( p = 0.02 ) . the corrected qt interval decreased , albeit nonsignificantly , from baseline to week 52 among bromocriptine - qr treated patients by 3.2 ms ( baseline average 418 ms ) and by 1.9 ms for the placebo arm ( baseline average 420 ms ) . there was no difference in the mean change in body weight from baseline to week 52 for either bromocriptine - qr ( + 0.2 kg ) or placebo ( + 0.1 kg ) . the study included a diverse population of patients with type 2 diabetes ( table 1 ) with the majority of patients receiving cardioprotective medications . on average for the study population , the baseline a1c , lipid , and blood pressure values indicated reasonably good control of hyperglycemia , dyslipidemia , and hypertension , respectively ; however , the presence of cardiovascular disease was still prevalent . overall , there were no differences in baseline characteristics between the groups that would not be expected by chance . saes occurred among 9.6% of placebo - treated patients versus 8.6% of bromocriptine - qr treated patients . aside from the cardiac soc class , the frequency of all remaining saes grouped by soc was similar between bromocriptine - qr and placebo . it should be noted that the cardiac soc includes a much broader definition comprising thirty - seven preferred terms ( i.e. , arrhythmia , chest pain , syncope ) as opposed to the prespecified cardiovascular end point . saes by soc and all - cause safety and composite cardiovascular end point * soc as determined by meddra coding of preferred term provided by study investigator . patients may appear in more than one system organ category if they experienced multiple aes belonging to different socs . * * the cardiac soc includes 37 different preferred terms such as arrhythmia , myocardial infarction , chest pain , and coronary artery disease ; however the composite cardiovascular end point comprised events that met the prespecified end point criteria set forth by an independent aeac that was blinded to treatment assignment . patients may appear in more than one component of the composite cardiovascular end point if they experienced multiple cardiovascular aes . in the analysis of time to first all - cause sae , the and upper bound of the one - sided confidence limit support noninferiority between bromocriptine - qr and placebo , ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . the treatment effect did not change appreciably with the addition of the baseline covariates of age , duration of diabetes , insulin usage , sex and race . the composite cvd end point occurred in 37 bromocriptine - qr treated ( 1.8% ) and 32 placebo - treated ( 3.2% ) patients resulting in a 40% cvd relative risk reduction ( hr 0.60 [ 95% two - sided ci 0.370.96 ] ; table 2 ) . the observed cvd risk reduction did not change appreciably with the addition of the baseline covariates of age , baseline a1c level , sex , race , and prior history of stroke or prior history of coronary revascularization . figure 1 shows the kaplan - meier estimates of the proportion of patients by treatment that experienced an event within the composite end point and demonstrates a clear separation in the curves that begins after 3 months of treatment . kaplan - meier curve of the occurrence of the prespecified composite cardiovascular end point among patients randomized to bromocriptine - qr or placebo . the composite cardiovascular end point consisted of the time to first myocardial infarction , stroke , coronary revascularization , hospitalization for unstable angina , or hospitalization for congestive heart failure that occurred after randomization . the hazard ratio of bromocriptine - qr versus placebo for the occurrence of the composite cardiovascular end point was 0.60 ( 95% ci 0.370.96 ) . the effect of treatment was estimated from the unadjusted cox proportional - hazard model that used all the available data . the frequency of deaths that occurred while on a study drug or within 30 days of stopping a study drug was similar between the treatment groups ( 0.19% bromocriptine - qr , 0.20% placebo ) . five patients randomized to bromocriptine - qr and one randomized to placebo died in excess of 30 days after stopping the study drug . saes occurred among 9.6% of placebo - treated patients versus 8.6% of bromocriptine - qr treated patients . aside from the cardiac soc class , the frequency of all remaining saes grouped by soc was similar between bromocriptine - qr and placebo . it should be noted that the cardiac soc includes a much broader definition comprising thirty - seven preferred terms ( i.e. , arrhythmia , chest pain , syncope ) as opposed to the prespecified cardiovascular end point . saes by soc and all - cause safety and composite cardiovascular end point * soc as determined by meddra coding of preferred term provided by study investigator . patients may appear in more than one system organ category if they experienced multiple aes belonging to different socs . * * the cardiac soc includes 37 different preferred terms such as arrhythmia , myocardial infarction , chest pain , and coronary artery disease ; however the composite cardiovascular end point comprised events that met the prespecified end point criteria set forth by an independent aeac that was blinded to treatment assignment . patients may appear in more than one component of the composite cardiovascular end point if they experienced multiple cardiovascular aes . in the analysis of time to first all - cause sae , the and upper bound of the one - sided confidence limit support noninferiority between bromocriptine - qr and placebo , ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . the treatment effect did not change appreciably with the addition of the baseline covariates of age , duration of diabetes , insulin usage , sex and race . the composite cvd end point occurred in 37 bromocriptine - qr treated ( 1.8% ) and 32 placebo - treated ( 3.2% ) patients resulting in a 40% cvd relative risk reduction ( hr 0.60 [ 95% two - sided ci 0.370.96 ] ; table 2 ) . the observed cvd risk reduction did not change appreciably with the addition of the baseline covariates of age , baseline a1c level , sex , race , and prior history of stroke or prior history of coronary revascularization . figure 1 shows the kaplan - meier estimates of the proportion of patients by treatment that experienced an event within the composite end point and demonstrates a clear separation in the curves that begins after 3 months of treatment . kaplan - meier curve of the occurrence of the prespecified composite cardiovascular end point among patients randomized to bromocriptine - qr or placebo . the composite cardiovascular end point consisted of the time to first myocardial infarction , stroke , coronary revascularization , hospitalization for unstable angina , or hospitalization for congestive heart failure that occurred after randomization . the hazard ratio of bromocriptine - qr versus placebo for the occurrence of the composite cardiovascular end point was 0.60 ( 95% ci 0.370.96 ) . the effect of treatment was estimated from the unadjusted cox proportional - hazard model that used all the available data . the frequency of deaths that occurred while on a study drug or within 30 days of stopping a study drug was similar between the treatment groups ( 0.19% bromocriptine - qr , 0.20% placebo ) . five patients randomized to bromocriptine - qr and one randomized to placebo died in excess of 30 days after stopping the study drug . saes occurred among 9.6% of placebo - treated patients versus 8.6% of bromocriptine - qr treated patients . aside from the cardiac soc class , the frequency of all remaining saes grouped by soc was similar between bromocriptine - qr and placebo . it should be noted that the cardiac soc includes a much broader definition comprising thirty - seven preferred terms ( i.e. , arrhythmia , chest pain , syncope ) as opposed to the prespecified cardiovascular end point . saes by soc and all - cause safety and composite cardiovascular end point * soc as determined by meddra coding of preferred term provided by study investigator . patients may appear in more than one system organ category if they experienced multiple aes belonging to different socs . * * the cardiac soc includes 37 different preferred terms such as arrhythmia , myocardial infarction , chest pain , and coronary artery disease ; however the composite cardiovascular end point comprised events that met the prespecified end point criteria set forth by an independent aeac that was blinded to treatment assignment . patients may appear in more than one component of the composite cardiovascular end point if they experienced multiple cardiovascular aes . in the analysis of time to first all - cause sae , the and upper bound of the one - sided confidence limit support noninferiority between bromocriptine - qr and placebo , ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . the treatment effect did not change appreciably with the addition of the baseline covariates of age , duration of diabetes , insulin usage , sex and race . the composite cvd end point occurred in 37 bromocriptine - qr treated ( 1.8% ) and 32 placebo - treated ( 3.2% ) patients resulting in a 40% cvd relative risk reduction ( hr 0.60 [ 95% two - sided ci 0.370.96 ] ; table 2 ) . the observed cvd risk reduction did not change appreciably with the addition of the baseline covariates of age , baseline a1c level , sex , race , and prior history of stroke or prior history of coronary revascularization . figure 1 shows the kaplan - meier estimates of the proportion of patients by treatment that experienced an event within the composite end point and demonstrates a clear separation in the curves that begins after 3 months of treatment . kaplan - meier curve of the occurrence of the prespecified composite cardiovascular end point among patients randomized to bromocriptine - qr or placebo . the composite cardiovascular end point consisted of the time to first myocardial infarction , stroke , coronary revascularization , hospitalization for unstable angina , or hospitalization for congestive heart failure that occurred after randomization . all cardiovascular events were independently adjudicated . the hazard ratio of bromocriptine - qr versus placebo for the occurrence of the composite cardiovascular end point was 0.60 ( 95% ci 0.370.96 ) . the effect of treatment was estimated from the unadjusted cox proportional - hazard model that used all the available data . the frequency of deaths that occurred while on a study drug or within 30 days of stopping a study drug was similar between the treatment groups ( 0.19% bromocriptine - qr , 0.20% placebo ) . five patients randomized to bromocriptine - qr and one randomized to placebo died in excess of 30 days after stopping the study drug . adverse events ( aes ) occurred in 89% of bromocriptine - qr treated and 83% of placebo - treated patients . aes were not commonly reported as severe in either treatment group ( 17% events reported as severe on bromocriptine - qr compared with 14% on placebo ) . more patients discontinued bromocriptine - qr than placebo due to an adverse event ( 24% vs. 11% , respectively ) . the most commonly reported adverse event among patients who discontinued bromocriptine - qr was nausea ( 7.6% bromocriptine - qr vs. 1% placebo ) . the only aes that occurred at a frequency rate of at least 5% and were numerically greater in the bromocriptine - qr arm were nausea , vomiting , dizziness , headache , and diarrhea . nausea was the most common adverse event ( 32.2% bromocriptine - qr vs. 7.6% placebo ) . the majority of the commonly occurring adverse events attributed to bromocriptine - qr occurred during the initial titration phase were dose related and transient in nature ( mean duration of < 14 days ) . after the initial titration phase , commonly occurring adverse events were reported at a frequency similar to that observed in the placebo treated arm . somnolence ( 4.3 bromocriptine - qr vs. 1.3% placebo ) and hypoesthesia ( 1.4 bromocriptine - qr vs. 1.1% placebo ) were the only aes within the nervous system organ class that were reported at a rate of < 5% and 1% and that occurred at a numerically greater frequency among bromocriptine - qr - treated patients . additionally , 15 ( 0.7% ) of patients on bromocriptine - qr and 14 ( 1.4% ) on placebo reported depression or depressed mood , and 13 ( 0.6% ) patients on bromocriptine - qr and 8 ( 0.8% ) on placebo reported anxiety . hypoglycemic aes occurred infrequently , with 6.9% bromocriptine - qr patients reporting an event versus 5.3% in the placebo - treated arm . there were no significant differences between the treatment arms in changes from baseline in hematology or chemistry laboratory values . there were minimal changes from baseline in lipid measures and blood pressure in both groups ( online appendix iv , table b ) . compared with those on placebo , heart rate among patients randomized to bromocriptine - qr decreased 1 bpm from a baseline study population mean heart rate of 68 bpm at week 52 ( p = 0.02 ) . the corrected qt interval decreased , albeit nonsignificantly , from baseline to week 52 among bromocriptine - qr treated patients by 3.2 ms ( baseline average 418 ms ) and by 1.9 ms for the placebo arm ( baseline average 420 ms ) . there was no difference in the mean change in body weight from baseline to week 52 for either bromocriptine - qr ( + 0.2 kg ) or placebo ( + 0.1 kg ) . the present studyis the first to assess , as a primary objective and end point , the overall and cardiovascular safety of a new oral antidiabetes therapy in a large population of patients with type 2 diabetes . the findings from this trial indicate that morning bromocriptine - qr therapy is noninferior to placebo for overall safety . the overall frequency of all - cause saes and the proportion of saes observed in each soc among patients taking bromocriptine - qr was noninferior to the frequency among patients in the placebo arm . furthermore , the frequency of the composite cardiovascular end point was statistically significantly reduced in the bromocriptine - qr group compared with the placebo group . the kaplan - meier estimates indicate that among 1,000 patients allocated to bromocriptine - qr , 13 first myocardial infarctions , strokes , coronary revascularizations , hospitalizations for congestive heart failure , or hospitalizations for unstable angina would be avoided over 1 year . simply stated , 79 patients would need to be treated for 1 year to avoid one first important cvd event . it should be appreciated that the reduction in cvd events occurred among a study population where the majority of the patients were receiving appropriate cardioprotective medications and where blood pressure , glucose and lipids on average were optimally controlled . furthermore , these results were demonstrated among patients with an average duration of type 2 diabetes of 8 years and in which a third had preexisting cardiovascular disease . bromocriptine - qr was developed to provide a discrete and brief daily interval of circulating bromocriptine at a particular time of day ( morning ) and thereby provide a timed pulse of dopamine activity centrally . this formulation / treatment feature differentiates bromocriptine - qr bioavailability and dosing from the multiple - times per day and higher overall dosing of traditional formulations of bromocriptine for other indications such as parkinson 's disease or acromegaly , which may deliver higher circulating drug levels throughout the day and as a consequence prolonged dopamine stimulation centrally . the mechanism by which such timed bromocriptine - qr may reduce cardiovascular outcomes is not clear . in this trial , treatment with bromocriptine - qr was associated with reductions in fasting triglyceride levels , blood pressure , and heart rate but not to the extent one would expect to explain the observed cvd risk reduction . improvements in a host of cvd risk factors in addition to glycemic control are likely necessary to truly attenuate the excessive risk of cvd events experienced by patients with type 2 diabetes ( 20 ) . the peripheral cardiometabolic effects of bromocriptine - qr on cardiovascular risk may be in part the consequence of its attenuation of central nervous system and hypothalamic functions potentiating sympathetic nervous system over - activity to the vasculature , visceral adipose , and liver as well as attenuation of increased hypothalamic - pituitary - adrenal axis activity , ( 14 ) which are known to increase cvd risk if overactive ( 21,22 ) . improvements in postprandial hyperglycemia and hyperlipidemia ( 16 ) have been observed with this treatment and may have contributed to the cvd event reduction in this trial . moreover , recent studies in animal models of insulin resistance have demonstrated marked improvements in both liver inflammatory pathways potentiating vascular damage and endothelial dysfunction during treatment with a parental formulation of bromocriptine ( a.c . , unpublished data ) . although increased sympathetic tone ( 23 ) , postprandial dysmetabolism ( 24 ) , endothelial dysfunction and inflammation ( 25 ) are important cvd risk factors that have been attenuated with bromocriptine therapy in previous clinical and preclinical studies , the impact of bromocriptine - qr on these measures was not assessed in this study . much more work is required to completely understand the complex biochemical and physiological mechanisms by which timed bromocriptine - qr produces its potential cardiovascular effects . a study of longer duration and sufficient size to provide a greater number of cardiovascular outcomes could allay any concerns that the results in this trial are a chance finding while further quantifying the potential long - term reduction in cardiovascular events . for the majority of patients experiencing nausea , the symptoms occurred during the initial titration of the drug and lasted less than 2 weeks . the gastrointestinal side effects associated with bromocriptine - qr are dose related and the forced titration scheme ( rechallenge with study drug after an ae at a given dose and attempt to continue to uptitrate to achieve a maximum tolerated dose of 4.8 mg ) likely contributed to a higher than expected discontinuation rate . the incidence rates of hypoglycemia were low and similar between bromocriptine - qr and placebo groups . importantly , the lack of hypoglycemic aes was observed even when bromocriptine - qr was used as add - on therapy to a variety of antidiabetes agents including insulin . this is most likely because bromocriptine - qr does not increase insulin levels and primarily reduces postprandial glucose by way of improving the body 's responsiveness to insulin ( 16 ) . bromocriptine - qr represents a new treatment modality for type 2 diabetes . in patients with type 2 diabetes , the addition of bromocriptine - qr to routine standard of care therapies was shown to be safe and associated with fewer cardiovascular outcomes . this first - in - class therapy may provide a new approach to addressing the comorbidities associated with type 2 diabetes .

objectivequick - release bromocriptine ( bromocriptine - qr ) , a d2 dopamine receptor agonist , is indicated as a treatment for type 2 diabetes . the cycloset safety trial , a 52-week , randomized , double - blind , multicenter trial , evaluated the overall safety and cardiovascular safety of this novel therapy for type 2 diabetes.research design and methodsa total of 3,095 patients with type 2 diabetes were randomized 2:1 to bromocriptine - qr or placebo in conjunction with the patient 's usual diabetes therapy ( diet controlled only or up to two antidiabetes medications , including insulin ) . the all - cause safety end point was the occurrence of any serious adverse event ( sae ) , with a hazard ratio ( hr ) noninferiority margin of 1.5 . in a prespecified analysis , the frequency of cardiovascular disease ( cvd ) events defined as a composite of myocardial infarction , stroke , coronary revascularization , and hospitalization for angina or congestive heart failure was evaluated using modified intent - to - treat analysis ( clinicaltrials.gov , nct00377676).resultsin the bromocriptine - qr group , 176 ( 8.6% ) people reported saes compared with 98 ( 9.6% ) in the placebo group ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . fewer people reported a cvd end point in the bromocriptine - qr group versus the placebo group ( 37 [ 1.8% ] vs. 32 [ 3.2% ] , respecively ) ( hr 0.60 [ 95% two - sided ci 0.350.96 ] ) . nausea was the most commonly reported adverse event in the bromocriptine - qr group.conclusionsthe frequency of saes was comparable between the treatment arms . compared with patients in the placebo arm , fewer patients taking bromocriptine - qr experienced a cardiovascular end point .

RESEARCH DESIGN AND METHODS Outcome measures Statistical analysis RESULTS Demographics Clinical outcomes None SAEs. CVD. Deaths. Overall adverse events summary Other AEs Laboratory measures, vital signs, and electrocardiograms CONCLUSIONS Supplementary Material

eligible patients had type 2 diabetes , as defined by the 2004 american diabetes association guidelines ( 18 ) , were between the ages of 30 and 80 years , had a bmi of < 43 kg / m , and an a1c level 10.0% . patients with nyhc i - ii congestive heart failure were allowed in the trial as were those with significant cardiovascular disease , including a cardiovascular event prior to 6 months before screening . patients were randomized in a 2:1 ratio to their usual antidiabetes regimen plus bromocriptine - qr or placebo taken with their morning meal . there were two main objectives of the cycloset safety trial : 1 ) assessment of overall safety of bromocriptine - qr by measuring the frequency of serious adverse events ( saes ) among patients taking bromocriptine - qr and placebo , and 2 ) cardiovascular safety assessed by determining the frequency of major cardiovascular events , defined as a composite of first myocardial infarction , stroke , coronary revascularization , or hospitalization for angina or congestive heart failure that occurred after randomization . an independent adverse event adjudication committee ( aeac ) consisting of two cardiologists and an endocrinologist , blinded to treatment assignment and medical dictionary for regulatory activities ( meddra ) coding of events by the study team , made the final sae system organ class ( soc ) classifications and assignment of an sae as a cardiovascular end point . an analysis of noninferiority tested the hypothesis that usual antidiabetes therapy ( uadt ) plus bromocriptine - qr is not inferior to that of uadt plus placebo in terms of the occurrence of all - cause saes using a one - sided = 0.05 level . in order to test the primary hypothesis at a one - sided = 0.05 and with a power of one = 0.90 when the noninferiority margin is 1.5 , the total number of patients with all - cause saes needed during the study was calculated to be 235 . statistical testing between the two groups was conducted using the two - sided 91.9% ci for the rate ratio of all - cause saes because this provides the same confidence boundaries as a one - sided 96% ci . the analysis of the composite cardiovascular endpoint used the modified intent - to - treat population of patients receiving at least one dose of a study drug . superiority or noninferiority between bromocriptine - qr and placebo for the composite cvd end point was defined as the upper bound of the two - sided 95% confidence limit being < 0 and < 1.5 , respectively . upon confirming noninferiority , a sequential superiority analysis of cvd end point based on the cox proportional - hazards regression with two - sided p values analysis of the cvd end point was conducted adjusting for baseline covariates including history of stroke and cardiovascular revascularization and center . there were two main objectives of the cycloset safety trial : 1 ) assessment of overall safety of bromocriptine - qr by measuring the frequency of serious adverse events ( saes ) among patients taking bromocriptine - qr and placebo , and 2 ) cardiovascular safety assessed by determining the frequency of major cardiovascular events , defined as a composite of first myocardial infarction , stroke , coronary revascularization , or hospitalization for angina or congestive heart failure that occurred after randomization . an independent adverse event adjudication committee ( aeac ) consisting of two cardiologists and an endocrinologist , blinded to treatment assignment and medical dictionary for regulatory activities ( meddra ) coding of events by the study team , made the final sae system organ class ( soc ) classifications and assignment of an sae as a cardiovascular end point . an analysis of noninferiority tested the hypothesis that usual antidiabetes therapy ( uadt ) plus bromocriptine - qr is not inferior to that of uadt plus placebo in terms of the occurrence of all - cause saes using a one - sided = 0.05 level . in order to test the primary hypothesis at a one - sided = 0.05 and with a power of one = 0.90 when the noninferiority margin is 1.5 , the total number of patients with all - cause saes needed during the study was calculated to be 235 . statistical testing between the two groups was conducted using the two - sided 91.9% ci for the rate ratio of all - cause saes because this provides the same confidence boundaries as a one - sided 96% ci . the ci was estimated by the corresponding estimate for the hazard ratio ( hr ) with cox regression analysis with consideration for possible center interaction . the analysis of the composite cardiovascular endpoint used the modified intent - to - treat population of patients receiving at least one dose of a study drug . superiority or noninferiority between bromocriptine - qr and placebo for the composite cvd end point was defined as the upper bound of the two - sided 95% confidence limit being < 0 and < 1.5 , respectively . upon confirming noninferiority , a sequential superiority analysis of cvd end point based on the cox proportional - hazards regression with two - sided p values analysis of the cvd end point was conducted adjusting for baseline covariates including history of stroke and cardiovascular revascularization and center . the study included a diverse population of patients with type 2 diabetes ( table 1 ) with the majority of patients receiving cardioprotective medications . on average for the study population , the baseline a1c , lipid , and blood pressure values indicated reasonably good control of hyperglycemia , dyslipidemia , and hypertension , respectively ; however , the presence of cardiovascular disease was still prevalent . saes occurred among 9.6% of placebo - treated patients versus 8.6% of bromocriptine - qr treated patients . aside from the cardiac soc class , the frequency of all remaining saes grouped by soc was similar between bromocriptine - qr and placebo . saes by soc and all - cause safety and composite cardiovascular end point * soc as determined by meddra coding of preferred term provided by study investigator . * * the cardiac soc includes 37 different preferred terms such as arrhythmia , myocardial infarction , chest pain , and coronary artery disease ; however the composite cardiovascular end point comprised events that met the prespecified end point criteria set forth by an independent aeac that was blinded to treatment assignment . in the analysis of time to first all - cause sae , the and upper bound of the one - sided confidence limit support noninferiority between bromocriptine - qr and placebo , ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . the composite cvd end point occurred in 37 bromocriptine - qr treated ( 1.8% ) and 32 placebo - treated ( 3.2% ) patients resulting in a 40% cvd relative risk reduction ( hr 0.60 [ 95% two - sided ci 0.370.96 ] ; table 2 ) . kaplan - meier curve of the occurrence of the prespecified composite cardiovascular end point among patients randomized to bromocriptine - qr or placebo . the composite cardiovascular end point consisted of the time to first myocardial infarction , stroke , coronary revascularization , hospitalization for unstable angina , or hospitalization for congestive heart failure that occurred after randomization . the hazard ratio of bromocriptine - qr versus placebo for the occurrence of the composite cardiovascular end point was 0.60 ( 95% ci 0.370.96 ) . the frequency of deaths that occurred while on a study drug or within 30 days of stopping a study drug was similar between the treatment groups ( 0.19% bromocriptine - qr , 0.20% placebo ) . aes were not commonly reported as severe in either treatment group ( 17% events reported as severe on bromocriptine - qr compared with 14% on placebo ) . more patients discontinued bromocriptine - qr than placebo due to an adverse event ( 24% vs. 11% , respectively ) . the most commonly reported adverse event among patients who discontinued bromocriptine - qr was nausea ( 7.6% bromocriptine - qr vs. 1% placebo ) . the only aes that occurred at a frequency rate of at least 5% and were numerically greater in the bromocriptine - qr arm were nausea , vomiting , dizziness , headache , and diarrhea . nausea was the most common adverse event ( 32.2% bromocriptine - qr vs. 7.6% placebo ) . additionally , 15 ( 0.7% ) of patients on bromocriptine - qr and 14 ( 1.4% ) on placebo reported depression or depressed mood , and 13 ( 0.6% ) patients on bromocriptine - qr and 8 ( 0.8% ) on placebo reported anxiety . hypoglycemic aes occurred infrequently , with 6.9% bromocriptine - qr patients reporting an event versus 5.3% in the placebo - treated arm . the study included a diverse population of patients with type 2 diabetes ( table 1 ) with the majority of patients receiving cardioprotective medications . on average for the study population , the baseline a1c , lipid , and blood pressure values indicated reasonably good control of hyperglycemia , dyslipidemia , and hypertension , respectively ; however , the presence of cardiovascular disease was still prevalent . saes occurred among 9.6% of placebo - treated patients versus 8.6% of bromocriptine - qr treated patients . aside from the cardiac soc class , the frequency of all remaining saes grouped by soc was similar between bromocriptine - qr and placebo . saes by soc and all - cause safety and composite cardiovascular end point * soc as determined by meddra coding of preferred term provided by study investigator . * * the cardiac soc includes 37 different preferred terms such as arrhythmia , myocardial infarction , chest pain , and coronary artery disease ; however the composite cardiovascular end point comprised events that met the prespecified end point criteria set forth by an independent aeac that was blinded to treatment assignment . in the analysis of time to first all - cause sae , the and upper bound of the one - sided confidence limit support noninferiority between bromocriptine - qr and placebo , ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . the composite cvd end point occurred in 37 bromocriptine - qr treated ( 1.8% ) and 32 placebo - treated ( 3.2% ) patients resulting in a 40% cvd relative risk reduction ( hr 0.60 [ 95% two - sided ci 0.370.96 ] ; table 2 ) . kaplan - meier curve of the occurrence of the prespecified composite cardiovascular end point among patients randomized to bromocriptine - qr or placebo . the composite cardiovascular end point consisted of the time to first myocardial infarction , stroke , coronary revascularization , hospitalization for unstable angina , or hospitalization for congestive heart failure that occurred after randomization . the hazard ratio of bromocriptine - qr versus placebo for the occurrence of the composite cardiovascular end point was 0.60 ( 95% ci 0.370.96 ) . the frequency of deaths that occurred while on a study drug or within 30 days of stopping a study drug was similar between the treatment groups ( 0.19% bromocriptine - qr , 0.20% placebo ) . saes occurred among 9.6% of placebo - treated patients versus 8.6% of bromocriptine - qr treated patients . aside from the cardiac soc class , the frequency of all remaining saes grouped by soc was similar between bromocriptine - qr and placebo . saes by soc and all - cause safety and composite cardiovascular end point * soc as determined by meddra coding of preferred term provided by study investigator . * * the cardiac soc includes 37 different preferred terms such as arrhythmia , myocardial infarction , chest pain , and coronary artery disease ; however the composite cardiovascular end point comprised events that met the prespecified end point criteria set forth by an independent aeac that was blinded to treatment assignment . in the analysis of time to first all - cause sae , the and upper bound of the one - sided confidence limit support noninferiority between bromocriptine - qr and placebo , ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . the composite cvd end point occurred in 37 bromocriptine - qr treated ( 1.8% ) and 32 placebo - treated ( 3.2% ) patients resulting in a 40% cvd relative risk reduction ( hr 0.60 [ 95% two - sided ci 0.370.96 ] ; table 2 ) . kaplan - meier curve of the occurrence of the prespecified composite cardiovascular end point among patients randomized to bromocriptine - qr or placebo . the composite cardiovascular end point consisted of the time to first myocardial infarction , stroke , coronary revascularization , hospitalization for unstable angina , or hospitalization for congestive heart failure that occurred after randomization . the hazard ratio of bromocriptine - qr versus placebo for the occurrence of the composite cardiovascular end point was 0.60 ( 95% ci 0.370.96 ) . the frequency of deaths that occurred while on a study drug or within 30 days of stopping a study drug was similar between the treatment groups ( 0.19% bromocriptine - qr , 0.20% placebo ) . aside from the cardiac soc class , the frequency of all remaining saes grouped by soc was similar between bromocriptine - qr and placebo . saes by soc and all - cause safety and composite cardiovascular end point * soc as determined by meddra coding of preferred term provided by study investigator . * * the cardiac soc includes 37 different preferred terms such as arrhythmia , myocardial infarction , chest pain , and coronary artery disease ; however the composite cardiovascular end point comprised events that met the prespecified end point criteria set forth by an independent aeac that was blinded to treatment assignment . in the analysis of time to first all - cause sae , the and upper bound of the one - sided confidence limit support noninferiority between bromocriptine - qr and placebo , ( hr 1.02 [ 96% one - sided ci 1.27 ] ) . the composite cvd end point occurred in 37 bromocriptine - qr treated ( 1.8% ) and 32 placebo - treated ( 3.2% ) patients resulting in a 40% cvd relative risk reduction ( hr 0.60 [ 95% two - sided ci 0.370.96 ] ; table 2 ) . kaplan - meier curve of the occurrence of the prespecified composite cardiovascular end point among patients randomized to bromocriptine - qr or placebo . the composite cardiovascular end point consisted of the time to first myocardial infarction , stroke , coronary revascularization , hospitalization for unstable angina , or hospitalization for congestive heart failure that occurred after randomization . the hazard ratio of bromocriptine - qr versus placebo for the occurrence of the composite cardiovascular end point was 0.60 ( 95% ci 0.370.96 ) . the frequency of deaths that occurred while on a study drug or within 30 days of stopping a study drug was similar between the treatment groups ( 0.19% bromocriptine - qr , 0.20% placebo ) . aes were not commonly reported as severe in either treatment group ( 17% events reported as severe on bromocriptine - qr compared with 14% on placebo ) . more patients discontinued bromocriptine - qr than placebo due to an adverse event ( 24% vs. 11% , respectively ) . the most commonly reported adverse event among patients who discontinued bromocriptine - qr was nausea ( 7.6% bromocriptine - qr vs. 1% placebo ) . the only aes that occurred at a frequency rate of at least 5% and were numerically greater in the bromocriptine - qr arm were nausea , vomiting , dizziness , headache , and diarrhea . nausea was the most common adverse event ( 32.2% bromocriptine - qr vs. 7.6% placebo ) . additionally , 15 ( 0.7% ) of patients on bromocriptine - qr and 14 ( 1.4% ) on placebo reported depression or depressed mood , and 13 ( 0.6% ) patients on bromocriptine - qr and 8 ( 0.8% ) on placebo reported anxiety . hypoglycemic aes occurred infrequently , with 6.9% bromocriptine - qr patients reporting an event versus 5.3% in the placebo - treated arm . compared with those on placebo , heart rate among patients randomized to bromocriptine - qr decreased 1 bpm from a baseline study population mean heart rate of 68 bpm at week 52 ( p = 0.02 ) . the corrected qt interval decreased , albeit nonsignificantly , from baseline to week 52 among bromocriptine - qr treated patients by 3.2 ms ( baseline average 418 ms ) and by 1.9 ms for the placebo arm ( baseline average 420 ms ) . the present studyis the first to assess , as a primary objective and end point , the overall and cardiovascular safety of a new oral antidiabetes therapy in a large population of patients with type 2 diabetes . the overall frequency of all - cause saes and the proportion of saes observed in each soc among patients taking bromocriptine - qr was noninferior to the frequency among patients in the placebo arm . furthermore , the frequency of the composite cardiovascular end point was statistically significantly reduced in the bromocriptine - qr group compared with the placebo group . the kaplan - meier estimates indicate that among 1,000 patients allocated to bromocriptine - qr , 13 first myocardial infarctions , strokes , coronary revascularizations , hospitalizations for congestive heart failure , or hospitalizations for unstable angina would be avoided over 1 year . furthermore , these results were demonstrated among patients with an average duration of type 2 diabetes of 8 years and in which a third had preexisting cardiovascular disease . improvements in a host of cvd risk factors in addition to glycemic control are likely necessary to truly attenuate the excessive risk of cvd events experienced by patients with type 2 diabetes ( 20 ) . importantly , the lack of hypoglycemic aes was observed even when bromocriptine - qr was used as add - on therapy to a variety of antidiabetes agents including insulin . bromocriptine - qr represents a new treatment modality for type 2 diabetes . in patients with type 2 diabetes , the addition of bromocriptine - qr to routine standard of care therapies was shown to be safe and associated with fewer cardiovascular outcomes .

the events of 11 september 2001 , and the subsequent mailings of anthraxladen envelopes within the usa , forever changed the way americans view public health and national security . as recently confirmed ( the wmd terrorism research center , 2011 ) , despite the investment of considerable financial and human resources since 2001 , the usa does not have the range of medical countermeasures ( mcms ) or established systems to rapidly and effectively respond to a deliberate chemical , biological , radiological or nuclear ( cbrn ) attack , or to a naturally occurring infectious disease outbreak . mcms are the drugs , vaccines and medical devices ( including diagnostic tests , equipment and supplies ) that will be needed to respond to a public health emergency , including products to prevent and respond to anthrax , smallpox , radiological / nuclear agents , pandemic influenza and other emerging diseases . in an effort to fill this gap in mcms , product developers and the us government face particular challenges , most of which fit into two major categories : ( i ) problems facing eager but relatively inexperienced companies conducting mcm research and development and ( ii ) unique scientific and regulatory issues and uncertainties facing developers and the government arising from the fact that many mcms can not ethically or feasibly be tested in humans , meaning that much , if not all , efficacy data must be derived from animal experimentation . the federal government has a crucial role to play in facilitating mcm development and acquisition and , therefore , in addressing the associated regulatory and scientific challenges . within the department of health and human services ( hhs ) the office of the assistant secretary for preparedness and response ( aspr ) leads the public health emergency medical countermeasures enterprise ( phemce or enterprise ) , a collaboration of agencies , such as the centers for disease control and prevention ( cdc ) , national institutes of health ( nih ) , food and drug administration ( fda ) , department of homeland security ( dhs ) and department of defense ( dod ) , which is working to support and encourage the development , procurement and stockpiling of mcms . in its role as regulator , evaluating medical products for their safety and efficacy , fda has a unique and critical part to play . in 2010 , hhs secretary sebelius released the findings of an extensive review of the enterprise and articulated a new strategic mcm vision ( hhs , 2010 ) . the review 's recommendations contributed to the august 2010 establishment of fda 's medical countermeasures initiative ( mcmi ) to facilitate development and availability of highpriority mcms and strengthen the mcm enterprise . implementation of the mcmi is being coordinated by the office of counterterrorism and emerging threats ( ocet ) in fda 's office of the commissioner . however , the mcmi involves close collaboration , both internally , among the medical product centres ( i.e. center for biologics evaluation and research , center for devices and radiological health , center for drug evaluation and research ) and externally , between fda and its federal partners and other relevant stakeholders . fda has taken a threepillar approach to fulfilling its overall mcmi mission : pillar i : enhance the mcm regulatory review process ; pillar ii : advance regulatory science for mcm development and evaluation ; and pillar iii : modernize the legal , regulatory and policy framework for an effective public health response ( fda , 2011a ) . this review presents a summary of the key scientific and regulatory challenges facing mcm development , approval and use . it also describes the approaches fda is taking through the mcmi to address these key challenges . the challenges confronting mcm development and availability are more complex than the already complicated process for developing medical products . generally , mcm development and approval must follow fda 's rigorous product review process . yet , in many cases , such as when limited human efficacy data are available , the scientific and regulatory hurdles may be greater than the challenges inherent in typical drug development . in addition , medical product development is very resourceintensive , with estimates of the costs of developing a new medical product ranging from $ 0.8 to more than 1.0 billion ( dimasi and grabowski , 2007 ) . although the project bioshield act funded the creation of a government market in 2004 to acquire certain mcms , including those that are not yet licensed or approved , the funds that are available are relatively small compared with the possible return on investment from a blockbuster drug . these challenges and the lack of a commercial market for many cbrn countermeasures have left most companies with extensive experience in meeting the complex regulatory requirements and a successful track record reluctant to take on mcm development . stepping into the void have been smaller biotechnology companies ( often startups ) , which , although often technically strong , have little or no experience advancing drug development through the fda review and approval process ( cohen , 2011 ) . it is therefore critical that fda provide more regulatory and scientific guidance to these companies earlier in and throughout the development process than might be the case with larger , more regulatoryexperienced pharmaceutical companies . however , it is extremely resource intensive , and the mcm scientific and technical expertise at fda who can provide this type of assistance and ensure equity in assistance among mcm sponsors is limited . this situation is exacerbated by the unprecedented scientific challenges and uncertainties fda faces when reviewing and evaluating mcm submissions . it is on these scientific challenges that fda has focused initial efforts during the first year of the mcmi . since the inception of mcmi in 2010 , fda has increased the human and fiscal resources it devotes to formal and informal meetings related to mcm development , including increasing preinvestigational new drug ( ind ) meetings and expanding the number of internal scientific and technical consultations on mcmrelated issues . , 2007 ; nbsb , 2010 ) that fda medical product centres may not be sufficiently consistent in their interpretation and implementation of the agency 's various regulations and policy for example in interpreting fda 's 2002 regulation new drug and biological drug products ; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible ( animal rule ) and its companion draft guidance , essential elements to address efficacy under the animal rule ( fda , 2009 ) . in other instances , issues that may often be misidentified as solely regulatory in nature are actually gaps in key scientific knowledge that are hindering fda 's regulatory guidance and decisionmaking abilities . to address these issues and uncertainties , fda has created crossdisciplinary , crossfunctional public health and security action teams ( action teams ) , comprising expertise from all fda medical product centres , including staff from the centre review divisions . the action teams have already increased intraagency collaboration and informational exchange , fostering more uniformity and consistency where possible . action teams are identifying and classifying the types of hurdles and gaps that are impeding mcm product development while providing a vehicle for harmonizing agency communications with federal partners and stakeholders . action team analysis has identified some impediments as primarily scientific knowledge gaps or statutory , regulatory , or policy limitations . scientific gaps can include limited knowledge about a threat agent 's disease process or a proposed mcm ( e.g. its safety , efficacy and/or performance ) being developed for a particular cbrn indication . as specific scientific gaps are identified , fda is working closely with federal partners in the enterprise to determine the best approach for addressing them , thus informing fda 's mcm regulatory science research agenda . legal , regulatory and policy limitations can include inconsistencies in interpretation and implementation of existing statutes , regulations or policies , or the lack of an appropriate framework for developing innovative mcm products and technologies . as specific limitations are identified , fda is working internally or with hhs , congress and other relevant partners to resolve them . fda has established a number of action teams , based on the highest research and development priorities determined by the enterprise . areas of focus include in vitro diagnostics , acute radiation syndrome , trauma and the warfighter , advanced manufacturing and development , and paediatric and maternal issues . fda is also developing an animal model qualification program to enable a productneutral evaluation and qualification of animal models within a context of use . the process for animal model qualification will be consistent with the process described in the guidance for industry , qualification process for drug development tools , once it has been finalized ( fda , 2010c ) . in 2010 , fda launched the advancing regulatory science initiative , and fda product centres established their regulatory science agendas and priorities to support more active participation in scientific research and to advance development of all fda regulated products ( fda , 2010a ) . regulatory science is the science of developing new tools , standards and approaches to assess the safety , efficacy , quality and performance of fdaregulated products . mcm regulatory science focuses on the development and approval of mcms for cbrn threats and emerging infectious diseases ( e.g. pandemic influenza , sars ) . mcmi 's pillar ii is providing oversight and direction for fda 's overall mcm regulatory science portfolio and serves as the conduit for obtaining stakeholder input to shape the research agenda . initially , pillar ii activities focused on addressing centrespecific priorities identified in their respective agendas as well as on mcm priorities determined by fda 's office of the chief scientist . areas were broadly identified as research related to animal models , biomarkers , and product quality and associated assay development , among others . pillar ii has already significantly strengthened and increased fda 's intramural mcm regulatory science research portfolio . first , at mcmi 's launch , fda cosponsored with the institute of medicine a workshop designed to provide a broad overview of existing regulatory science efforts , review the state of the science regarding mcm product development , and identify opportunities for regulatory science collaborations ( iom , 2011 ) . next , a steering committee was established to peer review centrespecific regulatory science research proposals seeking fda mcmi funding . the steering committee comprises the fda chief scientist , a scientific lead from each of fda 's three medical product centres , and representatives of enterprise partners ( dod , cdc , nih and aspr ) . proposals are assessed for significance , alignment with enterprise priorities , scientific feasibility and collaborative environment . it is important to note that the collaborative environment assessment includes consideration of current intraagency collaborations , current collaborations with enterprise partners and assessment of opportunities for potential collaboration , thereby leveraging synergies across proposals . third , building on these efforts , fda released a request for information that solicited further stakeholder input to enhance and refine the current mcm regulatory research agenda and shape the pillar ii regulatory science programme ( fda , 2011b ) . fda 's comprehensive regulatory science programme is intended to address the scientific challenges that are slowing the progress of mcms in the development pipeline and generate the data needed to advance products towards approval and availability . as already noted , one of the primary scientific challenges to mcm development is the infeasibility in many cases of conducting human efficacy studies either because there are insufficient or sporadic natural occurrences of a condition or because of ethical concerns ( e.g. associated morbidity / mortality ) . in such cases , product sponsors must pursue approval through nontraditional regulatory pathways , specifically using the animal rule . although it provides an alternative path to approval , the animal rule raises complicated scientific and regulatory questions as animal data are applied in a new way . the animal rule created a need for robust and relevant animal models for product development and evaluation , but in many cases , models do not exist . once models are developed that adequately represent the human condition for specific diseases , the models may need productspecific adaptation and , in some cases , may not be suitable for all products due to speciesspecific differences ( e.g. physiology , immunology , pharmacokinetics / pharmacodynamics ) or productspecific differences ( e.g. immune modulator targeting receptors not present in all species ) . targeted regulatory science research is needed to bridge interspecies gaps , identify acceptable correlates of protection or develop new methods , such as in vitro or in silico modelling , that will aid in advancing mcm product development . three products have had indications approved under the animal rule : pyridostigmine bromide , hydroxocobalamin and levofloxacin . however , in these cases , the animal rule approval was facilitated by prior approval for another indication or information available in another country . thus , sufficient safety and efficacy data had been developed to augment animal efficacy and other data submitted to fda for approval under the animal rule , underscoring the fact that substituting animal data for human data is not intended to be an easier route towards approval ( gronvall et al . , 2007 ) . in fact , experience has proven the contrary to be true : reliance on animal data exponentially increases the scientific complexities involved in mcm development , leading to increased regulatory uncertainties . the federal food , drug , and cosmetic act ( fd&c act ) gives fda various legal and regulatory authorities and mechanisms that can facilitate mcm development and regulatory review , and even allow emergency use of certain unapproved products under certain conditions . mcm development and approval must follow fda 's rigorous product regulatory review processes : for drugs and biologics , through the investigational new drug application phase and the new drug application or biologics licence application phase ; or , in the case of a device , through premarket approval or notification 510(k ) . however , in certain situations , accelerated processes or special mechanisms ( e.g. priority review ; special protocol assessments ; and the animal rule , as described in the previous section ) are needed . additionally , during or in anticipation of an actual emergency , fda can facilitate use of a needed mcm through expanded access mechanisms ( fd&c act , 561 , 21 u.s.c . 360bbb ) or through an emergency use authorization ( eua ) ( fd&c act , 564 , 21 u.s.c . some of the special legal and regulatory mechanisms for mcm development , approval , availability and use were established through emergency preparedness legislation enacted after the 2001 anthrax attacks . the animal rule was established in the public health security and bioterrorism preparedness and response act ( 2002 ) ; the emergency use authorities are provided for in the project bioshield act ( 2004 ) ; and technical assistance teams in the event of mcm shortages are provided for in the pandemic and allhazards preparedness act ( 2006 ) . as part of fda 's mcmi , legal and regulatory issues are coordinated and addressed through pillar iii activities , the goals of which are to support mcm development and availability by ensuring that us laws , regulations and policies enable the application of advances in regulatory science to the regulatory review process and adequately support us preparedness for and response to cbrn agents and emerging infectious disease threats . in addition , pillar iii staff have been assessing the strengths and weaknesses of the current legal , regulatory and policy environment regarding mcm development , distribution , availability and use . where changes are needed to better protect public health , fda is working with federal partners and relevant stakeholders to develop and propose new approaches to improve and modernize fda 's legal , regulatory and policy framework for effective public health emergency responses . most recently , fda proposed changes as part of the reauthorization of the pandemic and allhazards preparedness act , which would ( i ) provide enhanced clarity and flexibility for euas prior to a cbrn event to enhance rapid deployment , ( ii ) better facilitate preevent planning and positioning of medical products , ( iii ) clarify fda 's authority to extend the shelf life of stockpiled mcms and ( iv ) clarify that certain actions taken in preparation for or during an emergency will not violate fda laws . also , to support anthrax preparedness and response efforts based on stakeholder needs , fda collaborated with federal partners to issue a mass dispensing eua in july 2011 ( fda , 2011c ) and to amend the postal model eua in october 2011 ( fda , 2011d ) . in december 2010 , fda , in collaboration with federal partners , sponsored a legal and regulatory preparedness meeting to ( i ) inform state public health preparedness officials and legal counsel on fda 's legal authorities for mcm responses and ( ii ) become better informed about response challenges state and local public health officials and responders face . as already mentioned , fda is working to clarify and expand the agency 's interpretation and implementation of the animal rule and the companion draft guidance for industry , animal models essential elements to address efficacy under the animal rule ( fda , 2009 ) . fda has created a crosscentre , multidisciplinary team that is carefully considering the numerous comments received during the public comment period following publication of the draft guidance . additional comments were received during and after a subsequent public meeting on the draft guidance in november 2010 ( fda , 2010b ) . in response to the significant revisions requested by the community and expansive scope of the comments , fda intends to publish the guidance as a revised draft , enabling a second comment period . the revised and expanded guidance should provide additional scientific and regulatory information to support a better understanding of the specific expectations for animal data intended to support approval across the agency . since the inception of mcmi in 2010 , fda has increased the human and fiscal resources it devotes to formal and informal meetings related to mcm development , including increasing preinvestigational new drug ( ind ) meetings and expanding the number of internal scientific and technical consultations on mcmrelated issues . , 2007 ; nbsb , 2010 ) that fda medical product centres may not be sufficiently consistent in their interpretation and implementation of the agency 's various regulations and policy for example in interpreting fda 's 2002 regulation new drug and biological drug products ; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible ( animal rule ) and its companion draft guidance , essential elements to address efficacy under the animal rule ( fda , 2009 ) . in other instances , issues that may often be misidentified as solely regulatory in nature are actually gaps in key scientific knowledge that are hindering fda 's regulatory guidance and decisionmaking abilities . to address these issues and uncertainties , fda has created crossdisciplinary , crossfunctional public health and security action teams ( action teams ) , comprising expertise from all fda medical product centres , including staff from the centre review divisions . the action teams have already increased intraagency collaboration and informational exchange , fostering more uniformity and consistency where possible . action teams are identifying and classifying the types of hurdles and gaps that are impeding mcm product development while providing a vehicle for harmonizing agency communications with federal partners and stakeholders . action team analysis has identified some impediments as primarily scientific knowledge gaps or statutory , regulatory , or policy limitations . scientific gaps can include limited knowledge about a threat agent 's disease process or a proposed mcm ( e.g. its safety , efficacy and/or performance ) being developed for a particular cbrn indication . as specific scientific gaps are identified , fda is working closely with federal partners in the enterprise to determine the best approach for addressing them , thus informing fda 's mcm regulatory science research agenda . legal , regulatory and policy limitations can include inconsistencies in interpretation and implementation of existing statutes , regulations or policies , or the lack of an appropriate framework for developing innovative mcm products and technologies . as specific limitations are identified , fda is working internally or with hhs , congress and other relevant partners to resolve them . fda has established a number of action teams , based on the highest research and development priorities determined by the enterprise . areas of focus include in vitro diagnostics , acute radiation syndrome , trauma and the warfighter , advanced manufacturing and development , and paediatric and maternal issues . fda is also developing an animal model qualification program to enable a productneutral evaluation and qualification of animal models within a context of use . the process for animal model qualification will be consistent with the process described in the guidance for industry , qualification process for drug development tools , once it has been finalized ( fda , 2010c ) . in 2010 , fda launched the advancing regulatory science initiative , and fda product centres established their regulatory science agendas and priorities to support more active participation in scientific research and to advance development of all fda regulated products ( fda , 2010a ) . regulatory science is the science of developing new tools , standards and approaches to assess the safety , efficacy , quality and performance of fdaregulated products . mcm regulatory science focuses on the development and approval of mcms for cbrn threats and emerging infectious diseases ( e.g. pandemic influenza , sars ) . mcmi 's pillar ii is providing oversight and direction for fda 's overall mcm regulatory science portfolio and serves as the conduit for obtaining stakeholder input to shape the research agenda . initially , pillar ii activities focused on addressing centrespecific priorities identified in their respective agendas as well as on mcm priorities determined by fda 's office of the chief scientist . areas were broadly identified as research related to animal models , biomarkers , and product quality and associated assay development , among others . pillar ii has already significantly strengthened and increased fda 's intramural mcm regulatory science research portfolio . first , at mcmi 's launch , fda cosponsored with the institute of medicine a workshop designed to provide a broad overview of existing regulatory science efforts , review the state of the science regarding mcm product development , and identify opportunities for regulatory science collaborations ( iom , 2011 ) . next , a steering committee was established to peer review centrespecific regulatory science research proposals seeking fda mcmi funding . the steering committee comprises the fda chief scientist , a scientific lead from each of fda 's three medical product centres , and representatives of enterprise partners ( dod , cdc , nih and aspr ) . proposals are assessed for significance , alignment with enterprise priorities , scientific feasibility and collaborative environment . it is important to note that the collaborative environment assessment includes consideration of current intraagency collaborations , current collaborations with enterprise partners and assessment of opportunities for potential collaboration , thereby leveraging synergies across proposals . third , building on these efforts , fda released a request for information that solicited further stakeholder input to enhance and refine the current mcm regulatory research agenda and shape the pillar ii regulatory science programme ( fda , 2011b ) . fda 's comprehensive regulatory science programme is intended to address the scientific challenges that are slowing the progress of mcms in the development pipeline and generate the data needed to advance products towards approval and availability . as already noted , one of the primary scientific challenges to mcm development is the infeasibility in many cases of conducting human efficacy studies either because there are insufficient or sporadic natural occurrences of a condition or because of ethical concerns ( e.g. associated morbidity / mortality ) . in such cases , product sponsors must pursue approval through nontraditional regulatory pathways , specifically using the animal rule . although it provides an alternative path to approval , the animal rule raises complicated scientific and regulatory questions as animal data are applied in a new way . the animal rule created a need for robust and relevant animal models for product development and evaluation , but in many cases , models do not exist . once models are developed that adequately represent the human condition for specific diseases , the models may need productspecific adaptation and , in some cases , may not be suitable for all products due to speciesspecific differences ( e.g. physiology , immunology , pharmacokinetics / pharmacodynamics ) or productspecific differences ( e.g. immune modulator targeting receptors not present in all species ) . targeted regulatory science research is needed to bridge interspecies gaps , identify acceptable correlates of protection or develop new methods , such as in vitro or in silico modelling , that will aid in advancing mcm product development . three products have had indications approved under the animal rule : pyridostigmine bromide , hydroxocobalamin and levofloxacin . however , in these cases , the animal rule approval was facilitated by prior approval for another indication or information available in another country . thus , sufficient safety and efficacy data had been developed to augment animal efficacy and other data submitted to fda for approval under the animal rule , underscoring the fact that substituting animal data for human data is not intended to be an easier route towards approval ( gronvall et al . , 2007 ) . in fact , experience has proven the contrary to be true : reliance on animal data exponentially increases the scientific complexities involved in mcm development , leading to increased regulatory uncertainties . the federal food , drug , and cosmetic act ( fd&c act ) gives fda various legal and regulatory authorities and mechanisms that can facilitate mcm development and regulatory review , and even allow emergency use of certain unapproved products under certain conditions . mcm development and approval must follow fda 's rigorous product regulatory review processes : for drugs and biologics , through the investigational new drug application phase and the new drug application or biologics licence application phase ; or , in the case of a device , through premarket approval or notification 510(k ) . however , in certain situations , accelerated processes or special mechanisms ( e.g. priority review ; special protocol assessments ; and the animal rule , as described in the previous section ) are needed . additionally , during or in anticipation of an actual emergency , fda can facilitate use of a needed mcm through expanded access mechanisms ( fd&c act , 561 , 21 u.s.c . 360bbb ) or through an emergency use authorization ( eua ) ( fd&c act , 564 , 21 u.s.c . some of the special legal and regulatory mechanisms for mcm development , approval , availability and use were established through emergency preparedness legislation enacted after the 2001 anthrax attacks . the animal rule was established in the public health security and bioterrorism preparedness and response act ( 2002 ) ; the emergency use authorities are provided for in the project bioshield act ( 2004 ) ; and technical assistance teams in the event of mcm shortages are provided for in the pandemic and allhazards preparedness act ( 2006 ) . as part of fda 's mcmi , legal and regulatory issues are coordinated and addressed through pillar iii activities , the goals of which are to support mcm development and availability by ensuring that us laws , regulations and policies enable the application of advances in regulatory science to the regulatory review process and adequately support us preparedness for and response to cbrn agents and emerging infectious disease threats . in addition , pillar iii staff have been assessing the strengths and weaknesses of the current legal , regulatory and policy environment regarding mcm development , distribution , availability and use . where changes are needed to better protect public health , fda is working with federal partners and relevant stakeholders to develop and propose new approaches to improve and modernize fda 's legal , regulatory and policy framework for effective public health emergency responses . most recently , fda proposed changes as part of the reauthorization of the pandemic and allhazards preparedness act , which would ( i ) provide enhanced clarity and flexibility for euas prior to a cbrn event to enhance rapid deployment , ( ii ) better facilitate preevent planning and positioning of medical products , ( iii ) clarify fda 's authority to extend the shelf life of stockpiled mcms and ( iv ) clarify that certain actions taken in preparation for or during an emergency will not violate fda laws . also , to support anthrax preparedness and response efforts based on stakeholder needs , fda collaborated with federal partners to issue a mass dispensing eua in july 2011 ( fda , 2011c ) and to amend the postal model eua in october 2011 ( fda , 2011d ) . in december 2010 , fda , in collaboration with federal partners , sponsored a legal and regulatory preparedness meeting to ( i ) inform state public health preparedness officials and legal counsel on fda 's legal authorities for mcm responses and ( ii ) become better informed about response challenges state and local public health officials and responders face . as already mentioned , fda is working to clarify and expand the agency 's interpretation and implementation of the animal rule and the companion draft guidance for industry , animal models essential elements to address efficacy under the animal rule ( fda , 2009 ) . fda has created a crosscentre , multidisciplinary team that is carefully considering the numerous comments received during the public comment period following publication of the draft guidance . additional comments were received during and after a subsequent public meeting on the draft guidance in november 2010 ( fda , 2010b ) . in response to the significant revisions requested by the community and expansive scope of the comments , fda intends to publish the guidance as a revised draft , enabling a second comment period . the revised and expanded guidance should provide additional scientific and regulatory information to support a better understanding of the specific expectations for animal data intended to support approval across the agency . as identified in the 2010 enterprise review , fda has a crucial role to play in ensuring the success of the us enterprise mission and vision to create a nimble and flexible system to produce the mcms that will be needed quickly , should an attack occur . fda 's mcmi was launched to help address key challenges associated with the regulatory review process , the gaps in mcm regulatory science , and hurdles in the legal , regulatory and policy framework that may be slowing mcm development . filling the nation 's mcm gap is a longterm , complex effort that will require substantial collaboration among governmental entities at all levels , academia , industry and health professionals . in particular , success will require an appreciation of the long timelines , risks and high costs associated with developing mcms , a significant and ongoing investment of resources , and the commitment of our national leadership .

summarydespite substantial investments since the events of 2001 , much work remains to prepare the nation for a chemical , biological , radiological or nuclear ( cbrn ) attack or to respond to an emerging infectious disease threat . following a 2010 review of the us public health emergency medical countermeasures enterprise , fda launched its medical countermeasures initiative ( mcmi ) to facilitate the development and availability of medical products to counter cbrn and emerging disease threats . as a regulatory agency , fda has a unique and critical part to play in this national undertaking . using a threepillar approach , fda is addressing key challenges associated with the regulatory review process for medical countermeasures ; gaps in regulatory science for mcm development and evaluation ; and issues related to the legal , regulatory and policy framework for an effective public health response . filling the gaps in the mcm enterprise is a huge national undertaking , requiring the collaboration of all stakeholders , including federal partners , current and prospective developers of medical countermeasures , relevant research organizations , and state and local responders . especially critical to success are an appreciation of the long timelines , risks and high costs associated with developing medical countermeasures and the systems to deliver them and the requisite support of all stakeholders , including national leadership .

Introduction Key scientific and regulatory challenges to MCM development and availability Enhancing the MCM regulatory review process Advancing MCM regulatory science Modernizing the legal, regulatory and policy framework Conclusion

the events of 11 september 2001 , and the subsequent mailings of anthraxladen envelopes within the usa , forever changed the way americans view public health and national security . as recently confirmed ( the wmd terrorism research center , 2011 ) , despite the investment of considerable financial and human resources since 2001 , the usa does not have the range of medical countermeasures ( mcms ) or established systems to rapidly and effectively respond to a deliberate chemical , biological , radiological or nuclear ( cbrn ) attack , or to a naturally occurring infectious disease outbreak . mcms are the drugs , vaccines and medical devices ( including diagnostic tests , equipment and supplies ) that will be needed to respond to a public health emergency , including products to prevent and respond to anthrax , smallpox , radiological / nuclear agents , pandemic influenza and other emerging diseases . in an effort to fill this gap in mcms , product developers and the us government face particular challenges , most of which fit into two major categories : ( i ) problems facing eager but relatively inexperienced companies conducting mcm research and development and ( ii ) unique scientific and regulatory issues and uncertainties facing developers and the government arising from the fact that many mcms can not ethically or feasibly be tested in humans , meaning that much , if not all , efficacy data must be derived from animal experimentation . the federal government has a crucial role to play in facilitating mcm development and acquisition and , therefore , in addressing the associated regulatory and scientific challenges . within the department of health and human services ( hhs ) the office of the assistant secretary for preparedness and response ( aspr ) leads the public health emergency medical countermeasures enterprise ( phemce or enterprise ) , a collaboration of agencies , such as the centers for disease control and prevention ( cdc ) , national institutes of health ( nih ) , food and drug administration ( fda ) , department of homeland security ( dhs ) and department of defense ( dod ) , which is working to support and encourage the development , procurement and stockpiling of mcms . in its role as regulator , evaluating medical products for their safety and efficacy , fda has a unique and critical part to play . in 2010 , hhs secretary sebelius released the findings of an extensive review of the enterprise and articulated a new strategic mcm vision ( hhs , 2010 ) . the review 's recommendations contributed to the august 2010 establishment of fda 's medical countermeasures initiative ( mcmi ) to facilitate development and availability of highpriority mcms and strengthen the mcm enterprise . implementation of the mcmi is being coordinated by the office of counterterrorism and emerging threats ( ocet ) in fda 's office of the commissioner . fda has taken a threepillar approach to fulfilling its overall mcmi mission : pillar i : enhance the mcm regulatory review process ; pillar ii : advance regulatory science for mcm development and evaluation ; and pillar iii : modernize the legal , regulatory and policy framework for an effective public health response ( fda , 2011a ) . this review presents a summary of the key scientific and regulatory challenges facing mcm development , approval and use . it also describes the approaches fda is taking through the mcmi to address these key challenges . the challenges confronting mcm development and availability are more complex than the already complicated process for developing medical products . generally , mcm development and approval must follow fda 's rigorous product review process . although the project bioshield act funded the creation of a government market in 2004 to acquire certain mcms , including those that are not yet licensed or approved , the funds that are available are relatively small compared with the possible return on investment from a blockbuster drug . these challenges and the lack of a commercial market for many cbrn countermeasures have left most companies with extensive experience in meeting the complex regulatory requirements and a successful track record reluctant to take on mcm development . it is therefore critical that fda provide more regulatory and scientific guidance to these companies earlier in and throughout the development process than might be the case with larger , more regulatoryexperienced pharmaceutical companies . however , it is extremely resource intensive , and the mcm scientific and technical expertise at fda who can provide this type of assistance and ensure equity in assistance among mcm sponsors is limited . it is on these scientific challenges that fda has focused initial efforts during the first year of the mcmi . since the inception of mcmi in 2010 , fda has increased the human and fiscal resources it devotes to formal and informal meetings related to mcm development , including increasing preinvestigational new drug ( ind ) meetings and expanding the number of internal scientific and technical consultations on mcmrelated issues . , 2007 ; nbsb , 2010 ) that fda medical product centres may not be sufficiently consistent in their interpretation and implementation of the agency 's various regulations and policy for example in interpreting fda 's 2002 regulation new drug and biological drug products ; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible ( animal rule ) and its companion draft guidance , essential elements to address efficacy under the animal rule ( fda , 2009 ) . to address these issues and uncertainties , fda has created crossdisciplinary , crossfunctional public health and security action teams ( action teams ) , comprising expertise from all fda medical product centres , including staff from the centre review divisions . as specific scientific gaps are identified , fda is working closely with federal partners in the enterprise to determine the best approach for addressing them , thus informing fda 's mcm regulatory science research agenda . legal , regulatory and policy limitations can include inconsistencies in interpretation and implementation of existing statutes , regulations or policies , or the lack of an appropriate framework for developing innovative mcm products and technologies . as specific limitations are identified , fda is working internally or with hhs , congress and other relevant partners to resolve them . areas of focus include in vitro diagnostics , acute radiation syndrome , trauma and the warfighter , advanced manufacturing and development , and paediatric and maternal issues . the process for animal model qualification will be consistent with the process described in the guidance for industry , qualification process for drug development tools , once it has been finalized ( fda , 2010c ) . in 2010 , fda launched the advancing regulatory science initiative , and fda product centres established their regulatory science agendas and priorities to support more active participation in scientific research and to advance development of all fda regulated products ( fda , 2010a ) . mcm regulatory science focuses on the development and approval of mcms for cbrn threats and emerging infectious diseases ( e.g. areas were broadly identified as research related to animal models , biomarkers , and product quality and associated assay development , among others . pillar ii has already significantly strengthened and increased fda 's intramural mcm regulatory science research portfolio . first , at mcmi 's launch , fda cosponsored with the institute of medicine a workshop designed to provide a broad overview of existing regulatory science efforts , review the state of the science regarding mcm product development , and identify opportunities for regulatory science collaborations ( iom , 2011 ) . third , building on these efforts , fda released a request for information that solicited further stakeholder input to enhance and refine the current mcm regulatory research agenda and shape the pillar ii regulatory science programme ( fda , 2011b ) . fda 's comprehensive regulatory science programme is intended to address the scientific challenges that are slowing the progress of mcms in the development pipeline and generate the data needed to advance products towards approval and availability . as already noted , one of the primary scientific challenges to mcm development is the infeasibility in many cases of conducting human efficacy studies either because there are insufficient or sporadic natural occurrences of a condition or because of ethical concerns ( e.g. the animal rule created a need for robust and relevant animal models for product development and evaluation , but in many cases , models do not exist . in fact , experience has proven the contrary to be true : reliance on animal data exponentially increases the scientific complexities involved in mcm development , leading to increased regulatory uncertainties . the federal food , drug , and cosmetic act ( fd&c act ) gives fda various legal and regulatory authorities and mechanisms that can facilitate mcm development and regulatory review , and even allow emergency use of certain unapproved products under certain conditions . mcm development and approval must follow fda 's rigorous product regulatory review processes : for drugs and biologics , through the investigational new drug application phase and the new drug application or biologics licence application phase ; or , in the case of a device , through premarket approval or notification 510(k ) . priority review ; special protocol assessments ; and the animal rule , as described in the previous section ) are needed . some of the special legal and regulatory mechanisms for mcm development , approval , availability and use were established through emergency preparedness legislation enacted after the 2001 anthrax attacks . the animal rule was established in the public health security and bioterrorism preparedness and response act ( 2002 ) ; the emergency use authorities are provided for in the project bioshield act ( 2004 ) ; and technical assistance teams in the event of mcm shortages are provided for in the pandemic and allhazards preparedness act ( 2006 ) . as part of fda 's mcmi , legal and regulatory issues are coordinated and addressed through pillar iii activities , the goals of which are to support mcm development and availability by ensuring that us laws , regulations and policies enable the application of advances in regulatory science to the regulatory review process and adequately support us preparedness for and response to cbrn agents and emerging infectious disease threats . in addition , pillar iii staff have been assessing the strengths and weaknesses of the current legal , regulatory and policy environment regarding mcm development , distribution , availability and use . where changes are needed to better protect public health , fda is working with federal partners and relevant stakeholders to develop and propose new approaches to improve and modernize fda 's legal , regulatory and policy framework for effective public health emergency responses . most recently , fda proposed changes as part of the reauthorization of the pandemic and allhazards preparedness act , which would ( i ) provide enhanced clarity and flexibility for euas prior to a cbrn event to enhance rapid deployment , ( ii ) better facilitate preevent planning and positioning of medical products , ( iii ) clarify fda 's authority to extend the shelf life of stockpiled mcms and ( iv ) clarify that certain actions taken in preparation for or during an emergency will not violate fda laws . also , to support anthrax preparedness and response efforts based on stakeholder needs , fda collaborated with federal partners to issue a mass dispensing eua in july 2011 ( fda , 2011c ) and to amend the postal model eua in october 2011 ( fda , 2011d ) . in december 2010 , fda , in collaboration with federal partners , sponsored a legal and regulatory preparedness meeting to ( i ) inform state public health preparedness officials and legal counsel on fda 's legal authorities for mcm responses and ( ii ) become better informed about response challenges state and local public health officials and responders face . as already mentioned , fda is working to clarify and expand the agency 's interpretation and implementation of the animal rule and the companion draft guidance for industry , animal models essential elements to address efficacy under the animal rule ( fda , 2009 ) . fda has created a crosscentre , multidisciplinary team that is carefully considering the numerous comments received during the public comment period following publication of the draft guidance . in response to the significant revisions requested by the community and expansive scope of the comments , fda intends to publish the guidance as a revised draft , enabling a second comment period . since the inception of mcmi in 2010 , fda has increased the human and fiscal resources it devotes to formal and informal meetings related to mcm development , including increasing preinvestigational new drug ( ind ) meetings and expanding the number of internal scientific and technical consultations on mcmrelated issues . , 2007 ; nbsb , 2010 ) that fda medical product centres may not be sufficiently consistent in their interpretation and implementation of the agency 's various regulations and policy for example in interpreting fda 's 2002 regulation new drug and biological drug products ; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible ( animal rule ) and its companion draft guidance , essential elements to address efficacy under the animal rule ( fda , 2009 ) . to address these issues and uncertainties , fda has created crossdisciplinary , crossfunctional public health and security action teams ( action teams ) , comprising expertise from all fda medical product centres , including staff from the centre review divisions . as specific scientific gaps are identified , fda is working closely with federal partners in the enterprise to determine the best approach for addressing them , thus informing fda 's mcm regulatory science research agenda . legal , regulatory and policy limitations can include inconsistencies in interpretation and implementation of existing statutes , regulations or policies , or the lack of an appropriate framework for developing innovative mcm products and technologies . as specific limitations are identified , fda is working internally or with hhs , congress and other relevant partners to resolve them . areas of focus include in vitro diagnostics , acute radiation syndrome , trauma and the warfighter , advanced manufacturing and development , and paediatric and maternal issues . the process for animal model qualification will be consistent with the process described in the guidance for industry , qualification process for drug development tools , once it has been finalized ( fda , 2010c ) . in 2010 , fda launched the advancing regulatory science initiative , and fda product centres established their regulatory science agendas and priorities to support more active participation in scientific research and to advance development of all fda regulated products ( fda , 2010a ) . mcm regulatory science focuses on the development and approval of mcms for cbrn threats and emerging infectious diseases ( e.g. areas were broadly identified as research related to animal models , biomarkers , and product quality and associated assay development , among others . first , at mcmi 's launch , fda cosponsored with the institute of medicine a workshop designed to provide a broad overview of existing regulatory science efforts , review the state of the science regarding mcm product development , and identify opportunities for regulatory science collaborations ( iom , 2011 ) . the steering committee comprises the fda chief scientist , a scientific lead from each of fda 's three medical product centres , and representatives of enterprise partners ( dod , cdc , nih and aspr ) . third , building on these efforts , fda released a request for information that solicited further stakeholder input to enhance and refine the current mcm regulatory research agenda and shape the pillar ii regulatory science programme ( fda , 2011b ) . fda 's comprehensive regulatory science programme is intended to address the scientific challenges that are slowing the progress of mcms in the development pipeline and generate the data needed to advance products towards approval and availability . as already noted , one of the primary scientific challenges to mcm development is the infeasibility in many cases of conducting human efficacy studies either because there are insufficient or sporadic natural occurrences of a condition or because of ethical concerns ( e.g. the animal rule created a need for robust and relevant animal models for product development and evaluation , but in many cases , models do not exist . the federal food , drug , and cosmetic act ( fd&c act ) gives fda various legal and regulatory authorities and mechanisms that can facilitate mcm development and regulatory review , and even allow emergency use of certain unapproved products under certain conditions . mcm development and approval must follow fda 's rigorous product regulatory review processes : for drugs and biologics , through the investigational new drug application phase and the new drug application or biologics licence application phase ; or , in the case of a device , through premarket approval or notification 510(k ) . priority review ; special protocol assessments ; and the animal rule , as described in the previous section ) are needed . some of the special legal and regulatory mechanisms for mcm development , approval , availability and use were established through emergency preparedness legislation enacted after the 2001 anthrax attacks . the animal rule was established in the public health security and bioterrorism preparedness and response act ( 2002 ) ; the emergency use authorities are provided for in the project bioshield act ( 2004 ) ; and technical assistance teams in the event of mcm shortages are provided for in the pandemic and allhazards preparedness act ( 2006 ) . as part of fda 's mcmi , legal and regulatory issues are coordinated and addressed through pillar iii activities , the goals of which are to support mcm development and availability by ensuring that us laws , regulations and policies enable the application of advances in regulatory science to the regulatory review process and adequately support us preparedness for and response to cbrn agents and emerging infectious disease threats . in addition , pillar iii staff have been assessing the strengths and weaknesses of the current legal , regulatory and policy environment regarding mcm development , distribution , availability and use . where changes are needed to better protect public health , fda is working with federal partners and relevant stakeholders to develop and propose new approaches to improve and modernize fda 's legal , regulatory and policy framework for effective public health emergency responses . most recently , fda proposed changes as part of the reauthorization of the pandemic and allhazards preparedness act , which would ( i ) provide enhanced clarity and flexibility for euas prior to a cbrn event to enhance rapid deployment , ( ii ) better facilitate preevent planning and positioning of medical products , ( iii ) clarify fda 's authority to extend the shelf life of stockpiled mcms and ( iv ) clarify that certain actions taken in preparation for or during an emergency will not violate fda laws . also , to support anthrax preparedness and response efforts based on stakeholder needs , fda collaborated with federal partners to issue a mass dispensing eua in july 2011 ( fda , 2011c ) and to amend the postal model eua in october 2011 ( fda , 2011d ) . in december 2010 , fda , in collaboration with federal partners , sponsored a legal and regulatory preparedness meeting to ( i ) inform state public health preparedness officials and legal counsel on fda 's legal authorities for mcm responses and ( ii ) become better informed about response challenges state and local public health officials and responders face . as already mentioned , fda is working to clarify and expand the agency 's interpretation and implementation of the animal rule and the companion draft guidance for industry , animal models essential elements to address efficacy under the animal rule ( fda , 2009 ) . fda has created a crosscentre , multidisciplinary team that is carefully considering the numerous comments received during the public comment period following publication of the draft guidance . in response to the significant revisions requested by the community and expansive scope of the comments , fda intends to publish the guidance as a revised draft , enabling a second comment period . as identified in the 2010 enterprise review , fda has a crucial role to play in ensuring the success of the us enterprise mission and vision to create a nimble and flexible system to produce the mcms that will be needed quickly , should an attack occur . fda 's mcmi was launched to help address key challenges associated with the regulatory review process , the gaps in mcm regulatory science , and hurdles in the legal , regulatory and policy framework that may be slowing mcm development . filling the nation 's mcm gap is a longterm , complex effort that will require substantial collaboration among governmental entities at all levels , academia , industry and health professionals . in particular , success will require an appreciation of the long timelines , risks and high costs associated with developing mcms , a significant and ongoing investment of resources , and the commitment of our national leadership .

overweight and obesity is an increasing problem globally . the prevalence is often estimated by body mass index ( bmi ; weight in kg divided by squared height in meters ) . in epidemiological surveys , height and weight are often gathered using self - report questionnaires . this type of data collection is advantageous because it is cost - effective , rapid , and easy to administer when sampling large numbers of individuals , particularly when spread over large areas.1 however , the validity of self - reported data for body measurements has been questioned because some participants overestimate height and underestimate weight , resulting in a lower estimated bmi.15 a review including 64 studies published between 1979 and 2005 examined the validity of self - reported bmi and found that the mean errors varied , and the discrepancies were greatest in obese populations.1 weight status has been shown to predict misreporting of bmi in several large studies , with overweight and obese persons being more likely to under - report bmi.2,610 women have a greater tendency to underestimate bmi than men in many studies,9,11,12 but not all.2 age has been found to predict misreporting of weight and height , with younger women under - reporting weight more than older women , while the chance of over - reporting height increases with age.6,7,11,12 however , these observed associations of age with misreporting are not universal.5 the amount of discrepancy between self - reported bmi and measured bmi varies between studies , and average discrepancies of 0.670.80 kg / m among women have been reported.2,59 even though most studies find relatively small differences between self - reported and measured bmi,8,9 there is a risk that even small deviations can lead to misclassification of bmi and incorrectly estimate associations between bmi categories and incidence of disease.10 the world health organization classifies bmi into four categories : underweight , bmi < 18.5 kg / m ; normal weight , bmi 18.524.9 kg / m ; overweight , bmi 2529.9 kg / m ; and obese , bmi 30 kg / m.13 inaccurate reporting of weight and height may also result in incorrect estimates of the prevalence of overweight . for large studies covering geographically scattered participants , self - reported data are paramount , and examinations of the validity of these data are central to interpretation of the results . validity is a feature of the source population , and can not be inferred from studies from other countries or groups.14 it might change over time and as social and cultural norms about the phenomenon changes.1 to the best of our knowledge , the validity of self - reported height and weight has not been described in a norwegian population . the purpose of this study was to examine whether self - reported weight and height in a sample of women from a large norwegian cohort are valid indicators of bmi for use in epidemiological studies . the norwegian women and cancer ( nowac ) study is a large nationally representative prospective cohort study of women aged 3070 years at recruitment . started in 1991 , the original purpose of the nowac study was to explore the relationship between oral contraceptive use and breast cancer , and the cohort currently includes over 172,000 participants ( figure 1 ) . the study is described in detail by lund et al.15 during 20032006 , the nowac study collected blood samples and a new questionnaire ( hereafter referred to as the secondary questionnaire ) from recent participants born between 1943 and 1957 . the blood collection took place via mail , and the women were asked to contact their general practitioners for taking the blood sample . we focused on a random subsample of 4,498 of the women who were contacted to donate a sample of blood , and therefore were subject to a second data collection within a relatively short time span . of the women contacted , 71% complied ( n=3,194 , figure 1 ) . to limit the time between repeated questionnaires , this study includes the 1,837 women from the random subsample who were asked for and provided a blood sample within 1 year of their primary questionnaire . the primary questionnaire included questions on hormonal and reproductive factors , smoking , alcohol , diet , height , weight , physical activity , self - reported health and some diseases and medications , socioeconomic factors , and sun tanning habits . women were also asked to complete an additional two - page questionnaire , which accompanied the blood sample . this secondary questionnaire collected information that could influence biomarker measurements and updated some of the information from the primary questionnaire , eg , fasting status , menstruation / menopausal status , smoking , height , weight , use of dietary supplements , and medications taken in the previous 7 days . height and weight were self - reported in the primary questionnaire , but could be either self - reported or measured at the secondary questionnaire , and the participants were asked to indicate whether they had been measured by medical staff on the day of the blood sample . no instructions were given with regard to clothing or weight calibration to the participants or medical staff . values producing differences between the two measurements of more than 10 cm for height and 5 kg for weight were checked for data entry errors , but values entered on the original paper questionnaires were assumed to be accurate . missing values for the anthropometric measurements were rare in the primary and secondary questionnaires , with 3% missing for weight and 2% missing for height observed for each questionnaire . there appeared to be a slight tendency to round to the nearest 5 kg for self - reported weight measurements when comparing the frequency of weights ending in 0 or 5 in the primary questionnaire ( 35% ) and secondary questionnaire ( 31% ) , compared with weights measured by medical staff ( 25% ) . bmi was grouped into four categories according to the world health organization classification.13 information about age was based on birth year and provided by the national population register at statistics norway . education , marital status , and perceived health were reported as part of the nowac study . total years of education were categorized into basic or primary education ( 9 years ) , secondary school ( 1012 years ) , and college educated ( 13 years ) . marital status was categorized into married / living together , widowed or divorced , and unmarried . perceived health ( do you think of your own health as : ) was reported as very good , good , poor , or very poor . because few women in the blood sampling group reported very poor health ( n=3 ) equality of group means was tested with two - sample t - tests with pooled variance , while categorical variables were tested using a chi - square test of independence . statistical analyses were performed in sas version 9.4 ( sas institute , cary , nc , usa ) , and statistical significance was defined as a test resulting in a p - value less than 0.05 . repeated measurements of height , weight , and bmi were tested for equality using paired t - tests with pooled variance . differences between self - reported values on the primary questionnaire and values measured by medical staff when blood was sampled ( secondary questionnaire ) represent both changes over time and error . differences between repeated self - reported values are an estimate of changes in weight over time , assuming reporting bias is constant within 1 year . two - sample t - tests with pooled variance were used to test if the differences between types of measurements ( self - reported followed by medical staff - reported ) were larger than repeated self - reported measurements over time . the difference in differences since bmi is often categorized when used as a risk factor , categories were created for both measurements and cross - classified . the percentage correctly classified was calculated , and the agreement between repeated bmi classifications was assessed using a weighted cohen s kappa coefficient.16 this coefficient measures the agreement beyond what is expected by chance . the weighting penalizes errors based upon the level of disagreement ; for example , a misclassification of two categories has a greater penalty than a misclassification of only one category . altman analysis was used to compare the agreement between bmi based on self - reporting and values measured by medical staff , as well as between the two self - reported measurements.17 the average difference between the bmi values indicates the overall bias present in the data , while the limits of agreement ( mean 1.96 standard deviation ) indicate the precision of the measurements . for the comparison with values measured by medical staff , negative differences indicate that the bmi using medical staff values was higher than the self - reported bmi values , ie , negative differences indicate under - reporting . in order to examine representativeness , a comparison was made of basic demographic variables between the study sample group and the independent set of women remaining in the eligible cohort . specifically , the group of women who donated blood within a year of their questionnaire and supplied information on their anthropometric measurements ( n=1,723 ) were compared with those in the eligible cohort , who were willing to donate a blood sample , minus the random subsample ( n=91,828 ) using chi - square tests of independence and two sample t - tests . participants received mailed information about the study together with the questionnaires , and indicated their consent to participate . the study received approval from the regional committee for medical research ethics for the basic collection and storing of questionnaire information and blood sampling . equality of group means was tested with two - sample t - tests with pooled variance , while categorical variables were tested using a chi - square test of independence . statistical analyses were performed in sas version 9.4 ( sas institute , cary , nc , usa ) , and statistical significance was defined as a test resulting in a p - value less than 0.05 . repeated measurements of height , weight , and bmi were tested for equality using paired t - tests with pooled variance . differences between self - reported values on the primary questionnaire and values measured by medical staff when blood was sampled ( secondary questionnaire ) represent both changes over time and error . differences between repeated self - reported values are an estimate of changes in weight over time , assuming reporting bias is constant within 1 year . two - sample t - tests with pooled variance were used to test if the differences between types of measurements ( self - reported followed by medical staff - reported ) were larger than repeated self - reported measurements over time . the difference in differences provides an estimate of any self - report bias . since bmi is often categorized when used as a risk factor , categories were created for both measurements and cross - classified . the percentage correctly classified was calculated , and the agreement between repeated bmi classifications was assessed using a weighted cohen s kappa coefficient.16 this coefficient measures the agreement beyond what is expected by chance . the weighting penalizes errors based upon the level of disagreement ; for example , a misclassification of two categories has a greater penalty than a misclassification of only one category . altman analysis was used to compare the agreement between bmi based on self - reporting and values measured by medical staff , as well as between the two self - reported measurements.17 the average difference between the bmi values indicates the overall bias present in the data , while the limits of agreement ( mean 1.96 standard deviation ) indicate the precision of the measurements . for the comparison with values measured by medical staff , negative differences indicate that the bmi using medical staff values was higher than the self - reported bmi values , ie , negative differences indicate under - reporting . in order to examine representativeness , a comparison was made of basic demographic variables between the study sample group and the independent set of women remaining in the eligible cohort . specifically , the group of women who donated blood within a year of their questionnaire and supplied information on their anthropometric measurements ( n=1,723 ) were compared with those in the eligible cohort , who were willing to donate a blood sample , minus the random subsample ( n=91,828 ) using chi - square tests of independence and two sample t - tests . participants received mailed information about the study together with the questionnaires , and indicated their consent to participate . the study received approval from the regional committee for medical research ethics for the basic collection and storing of questionnaire information and blood sampling . the study sample consisted of 1,837 women who donated blood within a year of their primary questionnaire . of those , 280 women had their height and weight measured by medical staff at the time of their blood sample ( ie , secondary questionnaire ) . the rest either self - reported their height and weight ( n=1,443 ) or failed to indicate how the measurement was taken ( n=114 ) . women who self - reported their weight and height in the secondary questionnaire were similar to those who had their measurements taken by medical staff in terms of demographic characteristics ( table 1 ) . the women had an average age of 53.7 years at the time of their primary questionnaire , with a range of 4664 years . on average , they donated the blood sample 8 months after their primary questionnaire , with a range of 50365 days . comparisons of mean age and days between measurements failed to find any differences between those who self - reported anthropometric measurements at the secondary questionnaire and those who had their measurements taken by medical staff . there were no statistically significant differences between the two groups in terms of education level , marital status , or bmi category from the primary questionnaire . women who self - reported their weight and height at both questionnaires were more likely to have reported very good health status on the primary questionnaire than those who had their values measured at the secondary questionnaire ( p=0.05 ) , but both groups had few women reporting poor health . at the secondary measurement , the prevalence of the different bmi categories were : underweight 1% ( both groups ) , normal weight 51% in the self - reported group versus 49% in the measured group , overweight 34% ( self - reported ) versus 35% ( measured ) , and obesity 14% ( self - reported ) versus 15% ( measured ; see data in tables 3 and 5 ) . the majority of the study sample ( 79% ) self - reported their height and weight at the secondary questionnaire . the repeated self - measurements provide an estimate of changes over time , assuming any bias in reporting remains equal over the time period . on average , women reported little variation in their weight and height , with average differences of 0.6 kg and 0.1 cm ( table 2 ) . only 10% of women reported a weight difference of more than 5 kg ( gain or loss ) and only 10% reported a height difference of more than 1 cm . the mean differences in weight and bmi , although small in value , were statistically significant . for the women who self - reported values on both occasions ( n=1,380 ) , 11% ( n=152 ) were assigned a different bmi category at the secondary questionnaire when compared with the primary questionnaire ( table 3 ) , with 99% ( n=150 ) changing only one category . of those who changed category , the majority ( n=99 , 65% ) increased in bmi . when examining the women who changed bmi category either upward or downward , there were no statistically significant differences . women who increased a bmi category did not differ from those who were classified in the same category on both questionnaires in terms of education level , marital status , perceived health status , mean age , or mean days between measurements . however , there was a tendency for women who moved to a lower bmi category to report poorer health status ( 13% poor , 23% very good ) when compared with those who did not change in bmi category ( 7% and 34% respectively , p=0.07 ) . the normal weight bmi group at the secondary questionnaire had the highest percent agreement with the previously calculated bmi ( 95% ) , followed by the overweight ( 83% ) and obese ( 80% ) groups . few women had bmi values that corresponded to the underweight category . when examining the baseline classification ( row percentages ) in table 3 , there was not much variation with regard to how many women stayed in the same bmi category at the secondary questionnaire among those who self - reported their weight on both occasions , ie , 91% among the normal weight , 86% among the overweight , and 88% among the obese . when donating blood , 15% of the participating women ( n=280 ) had one of the medical staff measure their height and weight for the secondary questionnaire . mean weight as reported by medical staff was on average 1.5 kg higher than that self - reported by women in the primary questionnaire ( table 4 ) . height , on average , was 0.2 cm less than the self - reported values , and bmi calculated from the medical staff values was 0.6 kg / m higher than bmi calculated from self - reported values . while the differences are all small in value , they are statistically significant in paired t - tests . repeated measurements over time can be expected to change , and we estimated these changes for the study sample in the previous section ( table 2 ) . comparisons of the differences in reporting method ( self - reported , measured by medical staff ) and changes over time give an indication of self - report bias . weight measured by medical staff showed a larger increase on average than expected by changes over time ( 0.9 kg ) and the mean difference between reporting methods was significantly larger than that observed between repeated self - reports ( p<0.001 ) . height did not differ significantly more between reporting methods than expected through repeated self - reports . in line with the mean increase in weight , bmi measured by medical staff increased on average by 0.4 kg / m more than observed through repeated self - reports , which is a statistically significant increase ( p=0.002 ) . calculated bmi categories from self - reporting and values measured by medical staff differed for 20% of the women ( n=54 ) by one category ( table 5 ) . of those who changed category , the majority ( n=43 , 80% ) increased in bmi . women who increased one bmi category did not differ from those who were classified in the same category on both questionnaires in terms of education level , marital status , perceived health status , mean age , or mean days between measurements . while the majority of women in the sample were married ( table 1 ) , the 11 women who decreased in bmi category between self - reporting and values measured by medical staff were almost evenly split between married / cohabiting and unmarried ( fisher s exact test , p=0.04 ) . women with normal weight as measured by the medical staff had the highest agreement with the self - reported measure ( 94% ) , followed by obese women ( 80% ) . the highest level of misclassification was among overweight women , where 36% had self - reported values that corresponded to normal weight and 3% had self - reported bmi in the obese range ( table 5 ) . the weighted kappa assessing the agreement between values measured by medical staff and self - reported values was 0.73 ( 95% confidence interval 0.670.80 ) which corresponds to substantial agreement.18 in comparison , the expected agreement was 0.39 ( data not shown ) . when examining the baseline classification ( row percentages ) in table 5 , a slightly different picture emerges . among the self - reported obese , 91% were obese when objectively measured , while among the normal weight ( 78% ) and overweight ( 79% ) , this percentage was lower . in the self - reported normal weight group , most of the misreporters belonged to the overweight category when measured by medical staff ( 21% ) , while the misreporters among the overweight were fairly evenly spread among normal weight and obese when measured by medical staff ( 10% and 11% , respectively ) . the overall mean difference between the average of self - reported bmi and that measured by medical staff was 0.29 kg / m , indicating a small bias toward under - reporting of bmi in self - reported values . the 95% limits of agreement ( figure 2 ) for the differences between the two measurements demonstrate both high overall precision and higher variation between measurements for women in the obese bmi category compared with those in the normal range . women with an average bmi in the obese range were more likely to have under - reported their bmi beyond the 95% limits of agreement ( 18% , 6/33 ) than those with an average bmi in the overweight range ( 5% , 4/82 ) . over the entire range of bmi values , the level of agreement between the two measurements was substantial , as demonstrated by very similar cumulative distribution curves ( figure s1 ) . the under - reporting was slightly greater than that observed for repeated self - reports , which also showed greater variation in the obese bmi range , with 12% of women outside the 95% limits of agreement compared with only 6% outside the limits for the remaining women ( figure 2 ) . there were no statistically significant differences between the eligible cohort and those in the study sample in terms of self - reported weight or perceived health status . there were small but statistically significant differences in terms of mean age at the time of the primary questionnaire ( 53.9 years in the eligible cohort compared with 53.7 years in the study sample p=0.04 ) , marital status ( p=0.04 ) , and height ( 166.8 cm compared with 166.4 cm , p=0.008 ) . women in the study sample were more likely to be married ( 83% ) than those in the eligible cohort ( 81% ) . there was a significant difference between the two groups of women in level of education , with a higher proportion of women in the study sample having a college degree ( 52% ) than those in the comparison group ( 46% , p<0.001 ) . the majority of the study sample ( 79% ) self - reported their height and weight at the secondary questionnaire . the repeated self - measurements provide an estimate of changes over time , assuming any bias in reporting remains equal over the time period . on average , women reported little variation in their weight and height , with average differences of 0.6 kg and 0.1 cm ( table 2 ) . only 10% of women reported a weight difference of more than 5 kg ( gain or loss ) and only 10% reported a height difference of more than 1 cm . the mean differences in weight and bmi , although small in value , were statistically significant . for the women who self - reported values on both occasions ( n=1,380 ) , 11% ( n=152 ) were assigned a different bmi category at the secondary questionnaire when compared with the primary questionnaire ( table 3 ) , with 99% ( n=150 ) changing only one category . of those who changed category , the majority ( n=99 , 65% ) increased in bmi . when examining the women who changed bmi category either upward or downward , there were no statistically significant differences . women who increased a bmi category did not differ from those who were classified in the same category on both questionnaires in terms of education level , marital status , perceived health status , mean age , or mean days between measurements . however , there was a tendency for women who moved to a lower bmi category to report poorer health status ( 13% poor , 23% very good ) when compared with those who did not change in bmi category ( 7% and 34% respectively , p=0.07 ) . the normal weight bmi group at the secondary questionnaire had the highest percent agreement with the previously calculated bmi ( 95% ) , followed by the overweight ( 83% ) and obese ( 80% ) groups . few women had bmi values that corresponded to the underweight category . when examining the baseline classification ( row percentages ) in table 3 , there was not much variation with regard to how many women stayed in the same bmi category at the secondary questionnaire among those who self - reported their weight on both occasions , ie , 91% among the normal weight , 86% among the overweight , and 88% among the obese . when donating blood , 15% of the participating women ( n=280 ) had one of the medical staff measure their height and weight for the secondary questionnaire . mean weight as reported by medical staff was on average 1.5 kg higher than that self - reported by women in the primary questionnaire ( table 4 ) . height , on average , was 0.2 cm less than the self - reported values , and bmi calculated from the medical staff values was 0.6 kg / m higher than bmi calculated from self - reported values . while the differences are all small in value , they are statistically significant in paired t - tests . repeated measurements over time can be expected to change , and we estimated these changes for the study sample in the previous section ( table 2 ) . comparisons of the differences in reporting method ( self - reported , measured by medical staff ) and changes over time give an indication of self - report bias . weight measured by medical staff showed a larger increase on average than expected by changes over time ( 0.9 kg ) and the mean difference between reporting methods was significantly larger than that observed between repeated self - reports ( p<0.001 ) . height did not differ significantly more between reporting methods than expected through repeated self - reports . in line with the mean increase in weight , bmi measured by medical staff increased on average by 0.4 kg / m more than observed through repeated self - reports , which is a statistically significant increase ( p=0.002 ) . calculated bmi categories from self - reporting and values measured by medical staff differed for 20% of the women ( n=54 ) by one category ( table 5 ) . of those who changed category , the majority ( n=43 , 80% ) increased in bmi . women who increased one bmi category did not differ from those who were classified in the same category on both questionnaires in terms of education level , marital status , perceived health status , mean age , or mean days between measurements . while the majority of women in the sample were married ( table 1 ) , the 11 women who decreased in bmi category between self - reporting and values measured by medical staff were almost evenly split between married / cohabiting and unmarried ( fisher s exact test , p=0.04 ) . women with normal weight as measured by the medical staff had the highest agreement with the self - reported measure ( 94% ) , followed by obese women ( 80% ) . the highest level of misclassification was among overweight women , where 36% had self - reported values that corresponded to normal weight and 3% had self - reported bmi in the obese range ( table 5 ) . the weighted kappa assessing the agreement between values measured by medical staff and self - reported values was 0.73 ( 95% confidence interval 0.670.80 ) which corresponds to substantial agreement.18 in comparison , the expected agreement was 0.39 ( data not shown ) . when examining the baseline classification ( row percentages ) in table 5 , a slightly different picture emerges . among the self - reported obese , 91% were obese when objectively measured , while among the normal weight ( 78% ) and overweight ( 79% ) , this percentage was lower . in the self - reported normal weight group , most of the misreporters belonged to the overweight category when measured by medical staff ( 21% ) , while the misreporters among the overweight were fairly evenly spread among normal weight and obese when measured by medical staff ( 10% and 11% , respectively ) . the overall mean difference between the average of self - reported bmi and that measured by medical staff was 0.29 kg / m , indicating a small bias toward under - reporting of bmi in self - reported values . the 95% limits of agreement ( figure 2 ) for the differences between the two measurements demonstrate both high overall precision and higher variation between measurements for women in the obese bmi category compared with those in the normal range . women with an average bmi in the obese range were more likely to have under - reported their bmi beyond the 95% limits of agreement ( 18% , 6/33 ) than those with an average bmi in the overweight range ( 5% , 4/82 ) . over the entire range of bmi values , the level of agreement between the two measurements was substantial , as demonstrated by very similar cumulative distribution curves ( figure s1 ) . the under - reporting was slightly greater than that observed for repeated self - reports , which also showed greater variation in the obese bmi range , with 12% of women outside the 95% limits of agreement compared with only 6% outside the limits for the remaining women ( figure 2 ) . there were no statistically significant differences between the eligible cohort and those in the study sample in terms of self - reported weight or perceived health status . there were small but statistically significant differences in terms of mean age at the time of the primary questionnaire ( 53.9 years in the eligible cohort compared with 53.7 years in the study sample p=0.04 ) , marital status ( p=0.04 ) , and height ( 166.8 cm compared with 166.4 cm , p=0.008 ) . women in the study sample were more likely to be married ( 83% ) than those in the eligible cohort ( 81% ) . there was a significant difference between the two groups of women in level of education , with a higher proportion of women in the study sample having a college degree ( 52% ) than those in the comparison group ( 46% , p<0.001 ) . in this study , we estimated the misreporting of self - reported weight and height by comparing repeated self - reports with self - reported values followed by values measured by medical staff . the two groups did not differ except for better perceived health among those who gave repeated self - reports . the distribution of bmi categories did not differ between self - reported and measured values . weight and bmi were under - reported more by the repeated self - reporting group , but there was substantial agreement between self - reported values and those measured by medical staff ( weighted kappa for bmi 0.73 ) . under - reporting leading to misclassification of bmi category was most common among overweight women ( 36% ) , but the highest proportion of extreme under - reporters was found in the obese women ( 18% outside the 95% limits of agreement ) . the highest proportion of correctly classified women based on the primary self - reported measurement was found among the obese women . although the design of this validation study differs from that of most other studies , our findings are in accordance with studies comparing self - reported weight and height with weight and height measured by medical staff within a short time period . the difference in self - reported bmi and bmi measured by medical staff in our study was small but statistically significant , ie , 0.4 kg / m , and lower than what was found in women in the adventist health study ( 0.7 kg / m),2 epic - norfolk ( 0.92 kg / m),19 multiethnic cohort ( 0.67 kg / m),8 the sister study ( 0.7 kg / m),6 and in the skaraborg project ( 0.8 kg / m),7 women attending a us family medicine clinic ( 0.8 kg / m),5 and female participants in the national health and nutrition education survey iii ( 0.67 kg / m).9 the results were similar to those found for women in an austrian study ( 0.43 kg / m),20 but the difference was larger than what was found in australian women ( 0.12 kg / m).21 an earlier review found mean differences between self - reported and measured bmi of 0.9 to 1.2 kg / m in women from the general population.1 as in most other studies , the errors in bmi were due to under - reporting of weight , and there was no significant misreporting of height . in the national health and nutrition education survey iii , high correlations was found between self - reported and measured bmi , and also between self - reported and measured bmi and disease biomarkers.9 the correlations did not differ much by age , sex , or obesity status . further , the results were the same when the analyses were done with percent body fat rather than bmi as the measure of adiposity . stommel et al found that women aged 4255 years reported bmi more in accordance with direct measures than either their younger or older counterparts,22 and most of our participants were in that age range . a more recent paper found that there have been temporal changes in the precision of self - reported height and weight , leading to more accurate bmi estimations.23 as the opposite has also been found,24 there could be cultural differences in accuracy of self - reported anthropometric values , but we have not found other norwegian publications for comparison . correlations between self - reported and measured bmi are generally high ( > 0.90 in all ethnic groups9 ) , but they are not adequate for measuring reliability , because they are testing associations rather than agreement , and are not able to identify systematic errors.25 the percentage of agreement includes both the precision of the measurement and the frequency of errors , and has clinical meaning , while graphical presentations are useful for displaying distributions and the magnitude of error.25 in general , substantial agreement between bmi categorizations based on self - reporting and measured values has been found , but self - reported values tend to give a lower bmi category,1,2,7,11,20 especially in overweight and obese subjects.3,4,12,26,27 kappa values between 0.66 and 0.81 have been reported,2,12,19,21 and our value of 0.73 fits well with this . in our study , 80% of the women were correctly classified , similar to what others have found.2,5,21 studies vary as to whether the percentage of correctly classified women is lowest among the overweight5,21 or obese.68,12,19,20 in our study , the percentage was lowest among the overweight when the underweight group ( n=2 measured by medical staff ) was disregarded . when examining those who were measured by medical staff based on their self - reported bmi category at baseline , the highest percentage of correctly classified participants was found in the obese group ( 91% ) . hence , the lower percentage of correctly classified obese women based on the measured values was due to misclassification ( under - reporting ) among those who were self - reported overweight rather than misclassification ( over - reporting ) among the obese . the repeated self - reported values indicated a greater range of values for those with an average bmi in the obese range compared with other women . this may indicate errors in measurement or changes in reporting bias , but could also indicate greater variation in weight over time for women in the obese range . in a large public health study from spain , after adjusting for predictors of under - reporting of bmi , especially dissatisfaction with body size , the estimated overweight prevalence increased from 15.0% to 18.5%.3 a large validity study of self - reported bmi in the national health and nutrition education survey concluded that self - reports are sufficient for most epidemiological studies , but not for prevalence studies.9 in our study , the prevalence of overweight and obesity did not differ when using self - reported and measured values . since the validation subsample was representative , and there was no difference between the self - reported and measured group , except in perceived health status , it seems that the self - reported values may be utilized for estimating prevalence of overweight and , in particular , obesity . comparisons failed to find any differences between those whose anthropometric measurements were self - reported in the secondary questionnaire and those who were measured by medical staff , except that women who gave repeated self - reports were more likely to report very good health status on the primary questionnaire than those who had their values measured for the secondary questionnaire . it is likely that perceived health influences what kind of questions a woman asks the medical staff ( blood sample only , or also anthropometric measurements ) . also , women who perceived their health as good might not be interested in receiving feedback from medical staff or in spending any additional time in the office . there was no difference in the prevalence of self - perceived poor health between the two groups . we have previously shown that participants in the nowac study are representative of the female norwegian population as a whole,28 except for higher education than non - responders , and that cancer rates are the same in our cohort as in the general female population of the same age.15 in the current study , education was the only factor where significant differences were found between women who gave blood and the remaining cohort . there were no significant differences in bmi between the different education groups , so the validity of the present study is not threatened . the strengths of this study include the representativeness of the study sample and the unbiased study design . participants were not aware that their anthropometric measurements might be checked when providing their primary height and weight information . knowledge of future measurement of weight by medical staff may lead to more accurate reporting.27 this study has some limitations . different measurement instruments were used for different participants ; they were not calibrated nor were precise instructions provided . participants with values measured by medical staff would have used a different scale and measuring tape at home when providing the primary self - reported values . this variation in instruments undoubtedly increased the variability in the measurements and the measurement error . failure to calibrate instruments has been shown to increase the prevalence of overweight and obesity in population - based samples.29 however , digital home bathroom scales have been shown to provide sufficiently accurate and consistent weights for public health research purposes.30 further , the mean time lapse between measurements was quite long ( 8 months ) , but as the difference was equal in both groups , this complicates only the study design and not the final results . body weight may naturally shift up and down over time , so we studied differences in excess of what was found with repeated self - reports , assuming constant misreporting over the year . this could be questioned , but since the data collection took place over years , the results should be robust to seasonal variations . the results of this study show that self - reported data successfully distinguish between the obese and nonobese , and although there were some more misreporters among the overweight , the results are comparable with those of other studies . being able to correctly classify the obese is important , given that the association between bmi and mortality or morbidity is strongest for this group.31,32 women who had their weight measured after having self - reported had a significantly higher weight than those who self - reported twice . the tendency of under - reporting was largest among overweight women , while the most extreme under - reporters were found in the obese group . despite the under - reporting , the discrepancies between self - reported and directly measured bmi in women were small , and the agreement between self - reported and measured values was substantial , as demonstrated by the cumulative distribution of the bmi curves . our self - reported weight and height data provide a valid ranking of bmi for middle - aged norwegian women . cumulative distribution plot of self - reported and measured bmi . abbreviation : bmi , body mass index .

backgroundbody mass index ( bmi ) based on self - reported height and weight has been criticized as being biased because of an observed tendency for overweight and obese people to overestimate height and underestimate weight , resulting in higher misclassification for these groups . we examined the validity of bmi based on self - reported values in a sample of norwegian women aged 4464 years.methodsthe study sample of 1,837 participants in the norwegian women and cancer study self - reported height and weight , and then , within 1 year , either self - reported anthropometric again , or were measured by medical staff . demographic and anthropometric were compared using t - tests and chi - square tests of independence . misclassification of bmi categories was assessed by weighted cohen s kappa and bland altman plot.resultson average , the two measurements were taken 8 months apart , and self - reported weight increased by 0.6 kg ( p<0.05 ) , and bmi by 0.2 kg / m2 ( p<0.05 ) . the distribution of bmi categories did not differ between self - reported and measured values . there was substantial agreement between self - reported values and those measured by medical staff ( weighted kappa 0.73 ) . under - reporting resulting in misclassification of bmi category was most common among overweight women ( 36% ) , but the highest proportion of extreme under - reporting was found in obese women ( 18% outside the 95% limits of agreement ) . the cumulative distribution curves for the measured and self - reported values closely followed each other , but measurements by medical staff were shifted slightly toward higher bmi values.conclusionwhile there was substantial agreement between self - reported and measured bmi values , there was small but statistically significant under - reporting of weight and thus self - reported bmi . the tendency to under - report was largest among overweight women , while the largest degree of under - reporting was found in the obese group . self - reported weight and height provide a valid ranking of bmi for middle - aged norwegian women .

Background Subjects and methods Statistical analysis Ethical issues Results Variation in self-reported BMI over time Self-reporting versus measurement by medical staff Representativeness Discussion Conclusion Supplementary material

this type of data collection is advantageous because it is cost - effective , rapid , and easy to administer when sampling large numbers of individuals , particularly when spread over large areas.1 however , the validity of self - reported data for body measurements has been questioned because some participants overestimate height and underestimate weight , resulting in a lower estimated bmi.15 a review including 64 studies published between 1979 and 2005 examined the validity of self - reported bmi and found that the mean errors varied , and the discrepancies were greatest in obese populations.1 weight status has been shown to predict misreporting of bmi in several large studies , with overweight and obese persons being more likely to under - report bmi.2,610 women have a greater tendency to underestimate bmi than men in many studies,9,11,12 but not all.2 age has been found to predict misreporting of weight and height , with younger women under - reporting weight more than older women , while the chance of over - reporting height increases with age.6,7,11,12 however , these observed associations of age with misreporting are not universal.5 the amount of discrepancy between self - reported bmi and measured bmi varies between studies , and average discrepancies of 0.670.80 kg / m among women have been reported.2,59 even though most studies find relatively small differences between self - reported and measured bmi,8,9 there is a risk that even small deviations can lead to misclassification of bmi and incorrectly estimate associations between bmi categories and incidence of disease.10 the world health organization classifies bmi into four categories : underweight , bmi < 18.5 kg / m ; normal weight , bmi 18.524.9 kg / m ; overweight , bmi 2529.9 kg / m ; and obese , bmi 30 kg / m.13 inaccurate reporting of weight and height may also result in incorrect estimates of the prevalence of overweight . validity is a feature of the source population , and can not be inferred from studies from other countries or groups.14 it might change over time and as social and cultural norms about the phenomenon changes.1 to the best of our knowledge , the validity of self - reported height and weight has not been described in a norwegian population . the purpose of this study was to examine whether self - reported weight and height in a sample of women from a large norwegian cohort are valid indicators of bmi for use in epidemiological studies . height and weight were self - reported in the primary questionnaire , but could be either self - reported or measured at the secondary questionnaire , and the participants were asked to indicate whether they had been measured by medical staff on the day of the blood sample . there appeared to be a slight tendency to round to the nearest 5 kg for self - reported weight measurements when comparing the frequency of weights ending in 0 or 5 in the primary questionnaire ( 35% ) and secondary questionnaire ( 31% ) , compared with weights measured by medical staff ( 25% ) . because few women in the blood sampling group reported very poor health ( n=3 ) equality of group means was tested with two - sample t - tests with pooled variance , while categorical variables were tested using a chi - square test of independence . differences between self - reported values on the primary questionnaire and values measured by medical staff when blood was sampled ( secondary questionnaire ) represent both changes over time and error . altman analysis was used to compare the agreement between bmi based on self - reporting and values measured by medical staff , as well as between the two self - reported measurements.17 the average difference between the bmi values indicates the overall bias present in the data , while the limits of agreement ( mean 1.96 standard deviation ) indicate the precision of the measurements . for the comparison with values measured by medical staff , negative differences indicate that the bmi using medical staff values was higher than the self - reported bmi values , ie , negative differences indicate under - reporting . specifically , the group of women who donated blood within a year of their questionnaire and supplied information on their anthropometric measurements ( n=1,723 ) were compared with those in the eligible cohort , who were willing to donate a blood sample , minus the random subsample ( n=91,828 ) using chi - square tests of independence and two sample t - tests . differences between self - reported values on the primary questionnaire and values measured by medical staff when blood was sampled ( secondary questionnaire ) represent both changes over time and error . altman analysis was used to compare the agreement between bmi based on self - reporting and values measured by medical staff , as well as between the two self - reported measurements.17 the average difference between the bmi values indicates the overall bias present in the data , while the limits of agreement ( mean 1.96 standard deviation ) indicate the precision of the measurements . for the comparison with values measured by medical staff , negative differences indicate that the bmi using medical staff values was higher than the self - reported bmi values , ie , negative differences indicate under - reporting . specifically , the group of women who donated blood within a year of their questionnaire and supplied information on their anthropometric measurements ( n=1,723 ) were compared with those in the eligible cohort , who were willing to donate a blood sample , minus the random subsample ( n=91,828 ) using chi - square tests of independence and two sample t - tests . women who self - reported their weight and height in the secondary questionnaire were similar to those who had their measurements taken by medical staff in terms of demographic characteristics ( table 1 ) . women who self - reported their weight and height at both questionnaires were more likely to have reported very good health status on the primary questionnaire than those who had their values measured at the secondary questionnaire ( p=0.05 ) , but both groups had few women reporting poor health . at the secondary measurement , the prevalence of the different bmi categories were : underweight 1% ( both groups ) , normal weight 51% in the self - reported group versus 49% in the measured group , overweight 34% ( self - reported ) versus 35% ( measured ) , and obesity 14% ( self - reported ) versus 15% ( measured ; see data in tables 3 and 5 ) . when examining the baseline classification ( row percentages ) in table 3 , there was not much variation with regard to how many women stayed in the same bmi category at the secondary questionnaire among those who self - reported their weight on both occasions , ie , 91% among the normal weight , 86% among the overweight , and 88% among the obese . height , on average , was 0.2 cm less than the self - reported values , and bmi calculated from the medical staff values was 0.6 kg / m higher than bmi calculated from self - reported values . in line with the mean increase in weight , bmi measured by medical staff increased on average by 0.4 kg / m more than observed through repeated self - reports , which is a statistically significant increase ( p=0.002 ) . calculated bmi categories from self - reporting and values measured by medical staff differed for 20% of the women ( n=54 ) by one category ( table 5 ) . while the majority of women in the sample were married ( table 1 ) , the 11 women who decreased in bmi category between self - reporting and values measured by medical staff were almost evenly split between married / cohabiting and unmarried ( fisher s exact test , p=0.04 ) . women with normal weight as measured by the medical staff had the highest agreement with the self - reported measure ( 94% ) , followed by obese women ( 80% ) . the highest level of misclassification was among overweight women , where 36% had self - reported values that corresponded to normal weight and 3% had self - reported bmi in the obese range ( table 5 ) . the weighted kappa assessing the agreement between values measured by medical staff and self - reported values was 0.73 ( 95% confidence interval 0.670.80 ) which corresponds to substantial agreement.18 in comparison , the expected agreement was 0.39 ( data not shown ) . in the self - reported normal weight group , most of the misreporters belonged to the overweight category when measured by medical staff ( 21% ) , while the misreporters among the overweight were fairly evenly spread among normal weight and obese when measured by medical staff ( 10% and 11% , respectively ) . the overall mean difference between the average of self - reported bmi and that measured by medical staff was 0.29 kg / m , indicating a small bias toward under - reporting of bmi in self - reported values . the 95% limits of agreement ( figure 2 ) for the differences between the two measurements demonstrate both high overall precision and higher variation between measurements for women in the obese bmi category compared with those in the normal range . women with an average bmi in the obese range were more likely to have under - reported their bmi beyond the 95% limits of agreement ( 18% , 6/33 ) than those with an average bmi in the overweight range ( 5% , 4/82 ) . over the entire range of bmi values , the level of agreement between the two measurements was substantial , as demonstrated by very similar cumulative distribution curves ( figure s1 ) . the under - reporting was slightly greater than that observed for repeated self - reports , which also showed greater variation in the obese bmi range , with 12% of women outside the 95% limits of agreement compared with only 6% outside the limits for the remaining women ( figure 2 ) . there were no statistically significant differences between the eligible cohort and those in the study sample in terms of self - reported weight or perceived health status . there were small but statistically significant differences in terms of mean age at the time of the primary questionnaire ( 53.9 years in the eligible cohort compared with 53.7 years in the study sample p=0.04 ) , marital status ( p=0.04 ) , and height ( 166.8 cm compared with 166.4 cm , p=0.008 ) . for the women who self - reported values on both occasions ( n=1,380 ) , 11% ( n=152 ) were assigned a different bmi category at the secondary questionnaire when compared with the primary questionnaire ( table 3 ) , with 99% ( n=150 ) changing only one category . when examining the baseline classification ( row percentages ) in table 3 , there was not much variation with regard to how many women stayed in the same bmi category at the secondary questionnaire among those who self - reported their weight on both occasions , ie , 91% among the normal weight , 86% among the overweight , and 88% among the obese . height , on average , was 0.2 cm less than the self - reported values , and bmi calculated from the medical staff values was 0.6 kg / m higher than bmi calculated from self - reported values . in line with the mean increase in weight , bmi measured by medical staff increased on average by 0.4 kg / m more than observed through repeated self - reports , which is a statistically significant increase ( p=0.002 ) . calculated bmi categories from self - reporting and values measured by medical staff differed for 20% of the women ( n=54 ) by one category ( table 5 ) . while the majority of women in the sample were married ( table 1 ) , the 11 women who decreased in bmi category between self - reporting and values measured by medical staff were almost evenly split between married / cohabiting and unmarried ( fisher s exact test , p=0.04 ) . women with normal weight as measured by the medical staff had the highest agreement with the self - reported measure ( 94% ) , followed by obese women ( 80% ) . the highest level of misclassification was among overweight women , where 36% had self - reported values that corresponded to normal weight and 3% had self - reported bmi in the obese range ( table 5 ) . the weighted kappa assessing the agreement between values measured by medical staff and self - reported values was 0.73 ( 95% confidence interval 0.670.80 ) which corresponds to substantial agreement.18 in comparison , the expected agreement was 0.39 ( data not shown ) . in the self - reported normal weight group , most of the misreporters belonged to the overweight category when measured by medical staff ( 21% ) , while the misreporters among the overweight were fairly evenly spread among normal weight and obese when measured by medical staff ( 10% and 11% , respectively ) . the overall mean difference between the average of self - reported bmi and that measured by medical staff was 0.29 kg / m , indicating a small bias toward under - reporting of bmi in self - reported values . the 95% limits of agreement ( figure 2 ) for the differences between the two measurements demonstrate both high overall precision and higher variation between measurements for women in the obese bmi category compared with those in the normal range . women with an average bmi in the obese range were more likely to have under - reported their bmi beyond the 95% limits of agreement ( 18% , 6/33 ) than those with an average bmi in the overweight range ( 5% , 4/82 ) . over the entire range of bmi values , the level of agreement between the two measurements was substantial , as demonstrated by very similar cumulative distribution curves ( figure s1 ) . the under - reporting was slightly greater than that observed for repeated self - reports , which also showed greater variation in the obese bmi range , with 12% of women outside the 95% limits of agreement compared with only 6% outside the limits for the remaining women ( figure 2 ) . there were no statistically significant differences between the eligible cohort and those in the study sample in terms of self - reported weight or perceived health status . there were small but statistically significant differences in terms of mean age at the time of the primary questionnaire ( 53.9 years in the eligible cohort compared with 53.7 years in the study sample p=0.04 ) , marital status ( p=0.04 ) , and height ( 166.8 cm compared with 166.4 cm , p=0.008 ) . in this study , we estimated the misreporting of self - reported weight and height by comparing repeated self - reports with self - reported values followed by values measured by medical staff . the distribution of bmi categories did not differ between self - reported and measured values . weight and bmi were under - reported more by the repeated self - reporting group , but there was substantial agreement between self - reported values and those measured by medical staff ( weighted kappa for bmi 0.73 ) . under - reporting leading to misclassification of bmi category was most common among overweight women ( 36% ) , but the highest proportion of extreme under - reporters was found in the obese women ( 18% outside the 95% limits of agreement ) . the highest proportion of correctly classified women based on the primary self - reported measurement was found among the obese women . although the design of this validation study differs from that of most other studies , our findings are in accordance with studies comparing self - reported weight and height with weight and height measured by medical staff within a short time period . the difference in self - reported bmi and bmi measured by medical staff in our study was small but statistically significant , ie , 0.4 kg / m , and lower than what was found in women in the adventist health study ( 0.7 kg / m),2 epic - norfolk ( 0.92 kg / m),19 multiethnic cohort ( 0.67 kg / m),8 the sister study ( 0.7 kg / m),6 and in the skaraborg project ( 0.8 kg / m),7 women attending a us family medicine clinic ( 0.8 kg / m),5 and female participants in the national health and nutrition education survey iii ( 0.67 kg / m).9 the results were similar to those found for women in an austrian study ( 0.43 kg / m),20 but the difference was larger than what was found in australian women ( 0.12 kg / m).21 an earlier review found mean differences between self - reported and measured bmi of 0.9 to 1.2 kg / m in women from the general population.1 as in most other studies , the errors in bmi were due to under - reporting of weight , and there was no significant misreporting of height . in the national health and nutrition education survey iii , high correlations was found between self - reported and measured bmi , and also between self - reported and measured bmi and disease biomarkers.9 the correlations did not differ much by age , sex , or obesity status . a more recent paper found that there have been temporal changes in the precision of self - reported height and weight , leading to more accurate bmi estimations.23 as the opposite has also been found,24 there could be cultural differences in accuracy of self - reported anthropometric values , but we have not found other norwegian publications for comparison . correlations between self - reported and measured bmi are generally high ( > 0.90 in all ethnic groups9 ) , but they are not adequate for measuring reliability , because they are testing associations rather than agreement , and are not able to identify systematic errors.25 the percentage of agreement includes both the precision of the measurement and the frequency of errors , and has clinical meaning , while graphical presentations are useful for displaying distributions and the magnitude of error.25 in general , substantial agreement between bmi categorizations based on self - reporting and measured values has been found , but self - reported values tend to give a lower bmi category,1,2,7,11,20 especially in overweight and obese subjects.3,4,12,26,27 kappa values between 0.66 and 0.81 have been reported,2,12,19,21 and our value of 0.73 fits well with this . when examining those who were measured by medical staff based on their self - reported bmi category at baseline , the highest percentage of correctly classified participants was found in the obese group ( 91% ) . hence , the lower percentage of correctly classified obese women based on the measured values was due to misclassification ( under - reporting ) among those who were self - reported overweight rather than misclassification ( over - reporting ) among the obese . in a large public health study from spain , after adjusting for predictors of under - reporting of bmi , especially dissatisfaction with body size , the estimated overweight prevalence increased from 15.0% to 18.5%.3 a large validity study of self - reported bmi in the national health and nutrition education survey concluded that self - reports are sufficient for most epidemiological studies , but not for prevalence studies.9 in our study , the prevalence of overweight and obesity did not differ when using self - reported and measured values . since the validation subsample was representative , and there was no difference between the self - reported and measured group , except in perceived health status , it seems that the self - reported values may be utilized for estimating prevalence of overweight and , in particular , obesity . comparisons failed to find any differences between those whose anthropometric measurements were self - reported in the secondary questionnaire and those who were measured by medical staff , except that women who gave repeated self - reports were more likely to report very good health status on the primary questionnaire than those who had their values measured for the secondary questionnaire . the tendency of under - reporting was largest among overweight women , while the most extreme under - reporters were found in the obese group . despite the under - reporting , the discrepancies between self - reported and directly measured bmi in women were small , and the agreement between self - reported and measured values was substantial , as demonstrated by the cumulative distribution of the bmi curves . our self - reported weight and height data provide a valid ranking of bmi for middle - aged norwegian women .

however , their use for increasingly demanding applications in proteomics research and bioanalytics necessitates reliable size standards for instrument calibration . commonly employed standard reagents for esi and maldi mass spectrometry have to fulfil several requirements depending on the application , and they are expensive to produce . since in maldi - ms singly charged analyte ions are predominantly formed and a large m / z range can be observed , it is reasonable to use large polymers for calibration , thus matching the expected size range . to this end , a large variety of chemical polymers [ e.g. , polyethylene glycol , poly(methylmethacrylate ) , polystyrene , polydimethylsiloxane , polystyrene sulfonate ] [ 14 ] , as well as peptides and proteins ( e.g. , bradykinin fragments , aldolase , insulin ) are commercially available . the chemical polymer calibration kits are optimized with regard to homogeneous ionization properties , but they comprise polydisperse substances with a poorly controlled distribution of molecular weights . in contrast , peptides and proteins constitute intrinsically monodisperse compounds , but it takes considerable effort to prepare different proteins in sufficient purity and to combine them at a suitable ratio and composition for ms applications . furthermore , as the functionality of ionization sites in proteins is highly dependent on the chemical environment , which is defined by the amino acid sequence , use of conventional protein mixes as ms standards is hampered by uneven signal intensity distribution . apart from this , synthetic peptides are limited in length by chemical solid phase procedures , and molecular weights above 34 kda are hardly accessible at the required purity . on the other hand , esi - ms results in the formation of multiple - charged analyte ions with a characteristic m / z distribution in the raw spectrum . the relatively narrow experimentally measured m / z range allows the use of rather small , chemically synthesized compounds for calibration , each of which appears as a singly charged molecule ion in the raw spectrum . in this regard , a collection of perfluorinated hydrocarbons ( aliphatic and aromatic ) , trialkyl amines , as well as triazine and phosphazene derivatives , and also mixes thereof , are available , and even inorganic alkali salts may be used . amino compounds tend to carry desirable positive charges , whereas perfluorinated alkyl residues give rise to a mono - isotopic , simple mass increase of 50 da per cf2 moiety . a substituted phosphazene compound with six fluoroalkyl side chains , for example , yields a signal distribution with a plain difference of 300 da between the homologous compounds . furthermore , perfluorinated alkyl chains are largely protected from secondary fragmentation reactions following electrospray ionization , which normally result in rapid degradation of nonfluorinated hydrocarbon chains of equivalent length . a way to circumvent these problems would be a peptide standard with varying length but homogeneous composition , ideally prepared from a recombinant polypeptide comprising repetitive sequences of a small , defined set of amino acids . the pasylation technology , which was recently developed in our laboratory [ 10 , 11 ] , has inspired the design of an ms calibration standard that fulfils these requirements . pas polypeptides are composed of long stretches of repetitive sequences of the three small , hydrophilic residues , proline , alanine and serine . these pas sequences are natively disordered under physiological buffer conditions , and they adopt an expanded hydrodynamic volume , similar to the chemical polymer peg . initially , pas sequences were developed for genetic fusion with therapeutic proteins , thus effecting retarded kidney filtration in vivo and resulting in a similar extending effect on the plasma half - life of biopharmaceuticals as chemical conjugation with peg . with regard to application as an ms standard , the lack of secondary structure of the pas polypeptides and their exclusive composition of chemically stable amino acids without reactive side chains should result in a uniform ionization and protonation pattern for the peptide backbone , ensuring formation of multiple - charged ions especially suitable for esi detection . here , we report the design and synthesis of a pas - based ( poly)-peptide calibration standard which provides a simple m / z pattern , is easy to prepare , and should be useful for broad application in ms . the assembly of a synthetic gene encoding the pas - cal concatamer ( figure 1 ) was accomplished in several steps according to a previously developed strategy involving incomplete ligation of synthetic dna double strands that carry nonpalindromic three - nucleotide overhangs ( corresponding to an ala codon ) , followed by insertion into a plasmid that has a unique cleavage site for the type iis restriction enzyme sapi ( 5'-'nnn , ngaagagc ) . to this end , a pair of complementary oligodeoxynucleotides ( thermo fisher scientific , ulm , germany ) encoding the 24-residue pas#5 amino acid sequence with or without an additional arg residue at the c - terminal end leon - rot , germany ) and hybridized to obtain short gene cassettes , each with two 5'-overhangs of 3 nucleotides . these cassettes were ligated in the presence of a limiting amount of t4 dna ligase ( fermentas ) . the resulting pas - cal gene fragments , comprising two or three cassettes of either one or a mixture of both versions ( i.e. , with and without arg ) , were extracted from a preparative agarose gel and subcloned on a derivative of puc19 that carries a double ( inverse repeat ) sapi restriction site in its multiple cloning region , thus allowing both dna sequence analysis and , subsequently , precise excision.figure 1construction , expression , and purification of sumo / pas - cal . ( a ) the pas - cal polypeptide was expressed as a sumo fusion protein . after protein purification and digestion with sumo protease ( ulp1 ) , the pas - cal polypeptide concatamer yields a defined set of four standard peptides upon trypsin cleavage . lane m : low molecular weight marker ; lane 1 : uninduced e. coli cells ; lane 2 : induced cells after 3 h expression ; lane 3 : purified fusion protein ; lane 4 : sumo protease digest of sumo / pas - cal . ( c ) size exclusion chromatogram of the pas - cal polypeptide after sumo protease cleavage ( applying the reaction mixture ) construction , expression , and purification of sumo / pas - cal . ( a ) the pas - cal polypeptide was expressed as a sumo fusion protein . after protein purification and digestion with sumo protease ( ulp1 ) , the pas - cal polypeptide concatamer yields a defined set of four standard peptides upon trypsin cleavage . lane m : low molecular weight marker ; lane 1 : uninduced e. coli cells ; lane 2 : induced cells after 3 h expression ; lane 3 : purified fusion protein ; lane 4 : sumo protease digest of sumo / pas - cal . ( c ) size exclusion chromatogram of the pas - cal polypeptide after sumo protease cleavage ( applying the reaction mixture ) a derivative of the t7 expression vector prset5a [ 14 , 15 ] with its multiple cloning site flanked by unique ndei and hindiii restriction sites was used to insert the sumo gene ( smt3 ) . an additional sapi recognition site within the prset5a backbone was removed via quikchange site - directed mutagenesis ( stratagene , la jolla , ca , usa ) using primers 5'- gcgtattgggcgcttctccgcttcctcgctcac-3 ' and 5'-gtgagcgaggaagcggagaagcgcccaatacgc-3 ' . the structural gene for smt3 , which is devoid of introns , was amplified from chromosomal dna of s. cerevisiae using pcr primers 5'- atctagcatatgaaacatcaccaccatcaccattcggactcagaagtcaatcaag-3 ' ( forward ) and 5'-cctcataagcttgctcttcaggcgccaccaatctgttc-3 ' ( reverse ) , which also provided the sequences for an n - terminal his6-tag and a sapi recognition site at the c - terminus . suitable cloned pas - cal gene fragments from above were then inserted into the single sap i restriction site in a successive manner , finally resulting in the coding region for the fusion protein illustrated in figure 1 . for expression of the recombinant truncated sumo protease a gene fragment encoding the catalytic core of ulp1 ( aa 403621 ) was likewise amplified from s. cerevisiae chromosomal dna by pcr using primers 5'-atctagcatatgaaacatcaccaccatcaccatcttgttcctgaattaaatgaaaaagacg-3 ' and 5'-cttcataagcttattttaaagcgtcggttaaaatcaaatggg-3 ' , introducing an n - terminal his6-tag as above , followed by insertion into prset5a . the sumo / pas - cal fusion protein was expressed in the cytoplasm of e. coli bl21(de3 ) cotransformed with the expression vector and the plasmid plyse to ensure efficient repression of basal promoter activity . cells were grown at 37c in a 2 l shake flask culture with luria bertani medium and induced with 0.5 mm isopropyl -d - thiogalactopyranoside ( iptg ) at od550 0.6 for 3 h. the cell pellet was homogenized in a french pressure cell ( slm aminco , urbana , il , usa ) and clarified by centrifugation and sterile filtration . the resulting protein solution was loaded onto a ni / nta resin ( ni sepharose high performance ; ge healthcare , freiburg , germany ) ; then , an imidazole / hcl concentration gradient from 0 to 150 mm in 40 mm napi ph 7.5 , 500 mm nacl was applied , and elution fractions were analyzed by sds - page . selected fractions were combined and dialyzed against 20 mm tris / hcl ph 9.5 and applied to a 1 ml resource q ion exchange column ( ge healthcare ) . elution was performed with a nacl concentration gradient from 0 to 200 mm in the same buffer , and selected fractions were combined and concentrated by ultrafiltration . ten mg of sumo / pas - cal ( final concentration : 1.7 mg / ml ) was cleaved in the ion exchange running buffer with 10 g of the recombinant truncated sumo protease . this enzyme was produced in e. coli bl21(de3 ) according to a published procedure , purified via imac and sec , and stored in 1 mm -mercaptoethanol , 50% ( v / v ) glycerol , 0.2% ( v / v ) after digest for 1 h at 37c , the pas - cal polypeptide was separated from the sumo fragment and the protease by preparative sec on a superdex 200 26/60 column ( ge healthcare ) in the presence of 50 mm nh4hco3 , monitoring uv absorption at 225 nm . finally , for trypsin digestion , 30 g of the pas - cal polypeptide was first incubated with 0.5 g trypsin ( stock solution of 0.1 g / ml sequencing grade modified trypsin in 50 mm acetic acid ; promega , madison , wi , usa ) at 37c for 3 h , followed by addition of another 0.5 g trypsin and continued incubation at 30c overnight . the trypsin - digested or intact pas - cal polypeptide ( 0.28 mg / ml in 50 mm nh4hco3 ) was supplemented with 20% ( v / v ) acetonitrile ( lc - ms grade ; sigma - aldrich , steinheim , germany ) and 0.1% ( v / v ) formic acid ( lc - ms grade ; sigma - aldrich ) . analysis was performed on an maxis q - tof mass spectrometer ( bruker daltonics , bremen , germany ) equipped with an esi source ( capillary voltage : 4.5 kv ; end plate offset : 500 v ; nebulizer pressure : 0.4 bar ; dry gas flow : 4.0 l / min ; dry temperature : 180c ) . raw data were analyzed and deconvoluted with compass data analysis software ( version 4.0 ) . the observed m / z window was in the range of 500 to 3000 while best resolution was achieved between 500 and 1500 . electrospray calibrant solution ( # 63606 - 10ml ; fluka analytical / sigma aldrich , steinheim , germany ) was applied both for measuring the depicted esi mass spectra and also in a side by side comparison with the pas - cal calibration standard ( see text ) . for the latter experiment , a sample of the recombinant antigen - binding fragment ( fab ) of the antibody trastuzumab was prepared as previously described . solutions of both the intact and trypsin - digested pas - cal ( 0.28 mg / ml in 50 mm nh4hco3 ) were mixed in a ratio 3:1 to trigger the formation of dimeric and trimeric peptide adduct ions in the maldi spectrum . the resulting peptide mixture was supplemented with an equal volume of 2,5-dihydroxybenzoic acid ( dhb , 10 g / ml in 30% ( v / v ) acetonitrile in water , 0.2% ( v / v ) trifluoroacetic acid ) , and spotted onto a stainless steel target according to the dried droplet method . maldi - ms analysis was performed on an ultraflextreme instrument ( bruker ) in the linear positive mode ( source voltage : 25 kv ) . using 1000 shots per spectrum , an m / z range from 1000 to 23,000 was analyzed with ion suppression below m / z 1500 ; m / z values for the various expected peptide species were calculated based on the known amino acid sequences using compass isotopepattern software ( version 1.3 ) . the assembly of a synthetic gene encoding the pas - cal concatamer ( figure 1 ) was accomplished in several steps according to a previously developed strategy involving incomplete ligation of synthetic dna double strands that carry nonpalindromic three - nucleotide overhangs ( corresponding to an ala codon ) , followed by insertion into a plasmid that has a unique cleavage site for the type iis restriction enzyme sapi ( 5'-'nnn , ngaagagc ) . to this end , a pair of complementary oligodeoxynucleotides ( thermo fisher scientific , ulm , germany ) encoding the 24-residue pas#5 amino acid sequence with or without an additional arg residue at the c - terminal end leon - rot , germany ) and hybridized to obtain short gene cassettes , each with two 5'-overhangs of 3 nucleotides . these cassettes were ligated in the presence of a limiting amount of t4 dna ligase ( fermentas ) . the resulting pas - cal gene fragments , comprising two or three cassettes of either one or a mixture of both versions ( i.e. , with and without arg ) , were extracted from a preparative agarose gel and subcloned on a derivative of puc19 that carries a double ( inverse repeat ) sapi restriction site in its multiple cloning region , thus allowing both dna sequence analysis and , subsequently , precise excision.figure 1construction , expression , and purification of sumo / pas - cal . ( a ) the pas - cal polypeptide was expressed as a sumo fusion protein . after protein purification and digestion with sumo protease ( ulp1 ) , the pas - cal polypeptide concatamer yields a defined set of four standard peptides upon trypsin cleavage . lane m : low molecular weight marker ; lane 1 : uninduced e. coli cells ; lane 2 : induced cells after 3 h expression ; lane 3 : purified fusion protein ; lane 4 : sumo protease digest of sumo / pas - cal . ( c ) size exclusion chromatogram of the pas - cal polypeptide after sumo protease cleavage ( applying the reaction mixture ) construction , expression , and purification of sumo / pas - cal . ( a ) the pas - cal polypeptide was expressed as a sumo fusion protein . after protein purification and digestion with sumo protease ( ulp1 ) , the pas - cal polypeptide concatamer yields a defined set of four standard peptides upon trypsin cleavage . lane m : low molecular weight marker ; lane 1 : uninduced e. coli cells ; lane 2 : induced cells after 3 h expression ; lane 3 : purified fusion protein ; lane 4 : sumo protease digest of sumo / pas - cal . ( c ) size exclusion chromatogram of the pas - cal polypeptide after sumo protease cleavage ( applying the reaction mixture ) a derivative of the t7 expression vector prset5a [ 14 , 15 ] with its multiple cloning site flanked by unique ndei and hindiii restriction sites was used to insert the sumo gene ( smt3 ) . an additional sapi recognition site within the prset5a backbone was removed via quikchange site - directed mutagenesis ( stratagene , la jolla , ca , usa ) using primers 5'- gcgtattgggcgcttctccgcttcctcgctcac-3 ' and 5'-gtgagcgaggaagcggagaagcgcccaatacgc-3 ' . the structural gene for smt3 , which is devoid of introns , was amplified from chromosomal dna of s. cerevisiae using pcr primers 5'- atctagcatatgaaacatcaccaccatcaccattcggactcagaagtcaatcaag-3 ' ( forward ) and 5'-cctcataagcttgctcttcaggcgccaccaatctgttc-3 ' ( reverse ) , which also provided the sequences for an n - terminal his6-tag and a sapi recognition site at the c - terminus . suitable cloned pas - cal gene fragments from above were then inserted into the single sap i restriction site in a successive manner , finally resulting in the coding region for the fusion protein illustrated in figure 1 . for expression of the recombinant truncated sumo protease a gene fragment encoding the catalytic core of ulp1 ( aa 403621 ) was likewise amplified from s. cerevisiae chromosomal dna by pcr using primers 5'-atctagcatatgaaacatcaccaccatcaccatcttgttcctgaattaaatgaaaaagacg-3 ' and 5'-cttcataagcttattttaaagcgtcggttaaaatcaaatggg-3 ' , introducing an n - terminal his6-tag as above , followed by insertion into prset5a . the sumo / pas - cal fusion protein was expressed in the cytoplasm of e. coli bl21(de3 ) cotransformed with the expression vector and the plasmid plyse to ensure efficient repression of basal promoter activity . cells were grown at 37c in a 2 l shake flask culture with luria bertani medium and induced with 0.5 mm isopropyl -d - thiogalactopyranoside ( iptg ) at od550 0.6 for 3 h. the cell pellet was homogenized in a french pressure cell ( slm aminco , urbana , il , usa ) and clarified by centrifugation and sterile filtration . the resulting protein solution was loaded onto a ni / nta resin ( ni sepharose high performance ; ge healthcare , freiburg , germany ) ; then , an imidazole / hcl concentration gradient from 0 to 150 mm in 40 mm napi ph 7.5 , 500 mm nacl was applied , and elution fractions were analyzed by sds - page . selected fractions were combined and dialyzed against 20 mm tris / hcl ph 9.5 and applied to a 1 ml resource q ion exchange column ( ge healthcare ) . elution was performed with a nacl concentration gradient from 0 to 200 mm in the same buffer , and selected fractions were combined and concentrated by ultrafiltration . ten mg of sumo / pas - cal ( final concentration : 1.7 mg / ml ) was cleaved in the ion exchange running buffer with 10 g of the recombinant truncated sumo protease . this enzyme was produced in e. coli bl21(de3 ) according to a published procedure , purified via imac and sec , and stored in 1 mm -mercaptoethanol , 50% ( v / v ) glycerol , 0.2% ( v / v ) triton x100 at 20c prior to use . after digest for 1 h at 37c , the pas - cal polypeptide was separated from the sumo fragment and the protease by preparative sec on a superdex 200 26/60 column ( ge healthcare ) in the presence of 50 mm nh4hco3 , monitoring uv absorption at 225 nm . finally , for trypsin digestion , 30 g of the pas - cal polypeptide was first incubated with 0.5 g trypsin ( stock solution of 0.1 g / ml sequencing grade modified trypsin in 50 mm acetic acid ; promega , madison , wi , usa ) at 37c for 3 h , followed by addition of another 0.5 g trypsin and continued incubation at 30c overnight . the trypsin - digested or intact pas - cal polypeptide ( 0.28 mg / ml in 50 mm nh4hco3 ) was supplemented with 20% ( v / v ) acetonitrile ( lc - ms grade ; sigma - aldrich , steinheim , germany ) and 0.1% ( v / v ) formic acid ( lc - ms grade ; sigma - aldrich ) . analysis was performed on an maxis q - tof mass spectrometer ( bruker daltonics , bremen , germany ) equipped with an esi source ( capillary voltage : 4.5 kv ; end plate offset : 500 v ; nebulizer pressure : 0.4 bar ; dry gas flow : 4.0 l / min ; dry temperature : 180c ) . raw data were analyzed and deconvoluted with compass data analysis software ( version 4.0 ) . the observed m / z window was in the range of 500 to 3000 while best resolution was achieved between 500 and 1500 . electrospray calibrant solution ( # 63606 - 10ml ; fluka analytical / sigma aldrich , steinheim , germany ) was applied both for measuring the depicted esi mass spectra and also in a side by side comparison with the pas - cal calibration standard ( see text ) . for the latter experiment , a sample of the recombinant antigen - binding fragment ( fab ) of the antibody trastuzumab was prepared as previously described . solutions of both the intact and trypsin - digested pas - cal ( 0.28 mg / ml in 50 mm nh4hco3 ) were mixed in a ratio 3:1 to trigger the formation of dimeric and trimeric peptide adduct ions in the maldi spectrum . the resulting peptide mixture was supplemented with an equal volume of 2,5-dihydroxybenzoic acid ( dhb , 10 g / ml in 30% ( v / v ) acetonitrile in water , 0.2% ( v / v ) trifluoroacetic acid ) , and spotted onto a stainless steel target according to the dried droplet method . maldi - ms analysis was performed on an ultraflextreme instrument ( bruker ) in the linear positive mode ( source voltage : 25 kv ) . using 1000 shots per spectrum , an m / z range from 1000 to 23,000 was analyzed with ion suppression below m / z 1500 ; m / z values for the various expected peptide species were calculated based on the known amino acid sequences using compass isotopepattern software ( version 1.3 ) . to prepare a set of defined peptides for calibration purposes with sizes from 2 to 8 kda hence , extending the range of conventional chemical peptide synthesis we have designed a recombinant pas polypeptide concatamer that upon site - specific proteolysis yields four soluble ionizable peptides with a 2 kda mass spacing , ideally suitable for ms analysis ( figure 1 ) . after assembly from synthetic gene cassettes ( see the experimental section ) , this pas - cal polypeptide was expressed in e. coli as an n - terminal sumo - fusion protein , also equipped with a his6-tag , to ensure efficient translational initiation and accumulation in the bacterial cytoplasm . after protein purification to homogeneity via immobilized metal affinity chromatography ( imac ) and ion exchange chromatography ( iec ) , the pas - cal polypeptide was specifically cleaved from the fusion partner by means of sumo protease ulp1 [ 17 , 22 ] , and finally isolated via size exclusion chromatography ( sec ) . each peptide cassette within the pas - cal polymer is terminated by an arg residue , thus allowing efficient trypsin cleavage and resulting in four peptide fragments in equimolar ratio . during the molecular design of pas - cal , arg was chosen instead of lys since the more basic guanidinium group also provides one stable positive charge per peptide , thus ensuring high ion intensities in mass spectra obtained both by esi and maldi measurements . the sumo / pas - cal fusion protein was produced in high yield of about 7 mg per l e. coli shake flask culture ( figure 1b ) , where it was found exclusively in the soluble cell extract , owing to the high inherent hydrophilicity of pas polypeptides . due to the lack of hydrophobic side chains , the pas concatamer was not stainable with coomassie brilliant blue after cleavage from the fusion partner , but the progress of digest was visualized by the emerging low molecular weight protein band of the cleaved sumo moiety ( figure 1b ) . the liberated pas - cal polypeptide was detected during sec as a peak at an absorption wavelength of 225 nm ( i.e. , by detecting the peptide backbone ; see figure 1c ) . the purified homogeneous pas - cal polypeptide was subsequently digested with lc - ms grade trypsin in situ and directly applied to esi - ms analysis on a bruker maxis instrument ( figure 2 ) . indeed , a remarkably simple pattern of four peaks with masses of 2152.1 , 4130.1 , 6108.0 , and 8086.0 da was observed in the deconvoluted spectrum . the experimental data for both the intact concatamer ( before tryptic digest ) and the four resulting standard peptides perfectly matched the expected molecular masses ( see table 1 and figure 2).figure 2esi - ms raw spectra of pas - cal and its peptide fragments and deconvoluted spectra . ( a ) esi - ms spectrum of the intact isolated pas - cal polypeptide . ( b ) esi - ms spectrum of trypsin - digested pas - cal with annotated m / z species . the inset shows the deconvoluted spectrumtable 1esi - ms signals measured for pas - cal peptides ( average mass and m / z numbers)speciescalculated mass [ da]deconvoluted mass [ da ] observedm / z signalspeptide 1 ( a)2152.3712152.0806[m + 3h ] 718.3800[m + 4h ] 539.0368peptide 2 ( b)4130.5414130.0526[m + 4h ] 1033.5390[m + 5h ] 827.0327[m + 6h ] 689.3618[m + 7h ] 591.0254peptide 3 ( c)6108.7116108.0352[m + 8h ] 764.5251[m + 9h ] 679.6898peptide 4 ( d)8086.8818086.0095[m + 10h ] 809.6224[m + 11h ] 736.1119[m + 12h ] 674.8532pas - cal ( intact)20495.53620495.5[m + 6nh3 + 11h ] 1873.4[m + 7nh3 + 12h ] 1719.0[m + 7nh3 + 13h ] 1586.7[m + 8nh3 + 14h ] 1474.5[m + 8nh3 + 15h ] 1376.3[m + 7nh3 + 16h ] 1289.3[m + 6nh3 + 17h ] 1212.6[m + 4nh3 + 18h ] 1143.5[m + 2nh3 + 19h ] 1081.5[m + nh3 + 20h ] 1026.7[m + nh3 + 21h ] 977.8[m + nh3 + 22h ] 933.4[m + 23h ] 892.1[m + 24h ] 855.0[m + 25h ] 820.8[m + 26h ] 789.3[m + 27h ] 760.1[m + 28h ] 733.0[m + 29h ] 707.7[m + 30h ] 684.2[m + 31h ] 662.1 esi - ms raw spectra of pas - cal and its peptide fragments and deconvoluted spectra . ( a ) esi - ms spectrum of the intact isolated pas - cal polypeptide . ( b ) esi - ms spectrum of trypsin - digested pas - cal with annotated m / z species . the inset shows the deconvoluted spectrum esi - ms signals measured for pas - cal peptides ( average mass and m / z numbers ) interestingly , the longer the pas - cal peptide fragment , the higher was the number of peaks in the raw spectra and also the intensity of the integrated ms signal , clearly indicating that not only the single arg side chain and the n - terminal free amino group carry positive charges but that there are also contributions by sliding protons along the peptide backbone [ 24 , 25 ] . this results in multiple - charged species depending on the peptide length ( see table 1 ) despite the lack of ionizable side chains within the pas sequence itself . nevertheless , the homogenous amino acid composition of the pas ( poly)peptides seems to favor a surprisingly even distribution and quality of ionization sites . this behavior is most likely a result of the disordered random coil structure of the pas polypeptides . this also explains the high accessibility for proteolytic cleavage during the preparation of the peptide mix . in fact , the deconvoluted esi spectra of pas - cal before and after tryptic digest demonstrate not only the high purity of the pas - cal concatamer but also the absence of missed cleavage sites after proteolysis ( figure 2 ) . to illustrate the use of pas - cal as a calibrant for esi - ms , we compared its applicability to a commercial calibration standard , followed by measuring the same protein sample , a 49 kda recombinant fab fragment , in two independent esi experiments using a bruker maxis q - tof instrument ( figure 3 ) . in the first run , a commercially available fluoroalkyl phosphazene - based electrospray calibrant solution was used , whereas in the second measurement the mass spectrometer was calibrated with the intact pas - cal , exploiting the resulting regular m / z pattern for stringent signal assignment . with both calibration methods , the deconvoluted average mass of the fab fragment was very close ( within 1.2 da ) to the calculated mass of 49429.6 da , which validates the use of pas - cal for esi calibration , demonstrating that large proteins can be measured accurately . the isolated pas - cal polypeptide yielded stable m / z signals for several months when stored at 20c and for at least a couple of weeks when kept at 4c . proteolytic degradation could not be observed as long as the biopolymer was maintained sterile.figure 3comparison of esi - ms raw and corresponding deconvoluted spectra of a purified recombinant protein sample measured after instrument calibration with ( a ) a commercial standard ( fluka electrospray calibrant solution ) and ( b ) the pas - cal polypeptide . the mono - isotopic m / z signals of each standard that were utilized for calibration are listed in the inset table . the most prominent m / z signals in the raw spectra of the sample protein are annotated whereas deconvoluted spectra are depicted in the insets . for comparison , the calculated average mass of the measured fab fragment is 49429.6413 da comparison of esi - ms raw and corresponding deconvoluted spectra of a purified recombinant protein sample measured after instrument calibration with ( a ) a commercial standard ( fluka electrospray calibrant solution ) and ( b ) the pas - cal polypeptide . the mono - isotopic m / z signals of each standard that were utilized for calibration are listed in the inset table . the most prominent m / z signals in the raw spectra of the sample protein are annotated whereas deconvoluted spectra are depicted in the insets . for comparison , the calculated average mass of the measured fab fragment is 49429.6413 da in another application , maldi - ms analysis of a pas - cal peptide mixture comprising both the intact polypeptide and its tryptic peptide fragments revealed both the four standard peptides ( a , b , c , d ) and the singly ( cs 1 ) as well as doubly ( cs 2 ) charged states of the full - length concatamer at their expected m / z ratios ( figure 4 and table 2 ) . notably , by adjusting the relative amount of the tryptic peptide fragments to a 3-fold excess over the intact pas - cal , the formation of di- and trimeric adduct species among the peptides could be observed . it is well known from maldi experiments that several peptides can share one charge by coordinating the same proton , leading to m / z ratios derived from the combined masses of the individual molecular species involved in the complex . most likely , the bridging protons are shared by the basic arginine side chains that are present in each pas - cal peptide . exploiting this phenomenon of adduct ion formation , it was possible to complement the m / z gap between the singly and doubly charged states of the intact polypeptide ( see table 2 and figure 4 ) . note that due to the inherent sequence repetitivity of the concatamer , the m / z ratio 12218.5 can be assigned to two different dimeric peptide ion complexes . apparently , the overlap of these two signals results in an enhanced intensity for this m / z ratio . these additional features of the pas - cal standard in maldi experiments make it even better suited for the calibration of a broad m / z range.figure 4maldi - ms spectrum of pas - cal and its tryptic peptides acquired in linear mode . the inset shows an enlarged view of the charge states + 1 and + 2 of the intact polypeptide as well as the di- and trimeric species that are formed by the tryptic peptide fragmentstable 2maldi - ms signals measured for pas - cal peptides ( average m / z)speciescalculated m / z signalsobserved m / z signalspeptide 1 ( a)[m + h ] 2153.3792153.372peptide 2 ( b)[m + h ] 4131.5484131.618peptide 3 ( c)[m + h ] 6109.7186109.739peptide 4 ( d)[m + h ] 8087.8888088.431pas - cal ( intact)[m ] ( cs 2)10247.77510247.907[m ] ( cs 1)20495.53620495.361dimer cc or bd[m + m + h ] 12218.43912218.471dimer cd[m + m + h ] 14196.59914196.438dimer dd[m + m + h ] 16174.76916174.401trimer bcd[m + m + m + h ] 18327.14718327.671 maldi - ms spectrum of pas - cal and its tryptic peptides acquired in linear mode . the inset shows an enlarged view of the charge states + 1 and + 2 of the intact polypeptide as well as the di- and trimeric species that are formed by the tryptic peptide fragments maldi - ms signals measured for pas - cal peptides ( average m / z ) the use of an artificial polypeptide as ms calibration standard has been proposed before . the so - called qcal , which is composed of 22 concatenated peptides that are liberated in uniform stoichiometry by tryptic digest , was designed to assess maldi mass spectrometer performance for the separation and analysis of peptides . the cleaved peptides ought to yield a representative mixture of natural amino acids , including common sites for post - translational modifications , such as deamidation or methionine oxidation , to be optionally introduced by chemical treatment . the concatameric qconcat polypeptide contained a series of peptide fragments characteristic of proteins to be monitored in a proteomics study . if expressed under isotope - labeling conditions , the qconcat peptide fragments were applicable as internal standard for quantification of the corresponding sample proteins by ms analysis . however , as the tryptic qcal peptides fall into a rather low molecular weight range from 0.4 to 3.2 kda , their suitability for ms calibration is generally more limited than with our pas - cal standard . notably , both qconcat and qcal polypeptides were expressed in e. coli bl21(de3 ) and occurred exclusively in the insoluble cell fraction . this necessitated the use of 6 m guanidinium chloride to solubilize the inclusion bodies . the insolubility of the biosynthetic polypeptides also affected the efficiency of tryptic digest as not all predicted cleavage sites appeared to be equally accessible . in fact , only 10 of 20 predicted qconcat peptide fragments could be assigned unambiguously in the mass spectra and , among those , the detectable analyte peptides were rather short ( 10 to 15 residues ) . in comparison , the 22 qcal peptide fragments showed a broader range of lengths from 4 to 26 residues , but the closely similar molecular masses of some of the resulting peptides led to a narrow distribution of m / z ratios and complicated signal assignment . some of the expected peptides could not be detected in esi mass spectra or required chemical modification , like guanidination , to yield distinct signals . these findings may be due to impaired ionization properties of some of the peptide fragments as the local protonation efficiency in the mass spectrometer strongly depends on the molecular structure . in contrast , the pas - cal peptides described here show uniform composition and are highly soluble , which does not only ensure strong ionization propensity but also allows efficient expression as a sumo fusion protein as well as facile purification from the soluble cell extract , followed by quantitative protease cleavage . the esi mass spectrum of the resulting peptides revealed a simple ensemble of signals , which all could be assigned unambiguously in the raw spectrum . by deconvolution the four expected molecular sizes for the set of pas - cal peptides yield a broad mass range from 2 to 8 kda with very similar intensities . this makes pas - cal and its derived peptides a well - suited calibration standard , especially for the analysis of intact proteins . high mass accuracy is also an important goal in lc - ms experiments for proteomics studies , where peptide digest and extended measurements are usually involved . in this context compound for spiking the actual sample , generating the calibration peptide mix with known and distinct m / z peaks at high signal intensities upon tryptic hydrolysis in situ . thus , the variation between expected and measured m / z values can be used for calibration within the sample during prolonged measurement , while the pas - cal peak pattern may serve at the same time to assess the completeness of peptide cleavage . in general , the beneficial features of pas - cal can be explained by the unique amino acid composition of pas polypeptides . the designed mixture of the hydrophilic residues proline , alanine , and serine leads to a random coil conformation in aqueous solution that effects high solubility and excellent accessibility of trypsin cleavage sites , also providing uniform protonation sites at the amino and guanidine groups as well as along the peptide backbone . apart from these useful properties for ms applications , long pas polypeptides were successfully fused to biopharmaceutically active proteins without hampering their function in vitro or in vivo , which illustrates their biochemically inert behavior [ 10 , 11 ] . in this study , the biophysical advantages of the pasylation technology have been exploited to create a peptide calibration standard for mass spectrometry that shows ( 1 ) high expression yield in e. coli ( even though just tiny amounts are needed for ms analysis ) , ( 2 ) high tryptic cleavage efficiency , and ( 3 ) well detectable signals with rather uniform intensities in esi and maldi mass spectra . considering the flexible cloning strategy ( see the experimental section ) , the peptide standard range can be easily expanded or adjusted by inserting additional synthetic gene cassettes of desired lengths into the pas - cal coding region . the broad mass range from 2 to 8 kda covered by the present pas - cal standard preparation of the recombinant peptide standard may be further simplified by replacing the conformation - specific sumo protease cleavage site with another arg residue to allow liberation of all pas peptides upon tryptic digest in situ . apart from use as an isolated reagent , pas - cal may also be fused to other proteins than sumo , for example targets of biomedical interest . the resulting easily assignable ms signal signature should facilitate quantification in vivo , for example to measure intracellular expression levels in cell culture or plasma half - life in animal studies .

we describe the design , preparation , and mass - spectrometric characterization of a new recombinant peptide calibration standard with uniform biophysical and ionization characteristics for mass spectrometry . pas - cal is an artificial polypeptide concatamer of peptide cassettes with varying lengths , each composed of the three small , chemically stable amino acids pro , ala , and ser , which are interspersed by arg residues to allow site - specific cleavage with trypsin . pas - cal is expressed at high yields in escherichia coli as a small ubiquitin - like modifier ( sumo ) fusion protein , which is easily purified and allows isolation of the pas - cal moiety after sumo protease cleavage . upon subsequent in situ treatment with trypsin , the pas - cal polypeptide yields a set of four defined homogeneous peptides in the range from 2 to 8 kda with equal mass spacing . esi - ms analysis revealed a conveniently interpretable raw spectrum , which after deconvolution resulted in a very simple pattern of four peaks with similar ionization signals . maldi - ms analysis of a pas - cal peptide mixture comprising both the intact polypeptide and its tryptic fragments revealed not only the four standard peptides but also the singly and doubly charged states of the intact concatamer as well as di- and trimeric adduct ion species between the peptides , thus augmenting the observable m / z range . the advantageous properties of pas - cal are most likely a result of the strongly hydrophilic and conformationally disordered peg - like properties of the pas sequences . therefore , pas - cal offers an inexpensive and versatile recombinant peptide calibration standard for mass spectrometry in protein / peptide bioanalytics and proteomics research , the composition of which may be further adapted to fit individual needs.figure

Introduction Experimental PAS-cal Gene Construction PAS-cal Expression and Purification ESI-MS MALDI-MS Results and Discussion Conclusions

since in maldi - ms singly charged analyte ions are predominantly formed and a large m / z range can be observed , it is reasonable to use large polymers for calibration , thus matching the expected size range . on the other hand , esi - ms results in the formation of multiple - charged analyte ions with a characteristic m / z distribution in the raw spectrum . the relatively narrow experimentally measured m / z range allows the use of rather small , chemically synthesized compounds for calibration , each of which appears as a singly charged molecule ion in the raw spectrum . a way to circumvent these problems would be a peptide standard with varying length but homogeneous composition , ideally prepared from a recombinant polypeptide comprising repetitive sequences of a small , defined set of amino acids . pas polypeptides are composed of long stretches of repetitive sequences of the three small , hydrophilic residues , proline , alanine and serine . with regard to application as an ms standard , the lack of secondary structure of the pas polypeptides and their exclusive composition of chemically stable amino acids without reactive side chains should result in a uniform ionization and protonation pattern for the peptide backbone , ensuring formation of multiple - charged ions especially suitable for esi detection . here , we report the design and synthesis of a pas - based ( poly)-peptide calibration standard which provides a simple m / z pattern , is easy to prepare , and should be useful for broad application in ms . ( a ) the pas - cal polypeptide was expressed as a sumo fusion protein . after protein purification and digestion with sumo protease ( ulp1 ) , the pas - cal polypeptide concatamer yields a defined set of four standard peptides upon trypsin cleavage . ( c ) size exclusion chromatogram of the pas - cal polypeptide after sumo protease cleavage ( applying the reaction mixture ) construction , expression , and purification of sumo / pas - cal . ( a ) the pas - cal polypeptide was expressed as a sumo fusion protein . after protein purification and digestion with sumo protease ( ulp1 ) , the pas - cal polypeptide concatamer yields a defined set of four standard peptides upon trypsin cleavage . ( c ) size exclusion chromatogram of the pas - cal polypeptide after sumo protease cleavage ( applying the reaction mixture ) a derivative of the t7 expression vector prset5a [ 14 , 15 ] with its multiple cloning site flanked by unique ndei and hindiii restriction sites was used to insert the sumo gene ( smt3 ) . suitable cloned pas - cal gene fragments from above were then inserted into the single sap i restriction site in a successive manner , finally resulting in the coding region for the fusion protein illustrated in figure 1 . ten mg of sumo / pas - cal ( final concentration : 1.7 mg / ml ) was cleaved in the ion exchange running buffer with 10 g of the recombinant truncated sumo protease . this enzyme was produced in e. coli bl21(de3 ) according to a published procedure , purified via imac and sec , and stored in 1 mm -mercaptoethanol , 50% ( v / v ) glycerol , 0.2% ( v / v ) after digest for 1 h at 37c , the pas - cal polypeptide was separated from the sumo fragment and the protease by preparative sec on a superdex 200 26/60 column ( ge healthcare ) in the presence of 50 mm nh4hco3 , monitoring uv absorption at 225 nm . finally , for trypsin digestion , 30 g of the pas - cal polypeptide was first incubated with 0.5 g trypsin ( stock solution of 0.1 g / ml sequencing grade modified trypsin in 50 mm acetic acid ; promega , madison , wi , usa ) at 37c for 3 h , followed by addition of another 0.5 g trypsin and continued incubation at 30c overnight . electrospray calibrant solution ( # 63606 - 10ml ; fluka analytical / sigma aldrich , steinheim , germany ) was applied both for measuring the depicted esi mass spectra and also in a side by side comparison with the pas - cal calibration standard ( see text ) . solutions of both the intact and trypsin - digested pas - cal ( 0.28 mg / ml in 50 mm nh4hco3 ) were mixed in a ratio 3:1 to trigger the formation of dimeric and trimeric peptide adduct ions in the maldi spectrum . ( a ) the pas - cal polypeptide was expressed as a sumo fusion protein . after protein purification and digestion with sumo protease ( ulp1 ) , the pas - cal polypeptide concatamer yields a defined set of four standard peptides upon trypsin cleavage . ( c ) size exclusion chromatogram of the pas - cal polypeptide after sumo protease cleavage ( applying the reaction mixture ) construction , expression , and purification of sumo / pas - cal . ( a ) the pas - cal polypeptide was expressed as a sumo fusion protein . after protein purification and digestion with sumo protease ( ulp1 ) , the pas - cal polypeptide concatamer yields a defined set of four standard peptides upon trypsin cleavage . ( c ) size exclusion chromatogram of the pas - cal polypeptide after sumo protease cleavage ( applying the reaction mixture ) a derivative of the t7 expression vector prset5a [ 14 , 15 ] with its multiple cloning site flanked by unique ndei and hindiii restriction sites was used to insert the sumo gene ( smt3 ) . ten mg of sumo / pas - cal ( final concentration : 1.7 mg / ml ) was cleaved in the ion exchange running buffer with 10 g of the recombinant truncated sumo protease . after digest for 1 h at 37c , the pas - cal polypeptide was separated from the sumo fragment and the protease by preparative sec on a superdex 200 26/60 column ( ge healthcare ) in the presence of 50 mm nh4hco3 , monitoring uv absorption at 225 nm . electrospray calibrant solution ( # 63606 - 10ml ; fluka analytical / sigma aldrich , steinheim , germany ) was applied both for measuring the depicted esi mass spectra and also in a side by side comparison with the pas - cal calibration standard ( see text ) . solutions of both the intact and trypsin - digested pas - cal ( 0.28 mg / ml in 50 mm nh4hco3 ) were mixed in a ratio 3:1 to trigger the formation of dimeric and trimeric peptide adduct ions in the maldi spectrum . using 1000 shots per spectrum , an m / z range from 1000 to 23,000 was analyzed with ion suppression below m / z 1500 ; m / z values for the various expected peptide species were calculated based on the known amino acid sequences using compass isotopepattern software ( version 1.3 ) . to prepare a set of defined peptides for calibration purposes with sizes from 2 to 8 kda hence , extending the range of conventional chemical peptide synthesis we have designed a recombinant pas polypeptide concatamer that upon site - specific proteolysis yields four soluble ionizable peptides with a 2 kda mass spacing , ideally suitable for ms analysis ( figure 1 ) . after assembly from synthetic gene cassettes ( see the experimental section ) , this pas - cal polypeptide was expressed in e. coli as an n - terminal sumo - fusion protein , also equipped with a his6-tag , to ensure efficient translational initiation and accumulation in the bacterial cytoplasm . after protein purification to homogeneity via immobilized metal affinity chromatography ( imac ) and ion exchange chromatography ( iec ) , the pas - cal polypeptide was specifically cleaved from the fusion partner by means of sumo protease ulp1 [ 17 , 22 ] , and finally isolated via size exclusion chromatography ( sec ) . the sumo / pas - cal fusion protein was produced in high yield of about 7 mg per l e. coli shake flask culture ( figure 1b ) , where it was found exclusively in the soluble cell extract , owing to the high inherent hydrophilicity of pas polypeptides . the purified homogeneous pas - cal polypeptide was subsequently digested with lc - ms grade trypsin in situ and directly applied to esi - ms analysis on a bruker maxis instrument ( figure 2 ) . indeed , a remarkably simple pattern of four peaks with masses of 2152.1 , 4130.1 , 6108.0 , and 8086.0 da was observed in the deconvoluted spectrum . the experimental data for both the intact concatamer ( before tryptic digest ) and the four resulting standard peptides perfectly matched the expected molecular masses ( see table 1 and figure 2).figure 2esi - ms raw spectra of pas - cal and its peptide fragments and deconvoluted spectra . ( a ) esi - ms spectrum of the intact isolated pas - cal polypeptide . ( b ) esi - ms spectrum of trypsin - digested pas - cal with annotated m / z species . the inset shows the deconvoluted spectrumtable 1esi - ms signals measured for pas - cal peptides ( average mass and m / z numbers)speciescalculated mass [ da]deconvoluted mass [ da ] observedm / z signalspeptide 1 ( a)2152.3712152.0806[m + 3h ] 718.3800[m + 4h ] 539.0368peptide 2 ( b)4130.5414130.0526[m + 4h ] 1033.5390[m + 5h ] 827.0327[m + 6h ] 689.3618[m + 7h ] 591.0254peptide 3 ( c)6108.7116108.0352[m + 8h ] 764.5251[m + 9h ] 679.6898peptide 4 ( d)8086.8818086.0095[m + 10h ] 809.6224[m + 11h ] 736.1119[m + 12h ] 674.8532pas - cal ( intact)20495.53620495.5[m + 6nh3 + 11h ] 1873.4[m + 7nh3 + 12h ] 1719.0[m + 7nh3 + 13h ] 1586.7[m + 8nh3 + 14h ] 1474.5[m + 8nh3 + 15h ] 1376.3[m + 7nh3 + 16h ] 1289.3[m + 6nh3 + 17h ] 1212.6[m + 4nh3 + 18h ] 1143.5[m + 2nh3 + 19h ] 1081.5[m + nh3 + 20h ] 1026.7[m + nh3 + 21h ] 977.8[m + nh3 + 22h ] 933.4[m + 23h ] 892.1[m + 24h ] 855.0[m + 25h ] 820.8[m + 26h ] 789.3[m + 27h ] 760.1[m + 28h ] 733.0[m + 29h ] 707.7[m + 30h ] 684.2[m + 31h ] 662.1 esi - ms raw spectra of pas - cal and its peptide fragments and deconvoluted spectra . ( a ) esi - ms spectrum of the intact isolated pas - cal polypeptide . ( b ) esi - ms spectrum of trypsin - digested pas - cal with annotated m / z species . the inset shows the deconvoluted spectrum esi - ms signals measured for pas - cal peptides ( average mass and m / z numbers ) interestingly , the longer the pas - cal peptide fragment , the higher was the number of peaks in the raw spectra and also the intensity of the integrated ms signal , clearly indicating that not only the single arg side chain and the n - terminal free amino group carry positive charges but that there are also contributions by sliding protons along the peptide backbone [ 24 , 25 ] . this behavior is most likely a result of the disordered random coil structure of the pas polypeptides . in fact , the deconvoluted esi spectra of pas - cal before and after tryptic digest demonstrate not only the high purity of the pas - cal concatamer but also the absence of missed cleavage sites after proteolysis ( figure 2 ) . to illustrate the use of pas - cal as a calibrant for esi - ms , we compared its applicability to a commercial calibration standard , followed by measuring the same protein sample , a 49 kda recombinant fab fragment , in two independent esi experiments using a bruker maxis q - tof instrument ( figure 3 ) . in the first run , a commercially available fluoroalkyl phosphazene - based electrospray calibrant solution was used , whereas in the second measurement the mass spectrometer was calibrated with the intact pas - cal , exploiting the resulting regular m / z pattern for stringent signal assignment . with both calibration methods , the deconvoluted average mass of the fab fragment was very close ( within 1.2 da ) to the calculated mass of 49429.6 da , which validates the use of pas - cal for esi calibration , demonstrating that large proteins can be measured accurately . proteolytic degradation could not be observed as long as the biopolymer was maintained sterile.figure 3comparison of esi - ms raw and corresponding deconvoluted spectra of a purified recombinant protein sample measured after instrument calibration with ( a ) a commercial standard ( fluka electrospray calibrant solution ) and ( b ) the pas - cal polypeptide . for comparison , the calculated average mass of the measured fab fragment is 49429.6413 da comparison of esi - ms raw and corresponding deconvoluted spectra of a purified recombinant protein sample measured after instrument calibration with ( a ) a commercial standard ( fluka electrospray calibrant solution ) and ( b ) the pas - cal polypeptide . for comparison , the calculated average mass of the measured fab fragment is 49429.6413 da in another application , maldi - ms analysis of a pas - cal peptide mixture comprising both the intact polypeptide and its tryptic peptide fragments revealed both the four standard peptides ( a , b , c , d ) and the singly ( cs 1 ) as well as doubly ( cs 2 ) charged states of the full - length concatamer at their expected m / z ratios ( figure 4 and table 2 ) . notably , by adjusting the relative amount of the tryptic peptide fragments to a 3-fold excess over the intact pas - cal , the formation of di- and trimeric adduct species among the peptides could be observed . exploiting this phenomenon of adduct ion formation , it was possible to complement the m / z gap between the singly and doubly charged states of the intact polypeptide ( see table 2 and figure 4 ) . these additional features of the pas - cal standard in maldi experiments make it even better suited for the calibration of a broad m / z range.figure 4maldi - ms spectrum of pas - cal and its tryptic peptides acquired in linear mode . the inset shows an enlarged view of the charge states + 1 and + 2 of the intact polypeptide as well as the di- and trimeric species that are formed by the tryptic peptide fragmentstable 2maldi - ms signals measured for pas - cal peptides ( average m / z)speciescalculated m / z signalsobserved m / z signalspeptide 1 ( a)[m + h ] 2153.3792153.372peptide 2 ( b)[m + h ] 4131.5484131.618peptide 3 ( c)[m + h ] 6109.7186109.739peptide 4 ( d)[m + h ] 8087.8888088.431pas - cal ( intact)[m ] ( cs 2)10247.77510247.907[m ] ( cs 1)20495.53620495.361dimer cc or bd[m + m + h ] 12218.43912218.471dimer cd[m + m + h ] 14196.59914196.438dimer dd[m + m + h ] 16174.76916174.401trimer bcd[m + m + m + h ] 18327.14718327.671 maldi - ms spectrum of pas - cal and its tryptic peptides acquired in linear mode . the inset shows an enlarged view of the charge states + 1 and + 2 of the intact polypeptide as well as the di- and trimeric species that are formed by the tryptic peptide fragments maldi - ms signals measured for pas - cal peptides ( average m / z ) the use of an artificial polypeptide as ms calibration standard has been proposed before . in contrast , the pas - cal peptides described here show uniform composition and are highly soluble , which does not only ensure strong ionization propensity but also allows efficient expression as a sumo fusion protein as well as facile purification from the soluble cell extract , followed by quantitative protease cleavage . by deconvolution the four expected molecular sizes for the set of pas - cal peptides yield a broad mass range from 2 to 8 kda with very similar intensities . thus , the variation between expected and measured m / z values can be used for calibration within the sample during prolonged measurement , while the pas - cal peak pattern may serve at the same time to assess the completeness of peptide cleavage . in this study , the biophysical advantages of the pasylation technology have been exploited to create a peptide calibration standard for mass spectrometry that shows ( 1 ) high expression yield in e. coli ( even though just tiny amounts are needed for ms analysis ) , ( 2 ) high tryptic cleavage efficiency , and ( 3 ) well detectable signals with rather uniform intensities in esi and maldi mass spectra . the broad mass range from 2 to 8 kda covered by the present pas - cal standard preparation of the recombinant peptide standard may be further simplified by replacing the conformation - specific sumo protease cleavage site with another arg residue to allow liberation of all pas peptides upon tryptic digest in situ .

in general relativity , kinetic theory has been used relatively sparsely to model phenomenological matter in comparison to fluid models . from a mathematical point of view , however , there are fundamental advantages to using a kinetic description . in non - curved spacetimes kinetic theory has been studied intensively as a mathematical subject during several decades , and it has also played an important role from an engineering point of view . in the first part of this introduction , we will review kinetic theory in non - curved spacetimes and we will consider mainly the special relativistic case , but mathematical results in the nonrelativistic case will also be discussed . we believe that a good understanding of kinetic theory in non - curved spacetimes is fundamental to good comprehension of kinetic theory in general relativity . moreover , it is often the case that mathematical methods used to treat the einstein - vlasov system are carried over from methods developed in the special relativistic or nonrelativistic case . the purpose of kinetic theory is to model the time evolution of a collection of particles . for instance , the particles are atoms and molecules in a neutral gas or electrons and ions in a plasma . in stellar dynamics the particles are stars and in a cosmological case they are galaxies or even clusters of galaxies . a characteristic feature of kinetic theory is that its models are statistical and the particle systems are described by distribution functions f = f ( t , x , p ) , which represent the density of particles with given spacetime position ( t , x ) and momentum p . a distribution function contains a wealth of information , and macroscopical quantities are easily calculated from this function . in a fluid model the quantities that describe the system do not depend on the momentum p but only on the spacetime point ( t , x ) . a choice of model is usually made with regard to the physical properties of interest for the system or with regard to numerical considerations . it should be mentioned that a fluid model that is too naive may give rise to shell - crossing singularities , which are unphysical . in a kinetic description the time evolution of the system is determined by the interactions between the particles which depend on the physical situation . for instance , the driving mechanism for the time evolution of a neutral gas is the collision between particles ( the relativistic boltzmann equation ) . for a plasma the interaction is through the electric charges ( the vlasov - maxwell system ) , and in the stellar and cosmological cases the interaction is gravitational ( the einstein - vlasov system ) . of course , combinations of interaction processes are also considered but in many situations one of them is strongly dominating and the weaker processes are neglected . consider a collection of neutral particles in minkowski spacetime . let the signature of the metric be ( , + , + , + ) , let all the particles have rest mass m = 1 , and normalize the speed of light c to one . the four - momentum of a particle is denoted by p , a = 0 , 1 , 2 , 3 . since all particles have equal rest mass , the four - momentum for each particle is restricted to the mass shell , ppa = m = 1 . thus , by denoting the three - momentum by p , p may be written p = ( p , p ) , where |p| is the usual euclidean length of p and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p^0 } = \sqrt { 1 + \vert p{\vert ^2}}$\end{document } is the energy of a particle with three - momentum p. the relativistic velocity of a particle with momentum p is denoted by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat p}$\end{document } and is given by 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{p } = { p \over { \sqrt { 1 + \vert p{\vert ^2}}}}.$$\end{document } note that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vert\hat p\vert < 1 = c$\end{document}. the relativistic boltzmann equation models the spacetime behaviour of the one - particle distribution function f = f(t , x , p ) , and it has the form 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left({{\partial _ t } + { p \over { { p^0 } } } \cdot { \nabla _ x } } \right)\;f = q(f , f),$$\end{document } where the relativistic collision operator q(f , g ) is defined by 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(f , g ) = \int\nolimits_{{{\mathbb r}^3 } } { \int\nolimits_{{{\mathbb s}^2 } } { k(p , q,\omega ) [ f(p + a(p , q,\omega ) \omega ) g(q - a(p , q,\omega ) \omega ) - f(p)g(q)]\,dp\,d\omega . } } $ $ \end{document } ( note that g = f in equation ( 2 ) ) . here d is the element of surface area on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{\mathbb s}}^{\rm{2}}}$\end{document } and k(p , q , ) is the scattering kernel , which depends on the scattering cross - section in the interaction process . if two particles , with momentum p and q respectively , collide elastically ( no energy loss ) with scattering angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega \in { { \rm{\mathbb s}}^{\rm{2}}}$\end{document } , their momenta will change , p p and q q. the relation between p , q and p , q is 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^{\prime } = p + a(p , q,\omega)\omega , \quad q^{\prime } = q - a(p , q,\omega)\omega,$$\end{document } where 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a(p , q,\omega ) = { { 2({p^0 } + { q^0}){p^0}{q^0}(\omega \cdot ( \hat{q } - \hat{p } ) ) } \over { { { ( { p^0 } + { q^0})}^2 } - { { ( \omega \cdot ( p + q))}^2}}}.$$\end{document } this relation is a consequence of four - momentum conservation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^a } + { q^a } = { p^{a^\prime } } + { q^{a^\prime}},$$\end{document } or equivalently 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^0 } + { q^0 } = { p^{0^{\prime } } } + { q^{0^{\prime}}},$$\end{document } 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p + q = p^{\prime } + q^{\prime}.$$\end{document } these are the conservation equations for relativistic particle dynamics . in the classical case these equations read 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert p{\vert ^2 } + \vert q{\vert ^2 } = \vert p^{\prime}{\vert ^2 } + \vert q^{\prime}{\vert ^2},$$\end{document } 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p + q = p^{\prime } + q^{\prime}.$$\end{document } the function a(p , q , ) is the distance between p and p ( q and q ) , and the analogue function in the newtonian case has the form 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_{{\rm{cl}}}}(p , q,\omega ) = \omega \cdot ( q - p).$$\end{document } by inserting ac1 in place of a in equation ( 3 ) we obtain the classical boltzmann collision operator ( disregarding the scattering kernel , which is also different ) . in classical solutions to the relativistic boltzmann equations are studied as c , and it is proved that the limit as c of these solutions satisfy the classical boltzmann equation . the main result concerning the existence of solutions to the classical boltzmann equation is a theorem by diperna and lions that proves existence , but not uniqueness , of renormalized solutions ( i.e. solutions in a weak sense , which are even more general than distributional solutions ) . an analogous result holds in the relativistic case , as was shown by dudynsky and ekiel - jezewska . regarding classical solutions , illner and shinbrot have shown global existence of solutions to the nonrelativistic boltzmann equation for small initial data ( close to vacuum ) . at present there is no analogous result for the relativistic boltzmann equation and this must be regarded as an interesting open problem . there is however a recent result for the homogeneous relativistic boltzmann equation where global existence is shown for small initial data and bounded scattering kernel . when the data are close to equilibrium ( see below ) , global existence of classical solutions has been proved by glassey and strauss in the relativistic case and by ukai in the nonrelativistic case ( see also and ) . the collision operator q(f , g ) may be written in an obvious way as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(f , g ) = { q^ + } ( f , g ) - { q^ -}(f , g),$$\end{document } where q and q are called the gain and loss term , respectively . if the loss term is deleted the gain - term - only boltzmann equation is obtained . it is interesting to note that the methods of proof for the small data results mentioned above concentrate on gain - term - only equations , and once that is solved it is easy to include the loss term . in it is shown that the gain - term - only classical and relativistic boltzmann equations blow up for initial data not restricted to a small neighbourhood of trivial data . thus , if a global existence proof for unrestricted data will be given it will necessarily use the full collision operator . the gain term has a nice regularizing property in the momentum variable . in it is proved that given f l( ) and g l( ) with f , g 0 , then 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert \vert { q^ + } ( f , g)\vert { \vert _ { { h^1}({\mathbb r}_p^3 ) } } \leq c\vert \vert f\vert { \vert _ { { l^2}({\mathbb r}_p^3)}}\vert \vert g\vert { \vert _ { { l^1}({\mathbb r}_p^3)}},$$\end{document } under some technical requirements on the scattering kernel . here h is the usual sobolev space . the proof relies on the theory of fourier integral operators and on the method of stationary phase , and requires a careful analysis of the collision geometry , which is very different in the relativistic case . see also and for a simplified proof in the classical and relativistic case respectively . more precisely , lions used the regularizing theorem to prove that solutions to the ( classical ) boltzmann equation , with periodic boundary conditions , converge in l to a global maxwellian , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m = { e^ { - \alpha \vert p{\vert ^2 } + \beta \cdot p + \gamma } } \quad { \rm{with}}\,\alpha , \,\gamma \in r,\quad \alpha > 0,\quad \beta \in { { \mathbb r}^3},$$\end{document } as time goes to infinity . this result had first been obtained by arkeryd by using non - standard analysis . it should be pointed out that the convergence takes place through a sequence of times tending to infinity and it is not known whether the limit is unique or depends on the sequence . in the relativistic situation , the analogous question of convergence to a relativistic maxwellian , or a jttner equilibrium solution , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { e^{- \alpha \sqrt { 1 + \vert p{\vert ^2 } } + \beta \cdot p + \gamma}},\quad \alpha , \beta , \;{\rm{and}}\;\gamma \;{\rm{as}}\;{\rm{above}},\;{\rm{with}}\;\alpha > \vert \beta \vert,$$\end{document } had been studied by glassey and strauss [ 48 , 49 ] . in the periodic case they proved convergence in a variety of function spaces for initial data close to a jttner solution . having obtained the regularizing theorem for the relativistic gain term , it is a straightforward task to follow the method of lions and prove convergence to a local jttner solution for arbitrary data ( satisfying the natural bounds of finite energy and entropy ) that is periodic in the space variables . in it is next proved that the local jttner solution must be a global one , due to the periodicity of the solution . for more information on the relativistic boltzmann equation on minkowski space we refer to [ 41 , 34 , 110 ] and in the nonrelativistic case we refer to the excellent review paper by villani and the books [ 41 , 24 ] . let us consider a collisionless plasma , which is a collection of particles for which collisions are relatively rare and the interaction is through their charges . we assume below that the plasma consists only of one type of particle , whereas the results below also hold for plasmas with several particle species . the most general set of equations for modelling a collisionless plasma is the relativistic vlasov - maxwell system : 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + \hat{v}\cdot { \nabla _ x}f(e(t , x ) + \hat{v } \times b(t , x ) ) \cdot { \nabla _ v}f = 0$$\end{document } 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}e + j = c\nabla \times b,\quad \quad \nabla \cdot e = \rho,$$\end{document } 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}b = - c\nabla \times e,\quad \quad \nabla \cdot b = 0.$$\end{document } the notation follows the one already introduced with the exception that the momenta are now denoted by v instead of p. this has become a standard notation in this field . e and b are the electric and magnetic fields , and v is the relativistic velocity , 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{v } = { v \over { \sqrt { 1 + \vert v\vert ^{2}/{c^2}}}},$$\end{document } where c is the speed of light . the charge density and current j are given by 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho = \int\nolimits_{{{\mathbb r}^3 } } { fdv},\quad j = \int\nolimits_{{{\mathbb r}^3 } } { \hat{v}fdv.}$$\end{document } equation ( 12 ) is the relativistic vlasov equation and equations ( 13 , 14 ) are the maxwell equations . a special case in three dimensions is obtained by considering spherically symmetric initial data . for such data it can be shown that the solution will also be spherically symmetric , and that the magnetic field has to be constant . the maxwell equation e = tb then implies that the electric field is the gradient of a potential . hence , in the spherically symmetric case the relativistic vlasov - maxwell system takes the form 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + \hat{v } \cdot { \nabla _ x}f + \beta e(t , x ) \cdot { \nabla _ v}f = 0,$$\end{document } 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = \nabla \phi , \quad \nabla \phi = \rho.$$\end{document } here = 1 , and the constant magnetic field has been set to zero , since a constant field has no significance in this discussion . this system makes sense for any initial data , without symmetry constraints , and is called the relativistic vlasov - poisson equation . another special case of interest is the classical limit , obtained by letting c in equations ( 12 , 13 , 14 ) , yielding : 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + v \cdot { \nabla _ x}f + \beta e(t , x ) \cdot { \nabla _ v}f = 0,$$\end{document } 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = \nabla \phi , \quad \nabla \phi = \rho,$$\end{document } where = 1 . this is the ( nonrelativistic ) vlasov - poisson equation , and = 1 corresponds to repulsive forces ( the plasma case ) . taking = 1 means attractive forces and the vlasov - poisson equation is then a model for a newtonian self - gravitating system . one of the fundamental problems in kinetic theory is to find out whether or not spontaneous shock formations will develop in a collisionless gas , i.e. whether solutions to any of the equations above will remain smooth for all time , given smooth initial data . if the initial data are small this problem has an affirmative solution in all cases considered above ( see [ 42 , 47 , 12 , 13 ] ) . for initial data unrestricted in size the picture is more involved . in order to obtain smooth solutions globally in time , the main issue is to control the support of the momenta 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) : = \sup \{\vert v\vert:\exists ( s , x ) \in [ 0,t ] \times { { \mathbb r}^3}\;{\rm{such}}\;{\rm{that}}\;f(s , x , v ) \neq 0\},$$\end{document } i.e. to bound q(t ) by a continuous function so that q(t ) will not blow up in finite time . that such a control is sufficient for obtaining global existence of smooth solutions follows from well - known results in the different cases ( see [ 46 , 54 , 14 , 42 ] ) . for the full three - dimensional relativistic vlasov - maxwell system , this important problem of establishing whether or not solutions will remain smooth for all time is open . there has been an increased activity during the last years aiming at a solution of this problem . two new methods of proofs of the same theorem as in are given in [ 60 , 17 ] . a different sufficient criterion for global existence is given by pallard in and he also shows a new bound for the electromagnetic field in terms of q(t ) in . in two space and three momentum dimensions , glassey and schaeffer have shown that q(t ) can be controlled , which thus yields global existence of smooth solutions in that case ( see also ) . the nonrelativistic case batt gave an affirmative solution 1977 in the case of spherically symmetric data . pfaffelmoser ( see also schaeffer ) was the first one to give a proof for general smooth data . he obtained the bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c{(1 + t)^{(51 + \delta)/11}},$$\end{document } where > 0 could be taken arbitrarily small . the sharpest bound valid for = 1 and = 1 has been given by horst and reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c(1 + t)\log ( 2 + t).$$\end{document } in the case of repulsive forces ( = 1 ) rein has found the sharpest estimate by using a new identity for the vlasov - poisson equation , discovered independently by illner and rein and by perthame . rein s estimate reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c{(1 + t)^{2/3}}.$$\end{document } independently and about the same time as pfaffelmoser gave his proof , lions and perthame used a different method for proving global existence . to some extent their method seems to be more generally applicable to attack problems similar to the vlasov - poisson equation but which are still quite different ( see [ 3 , 61 ] ) . on the other hand , their method does not give such strong growth estimates on q(t ) as described above . for the relativistic vlasov - poisson equation , glassey and schaeffer showed in the case = 1 that if the data are spherically symmetric , q(t ) can be controlled , which is analogous to the result by batt mentioned above . also in the case of cylindrical symmetry they are able to control q(t ) ; see . if = 1 it was also shown in that blowup occurs in finite time for spherically symmetric data with negative total energy . this system , however , is unphysical in the sense that it is not a special case of the einstein - vlasov system . quite surprisingly , for general smooth initial data none of the techniques discussed above for the nonrelativistic vlasov - poisson equation apply in the relativistic case . this fact is annoying since it has been suggested that an understanding of this equation may be necessary for understanding the three - dimensional relativistic vlasov - maxwell equation . however , the relativistic vlasov - poisson equation lacks the lorentz invariance ; it is a hybrid of a classical galilei invariant field equation and a relativistic transport equation ( 17 ) . only for spherical symmetric data is the equation a fundamental physical equation . the classical vlasov - poisson equation is on the other hand galilean invariant . in a different equation for the field is introduced that is observer independent among lorentz observers . by coupling this equation for the field to the relativistic vlasov equation , the function q(t ) this is an indication that the transformation properties are important in studying existence of smooth solutions ( the situation is less subtle for weak solutions , where energy estimates and averaging are the main tools ( see [ 56 , 35 , 86 ] ) . hence , it is unclear whether or not the relativistic vlasov - poisson equation will play a central role in the understanding of the lorentz invariant relativistic vlasov - maxwell equation . we refer to the book by glassey for more information on the relativistic vlasov - maxwell system and the vlasov - poisson equation . before turning to the main theme of this review , i.e. the einstein - vlasov system , we introduce a system which has many mathematical features in common with the vlasov - maxwell system and which recently has been mathematically studied for the first time . it is based on an alternative theory of gravity which was given by nordstrom in 1913 . by coupling this model to a kinetic description of matter the nordstrm - vlasov system results . in nordstrm gravity the nordstrm - vlasov system reads 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\partial _ t^2\phi - { \delta _ x}\phi = - { e^{4\phi}}\int { { \mathfrak { f}}{{dp } \over { \sqrt { 1 + \vert p\vert ^{2}}}},}$$\end{document } 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}{\mathfrak { f } } + \widehat{p } \cdot { \nabla _ x}{\mathfrak { f } } - \left [ { ( { \partial _ t}\phi + \widehat{p } \cdot { \nabla _ x}\phi)p + { { ( 1 + \vert p\vert ^{2})}^{- 1/2}}{\nabla _ x}\phi } \right ] \cdot { \nabla _ p}{\mathfrak { f}}= 0.$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\widehat{p } = { p \over { \sqrt { 1 + \vert p{\vert ^2}}}}$$\end{document } denotes the relativistic velocity of a particle with momentum p. the mass of each particle , the gravitational constant , and the speed of light are all normalized to one . a solution ( f , ) of this system is interpreted as follows : the spacetime is a lorentzian manifold with a conformally flat metric which , in the coordinates ( t , x ) , takes the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } = { e^{2\phi}}{\rm{diag}}(- 1,\;1,\;1,\;1).$$\end{document } the particle distribution f defined on the mass shell in this metric is given by 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(t , x , p ) = { \mathfrak { f}}(t , x,{e^\phi } p).$$\end{document } the first mathematical study of this system was undertaken by calogero in , where static solutions are analyzed and where also more details on the derivation of the system are given . although the nordstrom - vlasov model of gravity does not describe physics correctly ( in the classical limit the vlasov - poisson system of newtonian gravity is however obtained ) it can nevertheless serve as a platform for developing new mathematical methods . the system has some common features with the einstein - vlasov system ( see next section 1.4 ) but seems in many respects to be less involved . the closest relationship from a mathematical point of view is the vlasov - maxwell system ; this is evident in where weak solutions are obtained in analogy with , in where a continuation criterion for existence of classical solutions is established in analogy with ( an alternative approach is given in ) , and in where global existence in the case of two space dimensions is proved in analogy with . the spherically symmetric case is studied in and can not be directly compared to the spherically symmetric vlasov - maxwell system ( i.e. the vlasov - poisson system ) since the hyperbolic nature of the equations is still present in the former system while it is lost in the latter case . in it is shown that classical solutions exist globally in time for compactly supported initial data under the additional condition that there is a lower bound on the modulus of the angular momentum of the initial particle system . it should be noted that this is not a smallness condition and that the result holds for arbitrary large initial data satisfying this hypothesis . in this section we will consider a self - gravitating collisionless gas and we will write down the einstein - vlasov system and describe its general mathematical features . we also refer to ehlers and stewart for more background on kinetic theory in general relativity . let m be a four - dimensional manifold and let gab be a metric with lorentz signature ( , + , + , + ) so that ( m , gab ) is a spacetime . we use the abstract index notation , which means that gab is a geometric object and not the components of a tensor . see for a discussion on this notation . the metric is assumed to be time - orientable so that there is a distinction between future and past directed vectors . the worldline of a particle with non - zero rest mass m is a timelike curve and the unit future - directed tangent vector v to this curve is the four - velocity of the particle . we assume that all particles have equal rest mass m and we normalize so that m = 1 . the possible values of the four - momentum are all future - directed unit timelike vectors and they constitute a hypersurface p in the tangent bundle tm , which is called the mass shell . the distribution function f that we introduced in the previous sections is a non - negative function on p. since we are considering a collisionless gas , the particles travel along geodesics in spacetime . the vlasov equation is an equation for f that exactly expresses this fact . to get an explicit expression for this equation we introduce local coordinates on the mass shell . we choose local coordinates on m such that the hypersurfaces t = x = constant are spacelike so that t is a time coordinate and x , j = 1 , 2 , 3 , are spatial coordinates ( letters in the beginning of the alphabet always take values 0 , 1 , 2 , 3 and letters in the middle take 1 , 2 , 3 ) . a timelike vector is future directed if and only if its zero component is positive . local coordinates on p can then be taken as x together with the spatial components of the four - momentum p in these coordinates . the vlasov equation then reads 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + { { { p^j } } \over { { p^0}}}{\partial _ { { x^j}}}f - { 1 \over { { p^0}}}\gamma _ { ab}^j{p^a}{p^b}{\partial _ { { p^j}}}f = 0.$$\end{document } here a , b = 0 , 1 , 2 , 3 and j = 1 , 2 , 3 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma _ { ab}^j$\end{document } are the christoffel symbols . it is understood that p is expressed in terms of p and the metric gab using the relation gabpp = 1 ( recall that m = 1 ) . in a fixed spacetime the vlasov equation is a linear hyperbolic equation for f and we can solve it by solving the characteristic system , 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{x^i } } \over { ds } } = { { { p^i } } \over { { p^0}}},$$\end{document } 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{p^i } } \over { ds } } = - \gamma _ { ab}^i{{{p^a}{p^b } } \over { { p^0}}}.$$\end{document } in terms of initial data f0 the solution to the vlasov equation can be written as 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f({x^a},{p^i } ) = { f_0}({x^i}(0,{x^a},{p^i}),{p^i}(0,{x^a},{p^i})),$$\end{document } where x(s , x , p ) and p(s , x , p ) solve equations ( 26 , 27 ) , and where x(t , x , p ) = x and p(t , x , p ) = p. in order to write down the einstein - vlasov system we need to define the energy - momentum tensor tab in terms of f and gab . in the coordinates ( x , p ) on p we define \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{ab } } = - \int\nolimits_{{{\mathbb r}^3 } } f { p_a}{p_b}\vert g\vert ^{{1/2}}{{d{p^1}d{p^2}d{p^3 } } \over { { p_0}}},$$\end{document } where as usual pa = gabp , and |g| denotes the absolute value of the determinant of g. equation ( 25 ) together with einstein s equations , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{ab } } : = { r_{ab } } - { 1 \over 2}r{g_{ab } } = 8\pi { t_{ab } } + \lambda { g_{ab}},$$\end{document } then form the einstein - vlasov system . here gab is the einstein tensor , rab the ricci tensor , r is the scalar curvature and is the cosmological constant . in most of this review we will assume that = 0 , but section 2.3 is devoted to the case of non - vanishing cosmological constant ( where also the case of adding a scalar field is discussed ) . we now define the particle current density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n^a } = - \int\nolimits_{{{\mathbb r}^3 } } f { p^a}\vert g\vert ^{{1/2}}{{d{p^1}d{p^2}d{p^3 } } \over { { p_0}}}.$$\end{document } using normal coordinates based at a given point and assuming that f is compactly supported it is not hard to see that tab is divergence - free which is a necessary compatability condition since gab is divergence - free by the bianchi identities . a computation in normal coordinates also shows that n is divergence - free , which expresses the fact that the number of particles is conserved . if v is a future directed timelike or null vector then we have nav 0 with equality if and only if f = 0 at the given point . moreover , if v and w are future directed timelike vectors then tabvw 0 , which is the dominant energy condition . together with the einstein equations imply that rabvv 0 for any timelike vector v , which is the strong energy condition . that the energy conditions hold for vlasov matter is one reason that the vlasov equation defines a well - behaved matter model in general relativity . another reason is the well - posedness theorem by choquet - bruhat for the einstein - vlasov system that we will state below . before stating that theorem the data in the cauchy problem for the einstein - vlasov system consist of the induced riemannian metric gij on the initial hypersurface s , the second fundamental form kij of s and matter data f. the relations between a given initial data set ( gij , kij ) on a three - dimensional manifold s and the metric gij on the spacetime manifold is that there exists an embedding of s into the spacetime such that the induced metric and second fundamental form of (s ) coincide with the result of transporting ( gij , kij ) with . for the relation of the distribution functions f and f we have to note that f is defined on the mass shell . the initial condition imposed is that the restriction of f to the part of the mass shell over (s ) should be equal to f d( ) ) , where sends each point of the mass shell over (s ) to its orthogonal projection onto the tangent space to (s ) . an initial data set for the einstein - vlasov system must satisfy the constraint equations , which read 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r - { k_{ij}}{k^{ij } } + ( { \rm{tr}}k)^{{2 } } = 16\pi \rho,$$\end{document } 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ i}k_\iota ^i - { \nabla _ \iota}({\rm{tr}}k ) = 8\pi j\iota.$$\end{document } here = tabnn and j = htbcn , where n is the future directed unit normal vector to the initial hypersurface and h = g + nn is the orthogonal projection onto the tangent space to the initial hypersurface . in terms of f we can express and j by ( j satisfies naj = 0 so it can naturally be identified with a vector intrinsic to s ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{\rho = \int\nolimits_{{{\mathbb r}^3 } } f { p^a}{p_a}{\vert ^{(3)}}g\vert ^{{1/2}}{{d{p^1}d{p^2}d{p^3 } } \over { 1 + { p_j}{p^j } } } , } \\ { { j_\iota } = \int\nolimits_{{{\mathbb r}^3 } } f { p_\iota}{\vert ^{(3)}}g\vert ^{{1/2}}d{p^1}d{p^2}d{p^3}. } \\ \end{array}$$\end{document } here |g| is the determinant of the induced riemannian metric on s. we can now state the local existence theorem by choquet - bruhat for the einstein - vlasov system . theorem 1 let s be a 3-dimensional manifold , gij a smooth riemannian metric on s , kij a smooth symmetric tensor on s and f a smooth non - negative function of compact support on the tangent bundle ts of s. suppose that these objects satisfy the constraint equations ( 29 , 30 ) . then there exists a smooth spacetime ( m , gab ) , a smooth distribution function f on the mass shell of this spacetime , and a smooth embedding of s into m which induces the given initial data on s such that gab and f satisfy the einstein - vlasov system and (s ) is a cauchy surface . moreover , given any other spacetime ( m , gab ) , distribution function f and embedding satisfying these conditions , there exists a diffeomorphism from an open neighbourhood of (s ) in m to an open neighbourhood of (s ) in m which satisfies = and carries gab and f to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{g ^\prime}_{ab}}$\end{document } and f , respectively . in this context we also mention that local existence has been proved for the yang - mills - vlasov system in , and that this problem for the einstein - maxwell - boltzmann system is treated in . this result is however not complete , the non - negativity of f is left unanswered . also , the hypotheses on the scattering kernel in this work leave some room for further investigation . this problem concerning physically reasonable assumptions on the scattering kernel seems not well understood in the context of the einstein - boltzmann system , and a careful study of this issue would be desirable . a main theme in the following sections is to discuss special cases for which the local existence theorem can be extended to a global one . there are interesting situations when this can be achieved , and such global existence theorems are not known for einstein s equations coupled to other forms of phenomenological matter models , i.e. fluid models ( see , however , ) . in this context it should be stressed that the results in the previous sections show that the mathematical understanding of kinetic equations on a flat background space is well - developed . on the other hand the mathematical understanding of fluid equations on a flat background space ( also in the absence of a newtonian gravitational field ) is not satisfying . it would be desirable to have a better mathematical understanding of these equations in the absence of gravity before coupling them to einstein s equations . this suggests that the vlasov equation is natural as matter model in mathematical general relativity . in general relativity two classes of initial data are distinguished . if an isolated matter distribution is studied , the data are called asymptotically flat . the initial hypersurface is topologically and ( since far away from the body one expects spacetime to be approximately flat ) appropriate fall off conditions are imposed . roughly , a smooth data set ( gij , kij , f0 ) on is said to be asymptotically flat if there exist global coordinates x such that as |x| tends to infinity the components gij in these coordinates tend to ij , the components kij tend to zero , f0 has compact support and certain weighted sobolev norms of gij and kij are finite ( see ) . one can also consider a case in which spacetime is asymptotically flat except in one direction , namely cylindrical spacetimes . a lot of work has been done on the spherically symmetric case and this will be reviewed below . in the case of cylindrical symmetry it has been shown that if singularities form , then the first one must occur at the axis of symmetry . spacetimes that possess a compact cauchy hypersurface are called cosmological spacetimes , and data are accordingly given on a compact 3-manifold . in this case in contrast to the asymptotically flat case , cosmological spacetimes admit a large number of symmetry classes . this gives one the possibility to study interesting special cases for which the difficulties of the full einstein equations are strongly reduced . we will discuss below cases for which the spacetime is characterized by the dimension of its isometry group together with the dimension of the orbit of the isometry group . the study of the global properties of solutions to the spherically symmetric einstein - vlasov system was initiated by rein and rendall in 1990 . they chose to work in coordinates where the metric takes the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { e^{2\mu ( t , r)}}d{t^2 } + { e^{2\lambda ( t , r)}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\varphi ^2}),$$\end{document } where t , r 0 , 0 [ 0 , ] , [ 0 , 2 ] . asymptotic flatness is expressed by the boundary conditions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{r \rightarrow \infty}{\lim}\lambda ( t , r ) = \underset{r \rightarrow \infty}{\lim } \mu ( t , r ) = 0,\quad \forall t \geq 0.$$\end{document } a regular centre is also required and is guaranteed by the boundary condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda ( t , 0 ) = 0.$$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x = ( r\sin \phi \cos \theta , r\sin \phi \sin \theta , r\cos \phi)$$\end{document } as spatial coordinate and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${v^j } = { p^j } + ( { e^\lambda } - 1){{x \cdot p } \over r}{{{x^j } } \over r}$$\end{document } as momentum coordinates , the einstein - vlasov system reads 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + { e^{\mu - \lambda}}{v \over { \sqrt { 1 + \vert v{\vert ^2 } } } } \cdot { \nabla _ x}f - \left({{\lambda _ t}{{x \cdot v } \over r } + { e^{\mu - \lambda}}{\mu _ r}\sqrt { 1 + \vert v{\vert ^2 } } } \right){x \over r } \cdot { \nabla _ x}f = 0,$$\end{document } 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}\rho,$$\end{document } 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}p.$$\end{document } the matter quantities are defined by 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( t , x ) = \int\nolimits_{{{\mathbb r}^3 } } { \sqrt { 1 + \vert v\vert ^{2 } } f(t , x , v)dv,}$$\end{document } 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(t , x ) = \int\nolimits_{{{\mathbb r}^3 } } { { { \left({{{x \cdot v } \over r } } \right)}^2}f(t , x , v){{dv } \over { \sqrt { 1 + \vert v\vert ^{2}}}}.}$$\end{document } let us point out that this system is not the full einstein - vlasov system . let the square of the angular momentum be denoted by l , i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l : = \vert x{\vert ^2}\vert v{\vert ^2 } - { ( x \cdot v)^2}.$$\end{document } a consequence of spherical symmetry is that angular momentum is conserved along the characteristics of equation ( 31 ) . introducing the variable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$w = { { x \cdot v } \over r},$$\end{document } the vlasov equation for f = f ( t , r , w , l ) becomes 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial t}w + { e^{\mu - \lambda}}{\mu _ r}e - { e^{\mu - \lambda}}{l \over { { r^3}e } } } \right){\partial _ w}f = 0,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = e(r , w , l ) = \sqrt { 1 + { w^2 } + l/{r^2}}.$$\end{document } the matter terms take the form 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( t , r ) = { \pi \over { { r^2}}}\int\nolimits_{- \infty}^\infty { \int\nolimits_0^\infty { ef(t , r , w , l)dw\;dl,}}$$\end{document } 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(t , r ) = { \pi \over { { r^2}}}\int\nolimits_{- \infty}^\infty { \int\nolimits_0^\infty { { { { w^2 } } \over e}f(t , r , w , l)dw\;dl.}}$$\end{document } let us write down a couple of known facts about the system ( 32 , 33 , 36 , 37 , 38 ) . a solution to the vlasov equation can be written as 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f{\text{(}}t{\text{,}}r{\text{,}}w{\text{,}}l{\text { ) = } } f_{\text{0 } } { \text{(}}r{\text{(0,}}t{\text{,}}r{\text{,}}w{\text{,}}l{\text { ) , } } w{\text{(0,}}t{\text{,}}r{\text{,}}w{\text{,}}l{\text{),}}l{\text{),}}$$\end{document } where r and w are solutions to the characteristic system 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dr } \over { ds } } = { e^{(\mu - \lambda)(s , r)}}{w \over { e(r , w , l)}},$$\end{document } 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dw } \over { ds } } = - { \lambda _ t}(s , r)w - { e^{(\mu - \lambda)(s , r)}}{\mu _ r}(s , r)e(r , w , l ) + { e^{(\mu - \lambda)(s , r)}}{l \over { { r^3}e(r , w , l)}},$$\end{document } such that the trajectory ( r(s , t , r , w , l ) , w(s , t , r , w , l ) , l ) goes through the point ( r , w , l ) when s = t. this representation shows that f is non - negative for all t 0 and that f f0. there are two known conservation laws for the einstein - vlasov system : conservation of the number of particles , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$4{\pi ^2}\int\nolimits_0^\infty { { e^{\lambda ( t , r)}}\left({\int\nolimits_{- \infty}^\infty { \int\nolimits_0^\infty { f(t , r , w , l)dl\;dw } } } \right)dr,}$$\end{document } and conservation of the adm mass 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m : = 4\pi \int\nolimits_0^\infty { { r^2}\rho ( t , r)dr.}$$\end{document } let us now review the global results concerning the cauchy problem that have been proved for the spherically symmetric einstein - vlasov system . as initial data we take a spherically symmetric , non - negative , and continuously differentiable function f0 with compact support that satisfies 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$4{\pi ^2}\int\nolimits_0^r { \int\nolimits_{- \infty}^\infty { \int\nolimits_0^\infty e { f_0}(r , w , l)dw } \;dl\;dr\ ; } < { r \over 2}.$$\end{document } this condition guarantees that no trapped surfaces are present initially . in it is shown that for such an initial datum there exists a unique , continuously differentiable solution f with f(0 ) = f0 on some right maximal interval [ 0 , t ) . if the solution blows up in finite time , i.e. if t < , then (t ) becomes unbounded as t t. moreover , a continuation criterion is shown that says that a local solution can be extended to a global one provided the v - support of f can be bounded on [ 0 , t ) . ( in they chose to work in the momentum variable v rather than w , l. ) this is analogous to the situation for the vlasov - maxwell system where the function q(t ) was introduced for the v - support . a control of the v - support immediately implies that and p are bounded in view of equations ( 34 , 35 ) . in the vlasov - maxwell case the field equations have a regularizing effect in the sense that derivatives can be expressed through spatial integrals , and it follows that the derivatives of f also can be bounded if the v - support is bounded . for the einstein - vlasov system such a regularization is less clear , since e.g. r depends on in a pointwise manner . however , certain combinations of second and first order derivatives of the metric components can be expressed in terms of matter components only , without derivatives ( a consequence of the geodesic deviation equation ) . this fact turns out to be sufficient for obtaining bounds also on the derivatives of f ( see for details ) . by considering initial data sufficiently close to zero , rein and rendall show that the v - support is bounded on [ 0 , t ) , and the continuation criterion then implies that t = . it should be stressed that even for small data no global existence result like this one is known for any other phenomenological matter model coupled to einstein s equations . the resulting spacetime in is geodesically complete , and the components of the energy momentum tensor as well as the metric quantities decay with certain algebraic rates in t. the mathematical method used by rein and rendall is inspired by the analogous small data result for the vlasov - poisson equation by bardos and degond . this should not be too surprising since for small data the gravitational fields are expected to be small and a newtonian spacetime should be a fair approximation . in this context we point out that in it is proved that the vlasov - poisson system is indeed the nonrelativistic limit of the spherically symmetric einstein - vlasov system , i.e. the limit when the speed of light c . ( in this issue is studied in the asymptotically flat case without symmetry assumptions . ) finally , we mention that there is an analogous small data result using a maximal time coordinate instead of a schwarzschild time coordinate . in these coordinates rendall has shown that there exist data leading to singular spacetimes as a consequence of penrose s singularity theorem . the schwarzschild time coordinate is expected to avoid the singularity , and by global existence we mean that solutions remain smooth as schwarzschild time tends to infinity . even though spacetime might be only partially covered in schwarzschild coordinates , a global existence theorem for general data would nevertheless be very important since it is likely that it would constitute a central step for proving weak cosmic censorship . indeed , if this coordinate system can be shown to cover the domain of outer communications and if null infinity could be shown to be complete , then weak cosmic censorship would follow . a partial investigation for general data in schwarzschild coordinates was done in and in maximal - isotropic coordinates in . in schwarzschild coordinates it is shown that if singularities form in finite time the first one must be at the centre . more precisely , if f(t , r , w , l ) = 0 when r > for some > 0 , and for all t , w , and l , then the solution remains smooth for all time . this rules out singularities of the shell - crossing type , which can be an annoying problem for other matter models ( e.g. dust ) . the main observation in is a cancellation property in the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda _ t}w + { e^{\mu - \lambda}}{\mu _ r}e$$\end{document } in the characteristic equation ( 41 ) . in a similar result is shown , but here also an assumption that one of the metric functions is bounded at the centre is assumed . however , with this assumption the result follows in a more direct way and the analysis of the vlasov equation is not necessary , which indicates that such a result might be true more generally . recently , dafermos and rendall have shown a similar result for the einstein - vlasov system in double - null coordinates . the main motivation for studying the system in these coordinates has its origin from the method of proof of the cosmic censorship conjecture for the einstein - scalar field system by christodoulou . an essential part of his method is based on the understanding of the formation of trapped surfaces . the presence of trapped surfaces ( for the relevant initial data ) is then crucial in proving the completeness of future null infinity in . in the relation between trapped surfaces and the completeness of null infinity was strengthened ; a single trapped surface or marginally trapped surface in the maximal development implies completeness of null infinty . the theorem holds true for any spherically symmetric matter spacetime if the matter model is such that first singularities necessarily emanate from the center ( where the notion of first is tied to the casual structure ) . the results in and in are not sufficient for concluding that the hypothesis of the matter needed in the theorem in is satisfied , since they concern a portion of the maximal development covered by particular coordinates . therefore , dafermos and rendall choose double - null coordinates which cover the maximal development , and they show that the mentioned hypothesis is satisfied for vlasov matter . in a numerical study was undertaken of the einstein - vlasov system in schwarzschild coordinates . a numerical scheme originally used for the vlasov - poisson system it has been shown by rein and rodewis that the numerical scheme has the desirable convergence properties . ( in the vlasov - poisson case convergence was proved in ; see also ) . it may even hold in a stronger sense than in the case of a massless scalar field ( see [ 29 , 31 ] ) . there may be no naked singularities formed for any regular initial data rather than just for generic data . this speculation is based on the fact that the naked singularities that occur in scalar field collapse appear to be associated with the existence of type ii critical collapse , while vlasov matter is of type i. this is indeed the primary goal of their numerical investigation : to analyze critical collapse and decide whether vlasov matter is type i or type ii . given small initial data no black holes are expected to form and matter will disperse ( which has been proved for a scalar field and for vlasov matter ) . for large data , black holes will form and consequently there is a transition regime separating dispersion of matter and formation of black holes . if we introduce a parameter a on the initial data such that for small a dispersion occurs and for large a a black hole is formed , we get a critical value ac separating these regions . if we take a > ac and denote by mb(a ) the mass of the black hole , then if mb(a ) 0 as a ac we have type ii matter , whereas for type i matter this limit is positive and there is a mass gap . for more information on critical collapse there is an independent numerical simulation by olabarrieta and choptuik ( using a maximal time coordinate ) and their conclusion agrees with the one in . critical collapse is related to self similar solutions ; martin - garcia and gundlach have presented a construction of such solutions for the massless einstein - vlasov system by using a method based partially on numerics . since such solutions often are related to naked singularities , it is important to note that their result is for the massless case ( in which case there is no known analogous result to the small data theorem in ) and their initial data are not in the class that we have described above . we end this section with a discussion of the spherically symmetric einstein - vlasov - maxwell system , i.e. the case considered above with charged particles . whereas the constraint equations in the uncharged case do not involve any problems to solve once the distribution function is given ( and satisfies equation ( 43 ) ) , the charged case is more challenging . in it is shown that solutions to the constraint equations do exist for the einstein - vlasov - maxwell system . in local existence is then shown together with a continuation criterion , and in the regularity theorem in is generalized to this case . in cosmology the whole universe is modelled , and the particles in the kinetic description are galaxies or even clusters of galaxies . the main goal again is to determine the global properties of the solutions to the einstein - vlasov system . in order to do so , a global time coordinate t must be found ( global existence ) and the asymptotic behaviour of the solutions when t tends to its limiting values has to be analyzed . this might correspond to approaching a singularity ( e.g. the big bang singularity ) or to a phase of unending expansion . since the general case is beyond the range of current mathematical techniques , all known results are for cases with symmetry ( see however where to some extent global properties are established in the case without symmetry ) . there are several existing results on global time coordinates for solutions of the einstein - vlasov system . in the spatially homogeneous case it is natural to choose a gaussian time coordinate based on a homogeneous hypersurface . the maximal range of a gaussian time coordinate in a solution of the einstein - vlasov system evolving from homogenous data on a compact manifold was determined in . it is finite in one time direction and infinite in the other for the other bianchi types . the asymptotic behaviour of solutions in the spatially homogeneous case has been analyzed in and . in , the case of massless particles both the nature of the initial singularity and the phase of unlimited expansion are analyzed . the main concern is the behaviour of bianchi models i , ii , and iii . a general conclusion is that the choice of matter model is very important since for all symmetry classes studied there are differences between the collisionless model and a perfect fluid model , both regarding the initial singularity and the expanding phase . the most striking example is for the bianchi ii models , where they find persistent oscillatory behaviour near the singularity , which is quite different from the known behaviour of bianchi type ii perfect fluid models . in it is also shown that solutions for massive particles are asymptotic to solutions with massless particles near the initial singularity . for bianchi i and ii it is also proved that solutions with massive particles are asymptotic to dust solutions at late times . all other results presently available on the subject concern spacetimes that admit a group of isometries acting on two - dimensional spacelike orbits , at least after passing to a covering manifold . the group may be two - dimensional ( local u(1 ) u(1 ) or t symmetry ) or three - dimensional ( spherical , plane , or hyperbolic symmetry ) . in all these cases , the quotient of spacetime by the symmetry group has the structure of a two - dimensional lorentzian manifold q. the orbits of the group action ( or appropriate quotients in the case of a local symmetry ) are called surfaces of symmetry . thus , there is a one - to - one correspondence between surfaces of symmetry and points of q. there is a major difference between the cases where the symmetry group is two- or three - dimensional . in the three - dimensional case no gravitational waves are admitted , in contrast to the two - dimensional case . in the former case , the field equations reduce to odes while in the latter their evolution part consists of nonlinear wave equations . three types of time coordinates that have been studied in the inhomogeneous case are cmc , areal , and conformal coordinates . a cmc time coordinate t is one where each hypersurface of constant time has constant mean curvature ( cmc ) and on each hypersurface of this kind the value of t is the mean curvature of that slice . in the case of areal coordinates , the time coordinate is a function of the area of the surfaces of symmetry ( e.g. proportional to the area or proportional to the square root of the area ) . in the case of conformal coordinates , let us first consider spacetimes ( m , g ) admitting a three - dimensional group of isometries . the topology of m is assumed to be s f , with f a compact two - dimensional manifold . the universal covering \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f}$\end{document } of f induces a spacetime \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\hat m,\hat g)$\end{document } by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat m , = \mathbb r \times { s^1 } \times \hat f$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat g = p*g$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p:\hat m \to m$\end{document } is the canonical projection . a three - dimensional group g of isometries is assumed to act on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\hat m,\hat g)$\end{document}. if f = s and g = so(3 ) , then ( m , g ) is called spherically symmetric ; if f = t and g = e2 ( euclidean group ) , then ( m , g ) is called plane symmetric ; and if f has genus greater than one and the connected component of the symmetry group g of the hyperbolic plane h acts isometrically on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat f = { h^2}$\end{document } , then ( m , g ) is said to have hyperbolic symmetry . in the case of spherical symmetry the existence of one compact cmc hypersurface implies that the whole spacetime can be covered by a cmc time coordinate that takes all real values [ 97 , 18 ] . the existence of one compact cmc hypersurface in this case was proved later by henkel using the concept of prescribed mean curvature ( pmc ) foliation . accordingly this gives a complete picture in the spherically symmetric case regarding cmc foliations . in the case of areal coordinates , rein has shown , under a size restriction on the initial data , that the past of an initial hypersurface can be covered . in the future direction it is shown that areal coordinates break down in finite time . in the case of plane and hyperbolic symmetry , rendall and rein showed in and , respectively , that the existence results ( for cmc time and areal time ) in the past direction for spherical symmetry also hold for these symmetry classes . the global cmc foliation results to the past imply that the past singularity is a crushing singularity , since the mean curvature blows up at the singularity . in addition , rein also proved in his special case with small initial data that the kretschmann curvature scalar blows up when the singularity is approached . hence , the singularity is both a crushing and a curvature singularity in this case . in both of these this gap was closed in , and global existence to the future was established in both cmc and areal coordinates . the global existence result for cmc time is partly a consequence of the global existence theorem in areal coordinates , together with a theorem by henkel that shows that there exists at least one hypersurface with ( negative ) constant mean curvature . also , the past direction was analyzed in areal coordinates and global existence was shown without any smallness condition on the data . it is , however , not concluded if the past singularity in this more general case without the smallness condition on the data is a curvature singularity as well . the question whether the areal time coordinate , which is positive by definition , takes all values in the range ( 0 , ) or only in ( r0 , ) for some positive r0 is also left open . in the special case in , it is indeed shown that r0 = 0 , but there is an example for vacuum spacetimes in the more general case of u(1 ) u(1 ) symmetry where r0 > 0 . she proves that if spacetime contains vlasov matter ( i.e. f 0 ) then r = 0 . her result applies to a more general case which we now turn to . for spacetimes admitting a two - dimensional isometry group , the first study was done by rendall in the case of local u(1 ) u(1 ) symmetry ( or local t symmetry ) . for a discussion of the topologies of these spacetimes we refer to the original paper . in the model case the spacetime is topologically of the form t , and to simplify our discussion later on we write down the metric in areal coordinates for this type of spacetime : 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{g = { e^{2(\eta - u)}}(- \alpha d{t^2 } + d{\theta ^2 } ) + { e^{- 2u}}{t^2}{{[dy + h\;d\theta + m\;dt]}^2 } } \\{\quad \quad + { e^{2u}}{{[dx + a\;dy + ( g + ah)d\theta + ( l + am)dt]}^2}. } \\\end{array}$$\end{document } here the metric coefficients , u , , a , h , l , and m depend on t and and , x , y s. in cmc coordinates are considered rather than areal coordinates . the advantage with a cmc approach is that the definition of a cmc hypersurface does not depend on any symmetry assumptions and it is possible that cmc foliations will exist for rather general spacetimes . the areal coordinate foliation , on the other hand , is adapted to the symmetry of spacetime but it has analytical advantages that we will see below . under the hypothesis that there exists at least one cmc hypersurface , rendall proves , without any smallness condition on the data , that the past of the given cmc hypersurface can be globally foliated by cmc hypersurfaces and that the mean curvature of these hypersurfaces blows up at the past singularity . the result in holds for vlasov matter and for matter described by a wave map ( which is not a phenomenological matter model ) . that the choice of matter model is important was shown by rendall who gives a non - global existence result for dust , which leads to examples of spacetimes that are not covered by a cmc foliation . the plane case where the symmetry group is three - dimensional is one subcase and the form of the metric in areal coordinates is obtained by letting a = g = h = l = m = 0 and u = log t/2 in equation ( 44 ) . another subcase , which still admits only two killing fields ( and which includes plane symmetry as a special case ) , is gowdy symmetry . it is obtained by letting g = h = l = m = 0 in equation ( 44 ) . in it is proved that the entire maximal globally hyperbolic spacetime can be foliated by constant areal time slices for arbitrary ( in size ) initial data . the areal coordinates are used in a direct way for showing global existence to the future whereas the analysis for the past direction is carried out in conformal coordinates . these coordinates are not fixed to the geometry of spacetime and it is not clear that the entire past has been covered . a chain of geometrical arguments then shows that areal coordinates indeed cover the entire spacetime . the method in was in turn inspired by the work for vacuum spacetimes where the idea of using conformal coordinates in the past direction was introduced . as pointed out in , the result by henkel guarantees the existence of one cmc hypersurface in the gowdy case and , together with the global areal foliation in , it follows that gowdy spacetimes with vlasov matter can be globally covered by cmc hypersurfaces as well ( also to the future ) . the general case of u(1 ) u(1 ) symmetry was considered in , where it is shown that there exist global cmc and areal time foliations which complete the picture . in this result as well as in the preceeding subcases mentioned above the question whether or not the areal time coordinate takes values in ( 0 , ) or in this issue was solved by weaver in where she concludes that r = 0 if the distribution function is not identically zero initially . a number of important questions remain open . to analyze the nature of the initial singularity , which at present is known only for small initial data in the case considered in , would be very interesting . the question of the asymptotics in the future direction is also an important issue where very little is known . the only situation where a result has been obtained is in the case with hyperbolic symmetry . under a certain size restriction on the initial data , the present cosmological observations indicate that the expansion of the universe is accelerating , and this has influenced the theoretical studies in the field during the last years . one way to produce models with accelerated expansion is to choose a positive cosmological constant in the einstein equations . another way is to include a nonlinear scalar field among the matter fields . in this section we will review the recent results for the einstein - vlasov system where a cosmological constant , or a linear or nonlinear scalar field have been included into the model . as in the previous section we start with the models with highest degree of symmetry , i.e. the locally spatially homogeneous models . in the case of a positive cosmological constant lee has shown global existence as well as future causal geodesic completeness for initial data which have bianchi symmetry . she also obtains the time decay of the components of the energy momentum tensor as t . the past direction for some spatially homogeneous models is considered in . existence back to the initial singularity is proved and the case with a negative cosmological constant is discussed . in lee considers the case with a nonlinear scalar field coupled to vlasov matter . the form of the energy momentum then reads 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{\alpha \beta } } = t_{\alpha \beta } ^{{\rm{vlasov } } } + { \nabla _ \alpha } \phi { \nabla _ \beta } \phi - \left ( { { 1 \over 2}{\nabla ^\gamma } \phi { \nabla _ \gamma } \phi + v(\phi ) } \right){g_{\alpha \beta } } .$$\end{document } here is the scalar field and v is a potential , and the bianchi identities lead to the following equation for the scalar field : 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla ^\gamma}\phi { \nabla _ \gamma}\phi = v^{\prime}(\phi).$$\end{document } under the assumption that v is non - negative and c , global existence to the future is obtained and if the potential is restricted to the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v(\phi ) = { v_0}{e^{- c\phi}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0 < c < 4\sqrt \pi$\end{document } then future geodesic completeness is proved . in the previous section 2.3 we discussed the situation when spacetime admits a three - dimensional group of isometries and we distinguished three cases : plane , spherical , and hyperbolic symmetry . in area time coordinates the metric takes the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { e^{\mu ( t , r)}}d{t^2 } + { e^{\lambda ( t , r)}}d{r^2 } + { t^2}(d{\theta ^2 } + \sin _ \kappa ^2\theta d{\varphi ^2}),$$\end{document } where k = 0 , 1 , + 1 correspond to the plane , spherical , and hyperbolic case , respectively , and where sin0 = 1 , sin1 = sin , and sin1 = sinh. in the einstein - vlasov system with a positive cosmological constant is investigated in the future ( expanding ) direction in the case of plane and hyperbolic symmetry . the authors prove global existence to the future in these coordinates and they also show future geodesic completeness . recall that in the case of = 0 , future geodesic completenss has only been established for hyperbolic symmetry under a smallness condition of the initial data . finally a form of the cosmic no - hair conjecture is obtained in for this class of spacetimes . indeed , here it is shown that the de sitter solution acts as a model for the dynamics of the solutions by proving that the generalized kasner exponents tend to 1/3 as t , which in the plane case is the de sitter solution . recall that when = 0 , rein showed that solutions can only exist for finite time in the future direction in area time coordinates . by adding a positive cosmological constant , global existence to the future is shown to hold true if initial data is given on t = t0 , where t02 > 1/. the asymptotic behaviour of the matter terms is also investigated and slightly stronger decay estimates are obtained in this case compared to the case of plane and hyperbolic symmetry . the results discussed so far in this section have concerned the future time direction and a positive cosmological constant . the past direction with a negative cosmological constant is analyzed in , where it is shown that for plane and spherical symmetry the areal time coordinate takes all positive values , which is in analogy with weaver s result for = 0 . if initial data are restricted by a smallness condition the theorem is proven also in the hyperbolic case , and for such data the result of the theorem holds true in all of the three symmetry classes when the cosmological constant is positive . the early - time asymptotics in the case of small initial data is also analyzed and is shown to be kasner - like . in the einstein - vlasov system with a linear scalar field . a local existence theorem and a continuation criterion , involving bounds on derivatives of the scalar field in addition to a bound on the support of one of the moment variables , is proven . for the einstein scalar field system , i.e. when f = 0 , the continuation criterion is shown to be satisfied in the future direction and global existence follows in that case . equilibrium states in galactic dynamics can be described as stationary solutions of the einstein - vlasov system , or of the vlasov - poisson system in the newtonian case . here we will consider the former case for which only static , spherically symmetric solutions have been constructed , but we mention that in the latter case also , stationary axially symmetric solutions have been found by rein . in the static , spherically symmetric case , let the spacetime metric have the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { e^{2\mu ( r)}}d{t^2 } + { e^{2\lambda ( r)}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\varphi ^2}),$$\end{document } where r > 0 , [ 0 , ] , [ 0 , 2 ] . as before , asymptotic flatness is expressed by the boundary conditions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathop { \lim}\limits_{r \rightarrow \infty } \lambda ( r ) = \mathop { \lim}\limits_{r \rightarrow \infty } \mu ( r ) = 0,\quad \forall t \geq 0,$$\end{document } and a regular centre requires \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda ( 0 ) = 0.$$\end{document } following the notation in section 2.1 , the time - independent einstein - vlasov system reads 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{\mu - \lambda}}{v \over { \sqrt { 1 + \vert v{\vert ^2}}}}\cdot{\nabla _ x}f - \sqrt { 1 + \vert v{\vert ^2 } } { e^{\mu - \lambda}}{\mu _ r}{x \over r}\cdot{\nabla _ v}f = 0,$$\end{document } 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}\rho,$$\end{document } 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}p.$$\end{document } the matter quantities are defined as before : 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( x ) = \int\nolimits_{{{\mathbb r}^3 } } { \sqrt { 1 + \vert v{\vert ^2 } } f(x , v)dv,}$$\end{document } 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(x ) = \int\nolimits_{{{\mathbb r}^3 } } { { { \left({{{x\cdot v } \over r } } \right)}^2}f(x , v){{dv } \over { \sqrt { 1 + \vert v{\vert ^2}}}}}.$$\end{document } the quantities \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e : = { e^{\mu ( r)}}\sqrt { 1 + \vert v{\vert ^2 } } , \quad l = \vert x{\vert ^2}\vert v{\vert ^2 } - { ( x \cdot v)^2 } = \vert x \times v{\vert ^2}$$\end{document } are conserved along characteristics . if we let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(x , v ) = \phi ( e , l)$$\end{document } for some function , the vlasov equation is automatically satisfied . the form of is usually restricted to 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi ( e , l ) = \phi ( e){(l - { l_0})^\iota},$$\end{document } where l > 1/2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${l_0 } \ge 0$\end{document}. if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi ( e ) = ( e - { e_0 } ) _ + ^k , k > - 1$\end{document } , for some positive constant e0 , this is called the polytropic ansatz . the case of isotropic pressure is obtained by letting l = 0 so that only depends on e. we refer to for information on the role of l0 . in passing , we mention that for the vlasov - poisson system it has been shown that every static spherically symmetric solution must have the form f = (e , l ) . it was an open question for some time to decide whether or not this was also true for the einstein - vlasov system . this was settled in 1999 by schaeffer , who found solutions that do not have this particular form globally on phase space , and consequently , jeans theorem is not valid in the relativistic case . however , almost all results in this field rest on this ansatz . by inserting the ansatz for f in the matter quantities and p , a nonlinear system for and is obtained , in which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{{e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}{g_\phi}(r,\mu ) , } \\ { { e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}{h_\phi}(r,\mu ) , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{{g_\phi}(r,\mu ) = { { 2\pi } \over { { r^2}}}\int\nolimits_1^\infty { \int\nolimits_0^{{r^{2({\epsilon^2 } - 1 ) } } } { \phi ( { e^{\mu ( r)}}\epsilon , l ) } { { { \epsilon^2 } } \over { \sqrt { { \epsilon^2 } - 1 - l/{r^2}}}}dl\;d\epsilon , } } \\ { { h_\phi}(r,\mu ) = { { 2\pi } \over { { r^2}}}\int\nolimits_1^\infty { \int\nolimits_0^{{r^{2({\epsilon^2 } - 1 ) } } } { \phi ( { e^{\mu ( r)}}\epsilon , l ) } \sqrt { { \epsilon^2 } - 1 - l/{r^2 } } dl\;d\epsilon . } } \\\end{array}$$\end{document } existence of solutions to this system was first proved in the case of isotropic pressure in and then extended to the general case in . the main problem is then to show that the resulting solutions have finite ( adm ) mass and compact support . this is accomplished in for a polytropic ansatz with isotropic pressure and in for a polytropic ansatz with possible anisotropic pressure . they use a perturbation argument based on the fact that the vlasov - poisson system is the limit of the einstein - vlasov system as the speed of light tends to infinity . two types of solutions are constructed , those with a regular centre [ 90 , 81 ] , and those with a schwarzschild singularity in the centre . in rendall and rein go beyond the polytropic ansatz and assume that satisfies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi ( e , l ) = c(e - { e_0 } ) _ + ^k{l^l } + o(({e_0 } - e ) _ + ^{\delta + k}{l^l}\quad { \rm{as}}\;e \rightarrow { e_0},$$\end{document } where k > 1 , l > 1/2 , k + l + 1/2 > 0 , k < l + 3/2 . they show that this assumption is sufficient for obtaining steady states with finite mass and compact support . the result is obtained in a more direct way and is not based on the perturbation argument mentioned above . their method is inspired by a work on stellar models by makino , in which he considers steady states of the euler - einstein system . in there is also an interesting discussion about steady states that appear in the astrophysics literature . they show that their result applies to most of these steady states , which proves that they have the desirable property of finite mass and compact support . all solutions described so far have the property that the support of p contains a ball about the centre . in rein shows that there exist steady states whose support is a finite , spherically symmetric shell , so that they have a vacuum region in the centre . at present , there are almost no known results concerning the stability properties of the steady states to the einstein - vlasov system . in the vlasov - poisson case , however , the nonlinear stability of stationary solutions has been investigated by guo and rein using the energy - casimir method . in the einstein - vlasov case , wolansky has applied the energy - casimir method and obtained some insights but the theory in this case is much less developed than in the vlasov - poisson case and the stability problem is essentially open .

the main purpose of this article is to provide a guide to theorems on global properties of solutions to the einstein - vlasov system . this system couples einstein s equations to a kinetic matter model . kinetic theory has been an important field of research during several decades in which the main focus has been on nonrelativistic and special relativistic physics , i.e. to model the dynamics of neutral gases , plasmas , and newtonian self - gravitating systems . in 1990 , rendall and rein initiated a mathematical study of the einstein - vlasov system . since then many theorems on global properties of solutions to this system have been established . the vlasov equation describes matter phenomenologically , and it should be stressed that most of the theorems presented in this article are not presently known for other such matter models ( i.e. fluid models ) . this paper gives introductions to kinetic theory in non - curved spacetimes and then the einstein - vlasov system is introduced . we believe that a good understanding of kinetic theory in non - curved spacetimes is fundamental to good comprehension of kinetic theory in general relativity .

Introduction to Kinetic Theory Global Existence Theorems for the Einstein-Vlasov System Stationary Solutions to the Einstein-Vlasov System

in general relativity , kinetic theory has been used relatively sparsely to model phenomenological matter in comparison to fluid models . in non - curved spacetimes kinetic theory has been studied intensively as a mathematical subject during several decades , and it has also played an important role from an engineering point of view . in the first part of this introduction , we will review kinetic theory in non - curved spacetimes and we will consider mainly the special relativistic case , but mathematical results in the nonrelativistic case will also be discussed . we believe that a good understanding of kinetic theory in non - curved spacetimes is fundamental to good comprehension of kinetic theory in general relativity . the purpose of kinetic theory is to model the time evolution of a collection of particles . for a plasma the interaction is through the electric charges ( the vlasov - maxwell system ) , and in the stellar and cosmological cases the interaction is gravitational ( the einstein - vlasov system ) . the main result concerning the existence of solutions to the classical boltzmann equation is a theorem by diperna and lions that proves existence , but not uniqueness , of renormalized solutions ( i.e. one of the fundamental problems in kinetic theory is to find out whether or not spontaneous shock formations will develop in a collisionless gas , i.e. this system , however , is unphysical in the sense that it is not a special case of the einstein - vlasov system . before turning to the main theme of this review , i.e. the einstein - vlasov system , we introduce a system which has many mathematical features in common with the vlasov - maxwell system and which recently has been mathematically studied for the first time . a solution ( f , ) of this system is interpreted as follows : the spacetime is a lorentzian manifold with a conformally flat metric which , in the coordinates ( t , x ) , takes the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } = { e^{2\phi}}{\rm{diag}}(- 1,\;1,\;1,\;1).$$\end{document } the particle distribution f defined on the mass shell in this metric is given by 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(t , x , p ) = { \mathfrak { f}}(t , x,{e^\phi } p).$$\end{document } the first mathematical study of this system was undertaken by calogero in , where static solutions are analyzed and where also more details on the derivation of the system are given . in this section we will consider a self - gravitating collisionless gas and we will write down the einstein - vlasov system and describe its general mathematical features . in a fixed spacetime the vlasov equation is a linear hyperbolic equation for f and we can solve it by solving the characteristic system , 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{x^i } } \over { ds } } = { { { p^i } } \over { { p^0}}},$$\end{document } 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{p^i } } \over { ds } } = - \gamma _ { ab}^i{{{p^a}{p^b } } \over { { p^0}}}.$$\end{document } in terms of initial data f0 the solution to the vlasov equation can be written as 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f({x^a},{p^i } ) = { f_0}({x^i}(0,{x^a},{p^i}),{p^i}(0,{x^a},{p^i})),$$\end{document } where x(s , x , p ) and p(s , x , p ) solve equations ( 26 , 27 ) , and where x(t , x , p ) = x and p(t , x , p ) = p. in order to write down the einstein - vlasov system we need to define the energy - momentum tensor tab in terms of f and gab . in the coordinates ( x , p ) on p we define \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{ab } } = - \int\nolimits_{{{\mathbb r}^3 } } f { p_a}{p_b}\vert g\vert ^{{1/2}}{{d{p^1}d{p^2}d{p^3 } } \over { { p_0}}},$$\end{document } where as usual pa = gabp , and |g| denotes the absolute value of the determinant of g. equation ( 25 ) together with einstein s equations , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{ab } } : = { r_{ab } } - { 1 \over 2}r{g_{ab } } = 8\pi { t_{ab } } + \lambda { g_{ab}},$$\end{document } then form the einstein - vlasov system . that the energy conditions hold for vlasov matter is one reason that the vlasov equation defines a well - behaved matter model in general relativity . before stating that theorem the data in the cauchy problem for the einstein - vlasov system consist of the induced riemannian metric gij on the initial hypersurface s , the second fundamental form kij of s and matter data f. the relations between a given initial data set ( gij , kij ) on a three - dimensional manifold s and the metric gij on the spacetime manifold is that there exists an embedding of s into the spacetime such that the induced metric and second fundamental form of (s ) coincide with the result of transporting ( gij , kij ) with . \\ \end{array}$$\end{document } here |g| is the determinant of the induced riemannian metric on s. we can now state the local existence theorem by choquet - bruhat for the einstein - vlasov system . then there exists a smooth spacetime ( m , gab ) , a smooth distribution function f on the mass shell of this spacetime , and a smooth embedding of s into m which induces the given initial data on s such that gab and f satisfy the einstein - vlasov system and (s ) is a cauchy surface . in this context we also mention that local existence has been proved for the yang - mills - vlasov system in , and that this problem for the einstein - maxwell - boltzmann system is treated in . this problem concerning physically reasonable assumptions on the scattering kernel seems not well understood in the context of the einstein - boltzmann system , and a careful study of this issue would be desirable . there are interesting situations when this can be achieved , and such global existence theorems are not known for einstein s equations coupled to other forms of phenomenological matter models , i.e. in this context it should be stressed that the results in the previous sections show that the mathematical understanding of kinetic equations on a flat background space is well - developed . the study of the global properties of solutions to the spherically symmetric einstein - vlasov system was initiated by rein and rendall in 1990 . }$$\end{document } let us point out that this system is not the full einstein - vlasov system . a solution to the vlasov equation can be written as 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f{\text{(}}t{\text{,}}r{\text{,}}w{\text{,}}l{\text { ) = } } f_{\text{0 } } { \text{(}}r{\text{(0,}}t{\text{,}}r{\text{,}}w{\text{,}}l{\text { ) , } } w{\text{(0,}}t{\text{,}}r{\text{,}}w{\text{,}}l{\text{),}}l{\text{),}}$$\end{document } where r and w are solutions to the characteristic system 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dr } \over { ds } } = { e^{(\mu - \lambda)(s , r)}}{w \over { e(r , w , l)}},$$\end{document } 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dw } \over { ds } } = - { \lambda _ t}(s , r)w - { e^{(\mu - \lambda)(s , r)}}{\mu _ r}(s , r)e(r , w , l ) + { e^{(\mu - \lambda)(s , r)}}{l \over { { r^3}e(r , w , l)}},$$\end{document } such that the trajectory ( r(s , t , r , w , l ) , w(s , t , r , w , l ) , l ) goes through the point ( r , w , l ) when s = t. this representation shows that f is non - negative for all t 0 and that f f0. there are two known conservation laws for the einstein - vlasov system : conservation of the number of particles , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$4{\pi ^2}\int\nolimits_0^\infty { { e^{\lambda ( t , r)}}\left({\int\nolimits_{- \infty}^\infty { \int\nolimits_0^\infty { f(t , r , w , l)dl\;dw } } } \right)dr,}$$\end{document } and conservation of the adm mass 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m : = 4\pi \int\nolimits_0^\infty { { r^2}\rho ( t , r)dr.}$$\end{document } let us now review the global results concerning the cauchy problem that have been proved for the spherically symmetric einstein - vlasov system . it should be stressed that even for small data no global existence result like this one is known for any other phenomenological matter model coupled to einstein s equations . in this context we point out that in it is proved that the vlasov - poisson system is indeed the nonrelativistic limit of the spherically symmetric einstein - vlasov system , i.e. in a numerical study was undertaken of the einstein - vlasov system in schwarzschild coordinates . we end this section with a discussion of the spherically symmetric einstein - vlasov - maxwell system , i.e. in it is shown that solutions to the constraint equations do exist for the einstein - vlasov - maxwell system . the main goal again is to determine the global properties of the solutions to the einstein - vlasov system . there are several existing results on global time coordinates for solutions of the einstein - vlasov system . the maximal range of a gaussian time coordinate in a solution of the einstein - vlasov system evolving from homogenous data on a compact manifold was determined in . in this section we will review the recent results for the einstein - vlasov system where a cosmological constant , or a linear or nonlinear scalar field have been included into the model . equilibrium states in galactic dynamics can be described as stationary solutions of the einstein - vlasov system , or of the vlasov - poisson system in the newtonian case . } \\\end{array}$$\end{document } existence of solutions to this system was first proved in the case of isotropic pressure in and then extended to the general case in . they use a perturbation argument based on the fact that the vlasov - poisson system is the limit of the einstein - vlasov system as the speed of light tends to infinity . at present , there are almost no known results concerning the stability properties of the steady states to the einstein - vlasov system . in the einstein - vlasov case , wolansky has applied the energy - casimir method and obtained some insights but the theory in this case is much less developed than in the vlasov - poisson case and the stability problem is essentially open .

benign prostatic hyperplasia ( bph ) is one of the most frequently occurring urologic diseases in men older than 50 . the bladder outlet obstruction causes a variety of urinary symptoms including nocturia , incomplete voiding , urgency , and hesitancy and comes to the fore as a significant health problem interrupting the quality of life of men over middle age . the prevalence rate of bph increases with age ; bph occurs in 40% to 70% of men aged 60 to 70 years . the current korean population is aging very rapidly compared with other countries ; with this , interest in health after middle age is increasing throughout society . however , owing to a lack of recognition about bph , the time until visiting the hospital is often delayed , and this tendency is truer in rural areas than in urban areas . in this situation , it is necessary to understand the prevalence rate of bph , but few systematic epidemiologic investigations have been carried out about bph in korea . also , there are limitations in the epidemiologic research conducted so far because the previous data were not collected by use of standardized diagnostic criteria or a specific clinical definition . therefore , in this study , the prevalence rate of bph was estimated among males living in the rural korean area of yangpyeong country . this study was conducted to investigate the prevalence rate of bph and its related factors on the basis of an epidemiologic investigation among males in one rural community in korea . the department of preventive medicine and the department of urology in hanyang university guri hospital planned the cohort research by targeting yangpyeong county . the participants of this study were males aged 40 and above in yangpyeong county , gyeonggi - do . the participants were recruited over august to september 2009 , august 2010 , and august 2011 . trained investigators collected the international prostate symptom score ( ipss ) , individual medical history , and demographic information for each participant . we excluded those already diagnosed with bph who were receiving treatment and taking health food supplements . prostate disease was assessed by measuring serum prostate - specific antigen ( psa ) , a doctor 's medical examination by interview , a digital rectal examination , and prostate volume by transrectal ultrasound ( sa6000ii , medison inc . , seoul , korea ) . a total of 799 males responded to all of the survey questions and underwent examinations . among them , 668 subjects remained for study after the exclusion of 1 person who did not consent to participate in the study , 1 person who had a prostate cancer history , and 109 subjects who were missing the digital rectal examination , transrectal ultrasonography , or other epidemiologic inf ormation . among these subjects , data were analyzed from the 599 subjects finally participating in this study after the exclusion of subjects in whom a nodule was perceived or who had a serum psa score of 4.0 or higher and thus who might have prostate cancer ( fig . 1 ) the severity of luts was evaluated by the ipss and quality of life and the participants were divided into four groups by age ( 40 - 49 , 50 - 59 , 60 - 69 , and 70 years and above ) . this study was approved by the institutional review board ( irb ) of hanyang university college of medicine . the irb numbers were hyuh irb 2010-r-38 and 2011 - 07 - 005 . in this study , bph was defined as a score of 8 or higher on the ipss and a prostate volume of 25 ml or above by transrectal ultrasonography , according to the criteria of the fourth edition of the textbook of urology published by the korean urological association . the prevalence rate of bph was compared by classifying the groups by age : 40 - 49 , 50 - 59 , 60 - 69 , and 70 years and above . in addition , the prevalence rate of each luts was checked by classifying the total ipss in each group into a mild symptom group ( score of 1 - 7 ) , a moderate symptom group ( score of 8 - 19 ) , and a severe symptom group ( score of 20 - 35 ) . additionally , the analysis was conducted on the correlations between the epidemiologic factors ( age , marital status , education level , smoking status , alcohol consumption status , regular exercise , body mass index [ bmi ] ) , comorbidities ( hypertension , diabetes ) , and serum psa and bph . , cary , nc , usa ) was used for the statistical analysis ; we also used the chisquare test , independent t - test , analysis of variance , and logistic regression analysis . the results of the statistical analysis were considered to be significant when the p - value was less than 0.05 . this study was conducted to investigate the prevalence rate of bph and its related factors on the basis of an epidemiologic investigation among males in one rural community in korea . the department of preventive medicine and the department of urology in hanyang university guri hospital planned the cohort research by targeting yangpyeong county . the participants of this study were males aged 40 and above in yangpyeong county , gyeonggi - do . the participants were recruited over august to september 2009 , august 2010 , and august 2011 . trained investigators collected the international prostate symptom score ( ipss ) , individual medical history , and demographic information for each participant . we excluded those already diagnosed with bph who were receiving treatment and taking health food supplements . prostate disease was assessed by measuring serum prostate - specific antigen ( psa ) , a doctor 's medical examination by interview , a digital rectal examination , and prostate volume by transrectal ultrasound ( sa6000ii , medison inc . , seoul , korea ) . a total of 799 males responded to all of the survey questions and underwent examinations . among them , 668 subjects remained for study after the exclusion of 1 person who did not consent to participate in the study , 1 person who had a prostate cancer history , and 109 subjects who were missing the digital rectal examination , transrectal ultrasonography , or other epidemiologic inf ormation . among these subjects , data were analyzed from the 599 subjects finally participating in this study after the exclusion of subjects in whom a nodule was perceived or who had a serum psa score of 4.0 or higher and thus who might have prostate cancer ( fig . 1 ) the severity of luts was evaluated by the ipss and quality of life and the participants were divided into four groups by age ( 40 - 49 , 50 - 59 , 60 - 69 , and 70 years and above ) . this study was approved by the institutional review board ( irb ) of hanyang university college of medicine . in this study , bph was defined as a score of 8 or higher on the ipss and a prostate volume of 25 ml or above by transrectal ultrasonography , according to the criteria of the fourth edition of the textbook of urology published by the korean urological association . the prevalence rate of bph was compared by classifying the groups by age : 40 - 49 , 50 - 59 , 60 - 69 , and 70 years and above . in addition , the prevalence rate of each luts was checked by classifying the total ipss in each group into a mild symptom group ( score of 1 - 7 ) , a moderate symptom group ( score of 8 - 19 ) , and a severe symptom group ( score of 20 - 35 ) . additionally , the analysis was conducted on the correlations between the epidemiologic factors ( age , marital status , education level , smoking status , alcohol consumption status , regular exercise , body mass index [ bmi ] ) , comorbidities ( hypertension , diabetes ) , and serum psa and bph . , cary , nc , usa ) was used for the statistical analysis ; we also used the chisquare test , independent t - test , analysis of variance , and logistic regression analysis . the results of the statistical analysis were considered to be significant when the p - value was less than 0.05 . there were 45 subjects ( 7.5% ) in the aged 40 - 49 years group , 165 subjects ( 27.5% ) in the aged 50 - 59 years group , 200 subjects ( 33.4% ) in the aged 60 - 69 years group , and 189 subjects ( 31.5% ) in the aged 70 years and above group . the prostate volume of the entire group of participants was 30.2510.14 g , and the average psa level was 1.200.79 ng / ml . the ipss and the quality of life score were 5.966.72 and 2.701.04 , respectively . in the age groups , the ipss was 2.223.76 in men aged 40 - 49 years , 3.505.06 in those aged 50 - 59 , 6.756.94 in those aged 60 - 69 , and 8.167.28 in men aged 70 or more . these changes with age were statistically significant ( p<0.001 ) . also , prostate volume was 25.365.22 g in men aged 40 - 49 years , 30.299.12 g in men aged 50 - 59 years , 30.549.7 g in men aged 60 - 69 years , and 31.0711.91 g in men aged 70 years and above , and these age - related increases were statistically significant ( p=0.008 ) . a total of 120 subjects ( 20.0% ) in the entire group of participants met the standard of bph as defined in this study . when we examined the prevalence rate according to age , we found that the prevalence rate increased with age . there were 2 subjects ( 4.4% ) with bph in the and aged 40 - 49 years group , 18 subjects ( 10.9% ) in the aged 50 - 59 years group , 44 subjects ( 22% ) in the aged 60 - 69 years group , and 56 subjects ( 26.6% ) in the aged 70 years and above group , and this increase with age was statistically significant ( p<0.001 ) ( table 1 ) . a total of 120 participants ( 20% ) met the definition of bph in this study . the average age of the bph group was 68.238.52 years , the average ipss was 14.675.95 , the average prostate volume was 37.0411.71 g , and the average psa level was 1.560.88 ng / ml ( table 2 ) . in the subscores of the ipss , the score for weak urinary stream was highest ( 3.372.08 ) ; the scores for incomplete emptying ( 2.432.18 ) , hesitancy ( 2.192.15 ) , and nocturia ( 2.051.31 ) were also high . in the survey questions about quality of life , the overall inconvenience of the patients due to their current urinary symptoms was used for the score ; the scores were divided from 0 as very satisfied to 6 as very dissatisfied . the quality of life score was significantly higher in the bph group ( 3.480.93 ) than in the non - bph group ( 2.500.91 ) . in this study , we conducted a correlation analysis to determine whether epidemiologic factors ( marital status , education level , smoking status , alcohol consumption status , bmi , and regular exercise ) and comorbidities ( hypertension and diabetes ) influenced the risk of bph . the non - bph group had a higher proportion of smokers than did the bph group ( table 3 ) . the reason the epidemiologic factors including lifestyle did not show a significant correlation may be that this study was based on a cross - sectional design that did not clearly show a sequential relationship by time . in the case of the bph group , the patients ' uncomfortable urinary status could have already influenced changes in health behavior . there were 45 subjects ( 7.5% ) in the aged 40 - 49 years group , 165 subjects ( 27.5% ) in the aged 50 - 59 years group , 200 subjects ( 33.4% ) in the aged 60 - 69 years group , and 189 subjects ( 31.5% ) in the aged 70 years and above group . the prostate volume of the entire group of participants was 30.2510.14 g , and the average psa level was 1.200.79 ng / ml . the ipss and the quality of life score were 5.966.72 and 2.701.04 , respectively . in the age groups , the ipss was 2.223.76 in men aged 40 - 49 years , 3.505.06 in those aged 50 - 59 , 6.756.94 in those aged 60 - 69 , and 8.167.28 in men aged 70 or more . these changes with age were statistically significant ( p<0.001 ) . also , prostate volume was 25.365.22 g in men aged 40 - 49 years , 30.299.12 g in men aged 50 - 59 years , 30.549.7 g in men aged 60 - 69 years , and 31.0711.91 g in men aged 70 years and above , and these age - related increases were statistically significant ( p=0.008 ) . a total of 120 subjects ( 20.0% ) in the entire group of participants met the standard of bph as defined in this study . when we examined the prevalence rate according to age , we found that the prevalence rate increased with age . there were 2 subjects ( 4.4% ) with bph in the and aged 40 - 49 years group , 18 subjects ( 10.9% ) in the aged 50 - 59 years group , 44 subjects ( 22% ) in the aged 60 - 69 years group , and 56 subjects ( 26.6% ) in the aged 70 years and above group , and this increase with age was statistically significant ( p<0.001 ) ( table 1 ) . a total of 120 participants ( 20% ) met the definition of bph in this study . the average age of the bph group was 68.238.52 years , the average ipss was 14.675.95 , the average prostate volume was 37.0411.71 g , and the average psa level was 1.560.88 ng / ml ( table 2 ) . in the subscores of the ipss , the score for weak urinary stream was highest ( 3.372.08 ) ; the scores for incomplete emptying ( 2.432.18 ) , hesitancy ( 2.192.15 ) , and nocturia ( 2.051.31 ) were also high . in the survey questions about quality of life , the overall inconvenience of the patients due to their current urinary symptoms was used for the score ; the scores were divided from 0 as very satisfied to 6 as very dissatisfied . the quality of life score was significantly higher in the bph group ( 3.480.93 ) than in the non - bph group ( 2.500.91 ) . in this study , we conducted a correlation analysis to determine whether epidemiologic factors ( marital status , education level , smoking status , alcohol consumption status , bmi , and regular exercise ) and comorbidities ( hypertension and diabetes ) influenced the risk of bph . the non - bph group had a higher proportion of smokers than did the bph group ( table 3 ) . the reason the epidemiologic factors including lifestyle did not show a significant correlation may be that this study was based on a cross - sectional design that did not clearly show a sequential relationship by time . in the case of the bph group , bph generally occurs in males in their 50s ; 80% of males in their 70s suffer from the luts related to bph . because in current korean society the population is aging , the older population is rapidly increasing . additionally , the incidence of metabolic syndrome , which is a risk factor for bph , and the number of patients with obesity are also annually increasing owing to westernized eating habits . bph and luts could lead to social problems caused by the increase in annual medical expenses related to serious complications such as falls , depression , and reduced quality of life , particularly in the elderly . in this situation , it is necessary to conduct systematic epidemiologic investigations of the prevalence rate of bph in korea . to investigate the prevalence rate of bph , it is f irst necessary to clarify the diagnostic criteria of bph . although methods exists for the diagnosis of bph , including taking the patient 's medical history and recording urinary symptoms , prostate volume measurement by digital rectal examination and transrectal ultrasonography , peak urinary flow rate ( qmax ) , and residual urine measurement , accurate diagnostic criteria have not been established . currently , bph is mainly defined clinically on the basis of the ipss , prostate volume , and qmax . garraway et al . defined bph as urinary symptoms , prostate volume of 20 ml or more as measured by transrectal ultrasonography , and qmax of 15 ml / s or less . in their study , they reported that the prevalence rate of bph was 14% in males in their 40s and 40% in males in their 70s . bosch et al . defined bph as a prostate volume of 30 ml or more and ipss of 8 points or higher and reported a bph prevalence rate of 19% . in a study targeting males aged 40 years and older in spain . in studies defining bph in korea , rhew et al . defined bph as an ipss of 8 points or higher and a qmax of 10 ml / s or less . defined it as an ipss of 8 points or higher , prostate volume by digital rectal examination of 30 ml or more , and qmax of 15 ml / s or less . in studies investigating the prevalence rate in seongnam city and jeju island , bph was defined as an ipss of 8 points or higher and prostate volume measured by transrectal ultrasonography of 30 ml or more . in this study , bph diagnosis was based on the symptom score and the prostate volume , and urof lowmetry examination was excluded . reported that the bph prevalence rate defined by two variables including symptom score and prostate volume and the bph prevalence rate defined by three variables including symptom score , prostate volume , and qmax were 12% and 10% , respectively , and that there was no statistically significant difference between the two rates . second , it was thought that there could be many errors when determining bph based on 10 ml / s or less or 15 ml / s or less in epidemiologic examinations because uroflowmetry can vary depending on the amount of urination . measured prostate volume through transrectal ultrasonography by targeting 175 males whose ages were between 27 and 70 years ; the average prostate volume of adenoma prostate was significantly higher than that of nonadenoma prostate : 28.0 ml ( range , 14.5 - 62.1 ml ) vs. 23.0 ml ( range , 11.3 - 39.1 ml ) . therefore , those authors proposed criteria for bph as an ipss of 8 points or higher and a prostate volume by transrectal ultrasonography of 25 ml or more . the bph prevalence rate was 20% ( 120/599 subjects ) in the present study and lower than the rate in other reports in korea . the reason for this could be that 210 of 599 males ( 35% ) were in their 40s to 50s and were thus relatively younger than the groups in the other studies in korea . bph prevalence rates reported in korea were 40% in the urban area of seongnam and 25.5% in busan . in addition , the prevalence rates were 23.2% in the rural area of yeoncheon , gyeonggi - do , and 27.7% in the inland area of chungcheongbuk - do . first , there were variables in the epidemiologic investigation , such as the selection of the target group and regional differences in the population . the prevalence rate in seongnam was much higher than in other regions because the study was an epidemiologic investigation targeting the elderly population ( 65 years old and above ) . although lee et al . conducted an epidemiologic investigation for the first time in yeoncheon area in 1995 , they only conducted a survey of the ipss and then estimated the prevalence rate of bph . reported that the bph prevalence rate as assessed by luts , digital rectal examination , and uroflowmetry examination was 11% ; here , the question about the accuracy of measuring prostate size could be raised because the prostate volume was measured by digital rectal examination . in this study , the survey and the epidemiologic investigation were performed by interviewers who were sufficiently trained ; unlike other authors ' measurement of the prostate volume by digital rectal examination , it could be thought that the reliability would be high because the prostate volume was accurately measured by transrectal ultrasonography . epidemiologic research on bph has greatly evolved over the past several years , and many epidemiologic data have been accumulated . even though age and genetics play an important role as causes of bph , it is notable that lifestyle factors such as metabolic syndrome or cardiovascular disease , which can be corrected , have a considerable impact on the natural progression of bph . one cohort study showed that luts prevalence increased by 80% in the group diagnosed with at least three components of the metabolic syndrome compared with the group without diagnosed components of the metabolic syndrome . additionally , other studies have shown that the risk of bph and luts is significantly increased in people with heart disease . in other previous studies , a correlation was observed in which the more the amount of fat increased , the more the prostate volume increased . in several research studies , weight , bmi , and waistline all showed a significant correlation with prostate volume . in this study , there was no significant difference in bmi between the bph group and the non - bph group . the reason for this may be that the cross - sectional design of this study did not clearly show the sequential relationship of time . the study criteria were also vague because there are few studies related to bmi and prostate volume in asia , including korea . in the longitudinal study of aging cohort in baltimore , maryland ( usa ) , each 1-kg / m increase in bmi corresponded to a 0.41-ml prostate volume increase . compared with nonobese participants ( bmi<25 kg / m ) , the obese participants ( bmi35 kg / m ) showed a 3.5-time higher bph risk . in addition , whereas some studies reported that there was a reverse protective effect of smoking on bph and luts , other studies reported that the risk level increased or showed no change . therefore , it is necessary to conduct more studies on the correlation between smoking and bph . first , the subjects were not a random sample of the population because we recruited subjects through a prostate health screening campaign . second , we did not consider the difference in the population distribution by age in yangpyeong county and the study group . third , although prevalence was the number of cases of the condition at a particular point in time , the participants of this study were recruited over 3 years ( august - september 2009 , august 2010 , and august 2011 ) in an effort to collect more participants . consequently , the results of this epidemiologic research investigating the bph prevalence rate in a rural korean area showed a lower value than in other countries . one reason for this may be that the ratio of relatively younger males in their 40s to 50s was somewhat higher in this epidemiologic investigation than in other studies . second , there may have been an influence of diet , with a focus on vegetables in the rural area compared with the westernized diet of high meat consumption in urban areas . although our study did not survey the eating habits of the individuals , in a study performed to evaluate food intake of the elderly residing in different regions of korea , the intake of meats was significantly higher in urban elderly than in rural elderly . third , the different diagnostic criteria of bph applied in each study could be a cause of the differences in prevalence rates . in this study , the prevalence rate of bph was estimated by targeting males aged 40 years and older in a rural korean area . the results showed that the prevalence rate of bph in males aged 40 years and older in yangpyeong county was 20.0% . when classifying the men according to age , none of the epidemiologic factors studied including lifestyle showed a statistically significant correlation with bph , except for smoking . this study showed a lower prevalence rate of bph in a rural area than reported in other studies in korea . unlike other studies conducted in rural areas , however , the reliability of this study can be considered to be high because prostate volume was measured by use of a more objective method .

purposewe investigated the prevalence rate of benign prostatic hyperplasia ( bph ) among korean males in a rural area through a cross - sectional , community - based epidemiologic survey and analyzed the correlation with epidemiologic factors.materials and methodsa total of 779 males who lived in yangpyeong county participated in a prostate examination campaign . targeting these men , we collected the international prostate symptom score ( ipss ) , medical history , demographic information , serum prostate - specific antigen , and prostate volume as measured by transrectal ultrasonography . the data for 599 participants were analyzed , excluding 180 men who had a possibility of prostate cancer . bph was defined as an ipss of 8 points or higher and a prostate volume of 25 ml or more.resultsthe prevalence rate of bph was 20.0% . the prevalence rate increased with age . there were 2 subjects ( 4.4% ) in the age group of 40 - 49 years , 18 subjects ( 10.9% ) in the age group of 50 - 59 years , 44 subjects ( 22% ) in the age group of 60 - 69 years , and 56 subjects ( 26.6% ) in the age group of over 70 years ; this increase with age was statistically significant ( p<0.001 ) . in the bph group , the average ipss was 14.675.95 , the average prostate volume was 37.0411.71 g , and the average prostate - specific antigen value was 1.560.88 ng / ml . in the analysis of correlations between the epidemiologic factors and the risk of bph , smoking was the only statistically significant factor.conclusionsthe total prevalence rate of bph in this study was 20.0% , which was a little lower than the rate reported in other cities or rural areas .

INTRODUCTION MATERIALS AND METHODS 1. Research participants 2. Criteria of epidemiologic investigation 3. Statistical analysis RESULTS 1. Prevalence rate of BPH 2. Distribution of factors associated With BPH 3. Analysis of the factors influencing BPH DISCUSSION CONCLUSIONS

benign prostatic hyperplasia ( bph ) is one of the most frequently occurring urologic diseases in men older than 50 . the prevalence rate of bph increases with age ; bph occurs in 40% to 70% of men aged 60 to 70 years . however , owing to a lack of recognition about bph , the time until visiting the hospital is often delayed , and this tendency is truer in rural areas than in urban areas . in this situation , it is necessary to understand the prevalence rate of bph , but few systematic epidemiologic investigations have been carried out about bph in korea . therefore , in this study , the prevalence rate of bph was estimated among males living in the rural korean area of yangpyeong country . this study was conducted to investigate the prevalence rate of bph and its related factors on the basis of an epidemiologic investigation among males in one rural community in korea . the participants of this study were males aged 40 and above in yangpyeong county , gyeonggi - do . the participants were recruited over august to september 2009 , august 2010 , and august 2011 . trained investigators collected the international prostate symptom score ( ipss ) , individual medical history , and demographic information for each participant . prostate disease was assessed by measuring serum prostate - specific antigen ( psa ) , a doctor 's medical examination by interview , a digital rectal examination , and prostate volume by transrectal ultrasound ( sa6000ii , medison inc . among them , 668 subjects remained for study after the exclusion of 1 person who did not consent to participate in the study , 1 person who had a prostate cancer history , and 109 subjects who were missing the digital rectal examination , transrectal ultrasonography , or other epidemiologic inf ormation . among these subjects , data were analyzed from the 599 subjects finally participating in this study after the exclusion of subjects in whom a nodule was perceived or who had a serum psa score of 4.0 or higher and thus who might have prostate cancer ( fig . 1 ) the severity of luts was evaluated by the ipss and quality of life and the participants were divided into four groups by age ( 40 - 49 , 50 - 59 , 60 - 69 , and 70 years and above ) . this study was approved by the institutional review board ( irb ) of hanyang university college of medicine . in this study , bph was defined as a score of 8 or higher on the ipss and a prostate volume of 25 ml or above by transrectal ultrasonography , according to the criteria of the fourth edition of the textbook of urology published by the korean urological association . the prevalence rate of bph was compared by classifying the groups by age : 40 - 49 , 50 - 59 , 60 - 69 , and 70 years and above . in addition , the prevalence rate of each luts was checked by classifying the total ipss in each group into a mild symptom group ( score of 1 - 7 ) , a moderate symptom group ( score of 8 - 19 ) , and a severe symptom group ( score of 20 - 35 ) . additionally , the analysis was conducted on the correlations between the epidemiologic factors ( age , marital status , education level , smoking status , alcohol consumption status , regular exercise , body mass index [ bmi ] ) , comorbidities ( hypertension , diabetes ) , and serum psa and bph . this study was conducted to investigate the prevalence rate of bph and its related factors on the basis of an epidemiologic investigation among males in one rural community in korea . the participants of this study were males aged 40 and above in yangpyeong county , gyeonggi - do . trained investigators collected the international prostate symptom score ( ipss ) , individual medical history , and demographic information for each participant . prostate disease was assessed by measuring serum prostate - specific antigen ( psa ) , a doctor 's medical examination by interview , a digital rectal examination , and prostate volume by transrectal ultrasound ( sa6000ii , medison inc . among them , 668 subjects remained for study after the exclusion of 1 person who did not consent to participate in the study , 1 person who had a prostate cancer history , and 109 subjects who were missing the digital rectal examination , transrectal ultrasonography , or other epidemiologic inf ormation . among these subjects , data were analyzed from the 599 subjects finally participating in this study after the exclusion of subjects in whom a nodule was perceived or who had a serum psa score of 4.0 or higher and thus who might have prostate cancer ( fig . 1 ) the severity of luts was evaluated by the ipss and quality of life and the participants were divided into four groups by age ( 40 - 49 , 50 - 59 , 60 - 69 , and 70 years and above ) . in this study , bph was defined as a score of 8 or higher on the ipss and a prostate volume of 25 ml or above by transrectal ultrasonography , according to the criteria of the fourth edition of the textbook of urology published by the korean urological association . the prevalence rate of bph was compared by classifying the groups by age : 40 - 49 , 50 - 59 , 60 - 69 , and 70 years and above . in addition , the prevalence rate of each luts was checked by classifying the total ipss in each group into a mild symptom group ( score of 1 - 7 ) , a moderate symptom group ( score of 8 - 19 ) , and a severe symptom group ( score of 20 - 35 ) . additionally , the analysis was conducted on the correlations between the epidemiologic factors ( age , marital status , education level , smoking status , alcohol consumption status , regular exercise , body mass index [ bmi ] ) , comorbidities ( hypertension , diabetes ) , and serum psa and bph . there were 45 subjects ( 7.5% ) in the aged 40 - 49 years group , 165 subjects ( 27.5% ) in the aged 50 - 59 years group , 200 subjects ( 33.4% ) in the aged 60 - 69 years group , and 189 subjects ( 31.5% ) in the aged 70 years and above group . the prostate volume of the entire group of participants was 30.2510.14 g , and the average psa level was 1.200.79 ng / ml . in the age groups , the ipss was 2.223.76 in men aged 40 - 49 years , 3.505.06 in those aged 50 - 59 , 6.756.94 in those aged 60 - 69 , and 8.167.28 in men aged 70 or more . these changes with age were statistically significant ( p<0.001 ) . also , prostate volume was 25.365.22 g in men aged 40 - 49 years , 30.299.12 g in men aged 50 - 59 years , 30.549.7 g in men aged 60 - 69 years , and 31.0711.91 g in men aged 70 years and above , and these age - related increases were statistically significant ( p=0.008 ) . a total of 120 subjects ( 20.0% ) in the entire group of participants met the standard of bph as defined in this study . when we examined the prevalence rate according to age , we found that the prevalence rate increased with age . there were 2 subjects ( 4.4% ) with bph in the and aged 40 - 49 years group , 18 subjects ( 10.9% ) in the aged 50 - 59 years group , 44 subjects ( 22% ) in the aged 60 - 69 years group , and 56 subjects ( 26.6% ) in the aged 70 years and above group , and this increase with age was statistically significant ( p<0.001 ) ( table 1 ) . a total of 120 participants ( 20% ) met the definition of bph in this study . the average age of the bph group was 68.238.52 years , the average ipss was 14.675.95 , the average prostate volume was 37.0411.71 g , and the average psa level was 1.560.88 ng / ml ( table 2 ) . in the subscores of the ipss , the score for weak urinary stream was highest ( 3.372.08 ) ; the scores for incomplete emptying ( 2.432.18 ) , hesitancy ( 2.192.15 ) , and nocturia ( 2.051.31 ) were also high . the quality of life score was significantly higher in the bph group ( 3.480.93 ) than in the non - bph group ( 2.500.91 ) . in this study , we conducted a correlation analysis to determine whether epidemiologic factors ( marital status , education level , smoking status , alcohol consumption status , bmi , and regular exercise ) and comorbidities ( hypertension and diabetes ) influenced the risk of bph . the non - bph group had a higher proportion of smokers than did the bph group ( table 3 ) . the reason the epidemiologic factors including lifestyle did not show a significant correlation may be that this study was based on a cross - sectional design that did not clearly show a sequential relationship by time . in the case of the bph group , the patients ' uncomfortable urinary status could have already influenced changes in health behavior . there were 45 subjects ( 7.5% ) in the aged 40 - 49 years group , 165 subjects ( 27.5% ) in the aged 50 - 59 years group , 200 subjects ( 33.4% ) in the aged 60 - 69 years group , and 189 subjects ( 31.5% ) in the aged 70 years and above group . the prostate volume of the entire group of participants was 30.2510.14 g , and the average psa level was 1.200.79 ng / ml . in the age groups , the ipss was 2.223.76 in men aged 40 - 49 years , 3.505.06 in those aged 50 - 59 , 6.756.94 in those aged 60 - 69 , and 8.167.28 in men aged 70 or more . these changes with age were statistically significant ( p<0.001 ) . also , prostate volume was 25.365.22 g in men aged 40 - 49 years , 30.299.12 g in men aged 50 - 59 years , 30.549.7 g in men aged 60 - 69 years , and 31.0711.91 g in men aged 70 years and above , and these age - related increases were statistically significant ( p=0.008 ) . a total of 120 subjects ( 20.0% ) in the entire group of participants met the standard of bph as defined in this study . when we examined the prevalence rate according to age , we found that the prevalence rate increased with age . there were 2 subjects ( 4.4% ) with bph in the and aged 40 - 49 years group , 18 subjects ( 10.9% ) in the aged 50 - 59 years group , 44 subjects ( 22% ) in the aged 60 - 69 years group , and 56 subjects ( 26.6% ) in the aged 70 years and above group , and this increase with age was statistically significant ( p<0.001 ) ( table 1 ) . a total of 120 participants ( 20% ) met the definition of bph in this study . the average age of the bph group was 68.238.52 years , the average ipss was 14.675.95 , the average prostate volume was 37.0411.71 g , and the average psa level was 1.560.88 ng / ml ( table 2 ) . in the subscores of the ipss , the score for weak urinary stream was highest ( 3.372.08 ) ; the scores for incomplete emptying ( 2.432.18 ) , hesitancy ( 2.192.15 ) , and nocturia ( 2.051.31 ) were also high . in the survey questions about quality of life , the overall inconvenience of the patients due to their current urinary symptoms was used for the score ; the scores were divided from 0 as very satisfied to 6 as very dissatisfied . the quality of life score was significantly higher in the bph group ( 3.480.93 ) than in the non - bph group ( 2.500.91 ) . in this study , we conducted a correlation analysis to determine whether epidemiologic factors ( marital status , education level , smoking status , alcohol consumption status , bmi , and regular exercise ) and comorbidities ( hypertension and diabetes ) influenced the risk of bph . the non - bph group had a higher proportion of smokers than did the bph group ( table 3 ) . the reason the epidemiologic factors including lifestyle did not show a significant correlation may be that this study was based on a cross - sectional design that did not clearly show a sequential relationship by time . in the case of the bph group , bph generally occurs in males in their 50s ; 80% of males in their 70s suffer from the luts related to bph . additionally , the incidence of metabolic syndrome , which is a risk factor for bph , and the number of patients with obesity are also annually increasing owing to westernized eating habits . in this situation , it is necessary to conduct systematic epidemiologic investigations of the prevalence rate of bph in korea . to investigate the prevalence rate of bph , it is f irst necessary to clarify the diagnostic criteria of bph . although methods exists for the diagnosis of bph , including taking the patient 's medical history and recording urinary symptoms , prostate volume measurement by digital rectal examination and transrectal ultrasonography , peak urinary flow rate ( qmax ) , and residual urine measurement , accurate diagnostic criteria have not been established . defined bph as urinary symptoms , prostate volume of 20 ml or more as measured by transrectal ultrasonography , and qmax of 15 ml / s or less . in their study , they reported that the prevalence rate of bph was 14% in males in their 40s and 40% in males in their 70s . defined bph as a prostate volume of 30 ml or more and ipss of 8 points or higher and reported a bph prevalence rate of 19% . defined bph as an ipss of 8 points or higher and a qmax of 10 ml / s or less . defined it as an ipss of 8 points or higher , prostate volume by digital rectal examination of 30 ml or more , and qmax of 15 ml / s or less . in studies investigating the prevalence rate in seongnam city and jeju island , bph was defined as an ipss of 8 points or higher and prostate volume measured by transrectal ultrasonography of 30 ml or more . in this study , bph diagnosis was based on the symptom score and the prostate volume , and urof lowmetry examination was excluded . reported that the bph prevalence rate defined by two variables including symptom score and prostate volume and the bph prevalence rate defined by three variables including symptom score , prostate volume , and qmax were 12% and 10% , respectively , and that there was no statistically significant difference between the two rates . measured prostate volume through transrectal ultrasonography by targeting 175 males whose ages were between 27 and 70 years ; the average prostate volume of adenoma prostate was significantly higher than that of nonadenoma prostate : 28.0 ml ( range , 14.5 - 62.1 ml ) vs. 23.0 ml ( range , 11.3 - 39.1 ml ) . therefore , those authors proposed criteria for bph as an ipss of 8 points or higher and a prostate volume by transrectal ultrasonography of 25 ml or more . the bph prevalence rate was 20% ( 120/599 subjects ) in the present study and lower than the rate in other reports in korea . in addition , the prevalence rates were 23.2% in the rural area of yeoncheon , gyeonggi - do , and 27.7% in the inland area of chungcheongbuk - do . first , there were variables in the epidemiologic investigation , such as the selection of the target group and regional differences in the population . the prevalence rate in seongnam was much higher than in other regions because the study was an epidemiologic investigation targeting the elderly population ( 65 years old and above ) . conducted an epidemiologic investigation for the first time in yeoncheon area in 1995 , they only conducted a survey of the ipss and then estimated the prevalence rate of bph . reported that the bph prevalence rate as assessed by luts , digital rectal examination , and uroflowmetry examination was 11% ; here , the question about the accuracy of measuring prostate size could be raised because the prostate volume was measured by digital rectal examination . in this study , the survey and the epidemiologic investigation were performed by interviewers who were sufficiently trained ; unlike other authors ' measurement of the prostate volume by digital rectal examination , it could be thought that the reliability would be high because the prostate volume was accurately measured by transrectal ultrasonography . even though age and genetics play an important role as causes of bph , it is notable that lifestyle factors such as metabolic syndrome or cardiovascular disease , which can be corrected , have a considerable impact on the natural progression of bph . additionally , other studies have shown that the risk of bph and luts is significantly increased in people with heart disease . in other previous studies , a correlation was observed in which the more the amount of fat increased , the more the prostate volume increased . in several research studies , weight , bmi , and waistline all showed a significant correlation with prostate volume . in this study , there was no significant difference in bmi between the bph group and the non - bph group . the reason for this may be that the cross - sectional design of this study did not clearly show the sequential relationship of time . in the longitudinal study of aging cohort in baltimore , maryland ( usa ) , each 1-kg / m increase in bmi corresponded to a 0.41-ml prostate volume increase . first , the subjects were not a random sample of the population because we recruited subjects through a prostate health screening campaign . second , we did not consider the difference in the population distribution by age in yangpyeong county and the study group . third , although prevalence was the number of cases of the condition at a particular point in time , the participants of this study were recruited over 3 years ( august - september 2009 , august 2010 , and august 2011 ) in an effort to collect more participants . consequently , the results of this epidemiologic research investigating the bph prevalence rate in a rural korean area showed a lower value than in other countries . one reason for this may be that the ratio of relatively younger males in their 40s to 50s was somewhat higher in this epidemiologic investigation than in other studies . in this study , the prevalence rate of bph was estimated by targeting males aged 40 years and older in a rural korean area . the results showed that the prevalence rate of bph in males aged 40 years and older in yangpyeong county was 20.0% . when classifying the men according to age , none of the epidemiologic factors studied including lifestyle showed a statistically significant correlation with bph , except for smoking . this study showed a lower prevalence rate of bph in a rural area than reported in other studies in korea . unlike other studies conducted in rural areas , however , the reliability of this study can be considered to be high because prostate volume was measured by use of a more objective method .

it can be accomplished by introduction of cell cycle molecules such as cyclin - dependent kinase 6 ( cdk6 ) and cyclin d1 , but their continuous overexpression raises oncogenic concerns . we attempted to mimic normal , transient , perinatal human -cell proliferation by delivering these molecules in a regulated and reversible manner . adult cadaveric islets were transduced with doxycycline ( dox)-inducible adenoviruses expressing cdk6 or cyclin d1 . end points were cdk6/cyclin d1 expression and human -cell proliferation , survival , and function . increasing doses of dox led to marked dose- and time - related increases in cdk6 and cyclin d1 , accompanied by a 20-fold increase in -cell proliferation . notably , dox withdrawal resulted in a reversal of both cdk6 and cyclin d1 expression as well as -cell proliferation . the adenoviral tetracycline ( tet)-on system has not been used previously to drive human -cell proliferation . human -cells can be induced to proliferate or arrest in a regulated , reversible manner , temporally and quantitatively mimicking the transient perinatal physiological proliferation that occurs in human -cells . human islets were obtained from the national institutes of health ( nih) and juvenile diabetes research foundation ( jdrf)supported integrated islet distribution program ( iidp ) and dr . tatsuya kin ( clinical islet laboratory , university of alberta , edmonton , alberta , canada ) . adenovirus preparation , immunoblots , glucose - stimulated insulin secretion , cdk and cyclin expression studies , and proliferation and survival studies were performed as described in detail previously ( 811,15,16 ) and in the figure legends . statistical differences were determined by two - tailed , unpaired student t test or by anova for repeated measures with post hoc analysis , as indicated in the figure legends . human islets were transduced with an adenovirus delivering the tet transactivator ( ad.tta ) and either a control adenovirus expressing green fluorescent protein ( ad.gfp ) or adenoviruses expressing cdk6 or cyclin d1 under the control of the tet response element ( ad.tre-cdk6 or ad.tre-cyclin d1 ) . increasing amounts of doxycycline ( dox ) ( 01 g / ml ) were added to the medium to define the dose responsiveness of the cdk-6 and cyclin d1 expression . figure 1 shows that cyclin d1 or cdk6 , or both , markedly increased with rising concentrations of dox , plateauing at 0.1 g / ml dox . close examination indicates that both cdk6 and cyclin d1 were expressed at low levels in the absence of dox . for example , control ( ad.gfp-transduced ) islets expressed 12.2% of the maximal amount of cdk6 expressed by ad.tre-cdk6 ( with 0.1 g / ml dox ) , whereas ad.tre-cdk6 without dox exposure expressed 23% of the maximum ; similarly , ad.gfp-transduced islets and ad.tre-cyclin d1 without dox expressed 2.7 and 13.9% of maximal cyclin d1 levels , respectively . this suggests some leakiness of the tre promoter , but below levels associated with activation of proliferation ( see below ) . dose - related , dox - induced expression of cdk6 or cyclin d1 from ad.tre in human islets . a and b : immunoblots of induced cdk6 or cyclin d1 expression , respectively , in intact human islets 72 h posttransduction with adenoviruses as indicated , and treated with or without increasing doses of dox . c and d : densitometric quantification of a and b ( n = 5 ) . p values refer to significance of indicated bars as compared with ad.gfp-ad.tta controls as determined by student unpaired t test . human islet donor information was as follows : mean age 43.7 2.1 years ; gender 16 men , 17 women ; bmi 27.5 0.9 ; purity 68.7 3.5% . islets were maintained in rpmi 1640 medium containing 5.5 mmol / l glucose , 10% tet - free fbs ( clontech , mountain view , ca ) , and 1% penicillin / streptomycin , unless otherwise stated . two types of adenoviruses were used in this tet - on system . in the first , the tta is driven by the cmv promoter . in the second , the cdnas encoding either human cyclin d1 or human cdk6 were controlled by the tre . the adenoviruses were generated using pjm17 ( 8,9 ) and a modified pac - cmvplpa ( 8,9 ) . in the ad.tre-cdk6 and ad.tre-cyclin d1 viruses , the cmv promoter in the pac - cmvplpa plasmid was replaced with the mfei - ecori promoter fragment of the ptre - shuttle2 from the tet - on system ( clontech ) , now referred to as pac - tre , and the cdk6-ha and ha - cyclin d1 cdnas were ligated into pac - tre . similarly , the cdna for the reverse tta from ptet - on advanced ( clontech ) was subcloned into paccmvplpa to produce the tta adenovirus . intact islets were transduced with adenovirus at a total of 250 multiplicities of infection ( moi ) ( 150 moi of ad.tre/100 moi of ad.tta ) in rpmi 1640 medium for 1 h. in some experiments , islets were dispersed by trypsinizing for 10 min at 37c . these single dispersed human islet cells were plated on 12-mm glass coverslips in 24-well plates and transduced at 150 moi ( 50 moi of each virus ) in complete media for 2 h. dox was added to the media immediately after transduction and replaced every other day as depicted . ( a high - quality color representation of this figure is available in the online issue . ) to determine the optimal time course for induction , human islets were transduced , placed in media containing 1 g / ml dox , and then harvested 24 , 48 , or 72 h later . 1 demonstrates that both cell cycle proteins increase rapidly over 72 h. there was no further increase after 72 h ( not shown ) . using laser confocal microscopy on isolated dispersed islets on coverslips , we examined brdu incorporation into insulin cells . in fig . 2a , negative controls ( ad.tre-cdk6 and ad . re - cyclin d1 without dox , or ad.gfp with dox ) did not show adenovirus - derived expression of cdk6 or cyclin d1 ( no white hemagglutinin [ ha ] epitope tag staining ) . on the other hand , cdk6/cyclin d1 staining ( white ha tag ) importantly , it is also clear that the adenoviruses were expressed in -cells , because the white ha tag colocalizes in many green insulin cells . most importantly , brdu staining can be seen prominently in human -cells expressing cdk6/cyclin d1 . in the bottom right panels , the ha staining has been separated from the merged images to more clearly demonstrate the brdu staining in insulin cells . a : dispersed human islets were plated and transduced with the indicated adenoviruses ( in combination with ad.tta ) without dox in the media ( white border ) or with 1 g / ml dox ( pink border ) . after 3 days in culture , brdu was added to the medium and the cells were fixed 18 h later . the dispersed islets were immunostained and visualized using laser confocal microscopy for insulin , brdu , and ha ( which is indicative of either adenovirally derived cdk6 or cyclin d1 , both of which contain an ha epitope tag ) . b : quantification of immunohistochemistry in a ( n = 13 ) . the numbers under each bar refer to the numbers of -cells counted per condition . p values refer to significance of indicated bars as compared with their ad.gfp/ad.tta controls using student unpaired t test . brdu ( 1:1,000 dilution ) ( ge healthcare , pittsburgh , pa ) was added to the islet media 18 h before fixation in 4% paraformaldehyde in phosphate - buffered saline for 10 min at room temperature . ma ) , anti - insulin antibody 1:1,000 ( dako , carpinteria , ca ) , anti - cdk6 antibody 1:100 ( santa cruz , santa cruz , ca ) , anti - cyclin d1 antibody 1:100 , and anti - ha antibody 1:50 . staining was visualized using alexa fluor secondary antibodies ( invitrogen , carlsbad , ca ) on an olympus fluoview 1000 confocal microscope . cell death was assessed by the deadend fluorometric tunel system ( promega , san luis obispo , ca ) according to manufacturer s instructions . for the positive controls , cells were exposed to 27 units dnase - i for 10 min at room temperature or , in the case of the cytokine treatment , 200 units / ml il-1 , 1,000 units / ml tnf- , and 1,000 units / ml ifn- for 24 h in serum - free medium ( 3 ) . only cells with brdu - stained nuclei that were completely surrounded by insulin staining were counted as brdu - positive -cells . ( a high - quality digital representation of this figure is available in the online issue . ) these experiments were repeated using 13 different human islet preparations and quantified , as shown in fig . 2b . as can be seen , although very few insulin cells were proliferating ( brdu ) when transduced either with the ad.gfp or with the ad.tta+ad.tre-cyclin d1/cdk6 in the absence of dox , transduction of -cells with ad.tre-cdk6/cyclin d1 in the presence of increasing amounts of dox led to marked , dose - related increases in -cell brdu incorporation , ranging from 2.5 up to 10% . thus , it is possible to activate adult human -cell proliferation by the introduction of dox in a dose - dependent manner . importantly , glucose - stimulated insulin secretion was not adversely affected by induction of proliferation , and insulin and pdx1 mrna expression also were not diminished ( supplementary fig . transferase - mediated dutp nick - end labeling ( tunel ) staining of transduced dispersed islets performed after 3 days of cdk6/cyclin d1 induction revealed no increase in the rare tunel - positive cells ( supplementary fig . 3a and b ) . cleaved caspase-9 , a product of the intrinsic apoptotic pathway , was not significantly elevated in islets overexpressing cdk6 and cyclin d1 . similarly , cleaved caspase-3 , activated in both the extrinsic and intrinsic pathways , independently confirmed that there was no increase in cell death , despite activation of cell cycle proliferation by cdk6/cyclin d1 . interleukin ( il)-1 treatment ( 100 units / ml ) had no effect on tunel immunostaining in ad.cdk6/cyclin d1treated islets in the presence of dox ( mean sem , 0.3 0.31% of insulin - positive cells ) vs. controls ( ad.gfp 1.4 0.8% , or ad.cdk6/cyclin d1 islets with no dox 0.95 0.94 ; p = ns , one - way anova ) . similarly , cell death as assessed using cleaved caspase-3 and -9 did not increase in response to il-1 in cdk6/cyclin d1expressing islets ( not shown ) . to mimic normal developmental events , and to avoid oncogenic concerns , we explored whether , and how quickly , the induced cdk6/cyclin d1 might decline after dox withdrawal . six different human islet preparations were transduced with adenoviruses and incubated for 3 days in complete medium containing 1 g / ml dox , at which time they were washed with phosphate - buffered saline and tet - free medium was added . islets were then either harvested immediately ( referred to as day 0 ) or at day 7 . figure 3 shows that 1 ) in six human islet preps , cdk6 and cyclin d1 return to baseline levels observed in control - transduced human islets ; and 2 ) when administered in combination ( as well as when expressed individually as in supplementary fig . 4 further indicates , using an additional six human islet preparations , that 1 ) the decline in human islets is progressive over the course of 1 week , declining by 82 and 94% , respectively ; and 2 ) the decline in cdk6 and cyclin d1 occurs with comparable timing whether administered in combination ( as in fig . a and b : the reversal of cdk6 and cyclin d1 overexpression , with return to endogenous levels observed in control human islets by day 7 , as seen both by cdk6 and cyclin d1 immunoblots as well as by immunoblots of the ha epitope tags . c and d : quantified results of six separate experiments with human islets , demonstrating that after dox withdrawal , cdk6 and cyclin d1 expression are indistinguishable from that seen in control human islets . also , note that in these experiments , in contrast to supplementary fig . 4 in which cdk6 and cyclin d1 adenoviruses were administered separately , here both were administered simultaneously to each human islet preparation . the decline in both cdk6 and cyclin d1 was significant by one - way anova ( p < 0.017 for cdk6 and p values shown over the bars indicate differences between dox - treated groups on day 7 versus day 0 . there were no statistically significant differences between the groups on day 7 . ( a high - quality color representation of this figure is available in the online issue . ) we next queried whether it was possible to reverse the proliferative effect in human -cells by withdrawing dox . 2 , showing that expressing cdk6 and cyclin d1 in -cells for 3 days leads to an increase in brdu incorporation ( top row ) . it further demonstrates that this proliferation continues for at least 10 days in the presence of dox ( second row ) . significantly , 3 days of dox exposure followed by a 7-day washout completely abolished the proliferation ( brdu immunostaining ) in -cells ( third row ) . proliferation in -cells promptly resumed when dox was reintroduced ( bottom row ) . in fig . 4b , the number of brdu and insulin cells over time is quantified , confirming that 3 days of dox induces robust proliferation , which disappears within 7 days of dox withdrawal , and can be reinduced to the full prior extent by re - exposure to dox for 3 days . reversibility of proliferation in human islets transduced with cdk6 and cyclin d1 adenovirus after dox removal . a : confocal microscopy of human islets ( nine different preparations ) dispersed to single cells , plated on coverslips , and transduced with the indicated adenoviruses without dox ( white boxes ) in the media or with 1 g / ml dox ( pink boxes ) for 3 ( top row ) , 10 ( second and third rows ) , or 13 days ( bottom row ) . islet cells were incubated with dox for 3 days followed by a 7-day washout period ( third row ) and then re - exposure to dox for an additional 3 days ( bottom row ) . the dispersed islets were immunostained for insulin ( green ) , brdu ( red ) , and adenovirally delivered cdk6 or cyclin d1 ( indicated by the white ha epitope tag ) . b : quantification of immunohistochemistry in a. the numbers under each bar refer to the numbers of -cells counted per condition . comparisons were made using unpaired student t test ( * p < 0.05 ) or by one - way anova with tukey post hoc analysis ( * * p < 0.05 ) . ( a high - quality digital representation of this figure is available in the online issue . ) human islets were transduced with an adenovirus delivering the tet transactivator ( ad.tta ) and either a control adenovirus expressing green fluorescent protein ( ad.gfp ) or adenoviruses expressing cdk6 or cyclin d1 under the control of the tet response element ( ad.tre-cdk6 or ad.tre-cyclin d1 ) . increasing amounts of doxycycline ( dox ) ( 01 g / ml ) were added to the medium to define the dose responsiveness of the cdk-6 and cyclin d1 expression . figure 1 shows that cyclin d1 or cdk6 , or both , markedly increased with rising concentrations of dox , plateauing at 0.1 g / ml dox . close examination indicates that both cdk6 and cyclin d1 were expressed at low levels in the absence of dox . for example , control ( ad.gfp-transduced ) islets expressed 12.2% of the maximal amount of cdk6 expressed by ad.tre-cdk6 ( with 0.1 g / ml dox ) , whereas ad.tre-cdk6 without dox exposure expressed 23% of the maximum ; similarly , ad.gfp-transduced islets and ad.tre-cyclin d1 without dox expressed 2.7 and 13.9% of maximal cyclin d1 levels , respectively . this suggests some leakiness of the tre promoter , but below levels associated with activation of proliferation ( see below ) . dose - related , dox - induced expression of cdk6 or cyclin d1 from ad.tre in human islets . a and b : immunoblots of induced cdk6 or cyclin d1 expression , respectively , in intact human islets 72 h posttransduction with adenoviruses as indicated , and treated with or without increasing doses of dox . c and d : densitometric quantification of a and b ( n = 5 ) . p values refer to significance of indicated bars as compared with ad.gfp-ad.tta controls as determined by student unpaired t test . human islet donor information was as follows : mean age 43.7 2.1 years ; gender 16 men , 17 women ; bmi 27.5 0.9 ; purity 68.7 3.5% . islets were maintained in rpmi 1640 medium containing 5.5 mmol / l glucose , 10% tet - free fbs ( clontech , mountain view , ca ) , and 1% penicillin / streptomycin , unless otherwise stated . two types of adenoviruses were used in this tet - on system . in the first , the tta is driven by the cmv promoter . in the second , the cdnas encoding either human cyclin d1 or human cdk6 were controlled by the tre . the adenoviruses were generated using pjm17 ( 8,9 ) and a modified pac - cmvplpa ( 8,9 ) . in the ad.tre-cdk6 and ad.tre-cyclin d1 viruses , the cmv promoter in the pac - cmvplpa plasmid was replaced with the mfei - ecori promoter fragment of the ptre - shuttle2 from the tet - on system ( clontech ) , now referred to as pac - tre , and the cdk6-ha and ha - cyclin d1 cdnas were ligated into pac - tre . similarly , the cdna for the reverse tta from ptet - on advanced ( clontech ) was subcloned into paccmvplpa to produce the tta adenovirus . intact islets were transduced with adenovirus at a total of 250 multiplicities of infection ( moi ) ( 150 moi of ad.tre/100 moi of ad.tta ) in rpmi 1640 medium for 1 h. in some experiments , islets were dispersed by trypsinizing for 10 min at 37c . these single dispersed human islet cells were plated on 12-mm glass coverslips in 24-well plates and transduced at 150 moi ( 50 moi of each virus ) in complete media for 2 h. dox was added to the media immediately after transduction and replaced every other day as depicted . ( a high - quality color representation of this figure is available in the online issue . ) to determine the optimal time course for induction , human islets were transduced , placed in media containing 1 g / ml dox , and then harvested 24 , 48 , or 72 h later . 1 demonstrates that both cell cycle proteins increase rapidly over 72 h. there was no further increase after 72 h ( not shown ) . using laser confocal microscopy on isolated dispersed islets on coverslips , we examined brdu incorporation into insulin cells . in fig . 2a , negative controls ( ad.tre-cdk6 and ad . re - cyclin d1 without dox , or ad.gfp with dox ) did not show adenovirus - derived expression of cdk6 or cyclin d1 ( no white hemagglutinin [ ha ] epitope tag staining ) . on the other hand , cdk6/cyclin d1 staining ( white ha tag ) importantly , it is also clear that the adenoviruses were expressed in -cells , because the white ha tag colocalizes in many green insulin cells . most importantly , brdu staining can be seen prominently in human -cells expressing cdk6/cyclin d1 . in the bottom right panels , the ha staining has been separated from the merged images to more clearly demonstrate the brdu staining in insulin cells . a : dispersed human islets were plated and transduced with the indicated adenoviruses ( in combination with ad.tta ) without dox in the media ( white border ) or with 1 g / ml dox ( pink border ) . after 3 days in culture , brdu was added to the medium and the cells were fixed 18 h later . the dispersed islets were immunostained and visualized using laser confocal microscopy for insulin , brdu , and ha ( which is indicative of either adenovirally derived cdk6 or cyclin d1 , both of which contain an ha epitope tag ) . p values refer to significance of indicated bars as compared with their ad.gfp/ad.tta controls using student unpaired t test . brdu ( 1:1,000 dilution ) ( ge healthcare , pittsburgh , pa ) was added to the islet media 18 h before fixation in 4% paraformaldehyde in phosphate - buffered saline for 10 min at room temperature . cells were immunostained with anti - brdu antibody 1:500 ( abcam , cambridge , ma ) , anti - insulin antibody 1:1,000 ( dako , carpinteria , ca ) , anti - cdk6 antibody 1:100 ( santa cruz , santa cruz , ca ) , anti - cyclin d1 antibody 1:100 , and anti - ha antibody 1:50 . staining was visualized using alexa fluor secondary antibodies ( invitrogen , carlsbad , ca ) on an olympus fluoview 1000 confocal microscope . cell death was assessed by the deadend fluorometric tunel system ( promega , san luis obispo , ca ) according to manufacturer s instructions . for the positive controls , cells were exposed to 27 units dnase - i for 10 min at room temperature or , in the case of the cytokine treatment , 200 units / ml il-1 , 1,000 units / ml tnf- , and 1,000 units / ml ifn- for 24 h in serum - free medium ( 3 ) . only cells with brdu - stained nuclei that were completely surrounded by insulin staining were counted as brdu - positive -cells . ( a high - quality digital representation of this figure is available in the online issue . ) these experiments were repeated using 13 different human islet preparations and quantified , as shown in fig . 2b . as can be seen , although very few insulin cells were proliferating ( brdu ) when transduced either with the ad.gfp or with the ad.tta+ad.tre-cyclin d1/cdk6 in the absence of dox , transduction of -cells with ad.tre-cdk6/cyclin d1 in the presence of increasing amounts of dox led to marked , dose - related increases in -cell brdu incorporation , ranging from 2.5 up to 10% . thus , it is possible to activate adult human -cell proliferation by the introduction of dox in a dose - dependent manner . importantly , glucose - stimulated insulin secretion was not adversely affected by induction of proliferation , and insulin and pdx1 mrna expression also were not diminished ( supplementary fig . transferase - mediated dutp nick - end labeling ( tunel ) staining of transduced dispersed islets performed after 3 days of cdk6/cyclin d1 induction revealed no increase in the rare tunel - positive cells ( supplementary fig . 3a and b ) . cleaved caspase-9 , a product of the intrinsic apoptotic pathway , was not significantly elevated in islets overexpressing cdk6 and cyclin d1 . similarly , cleaved caspase-3 , activated in both the extrinsic and intrinsic pathways , independently confirmed that there was no increase in cell death , despite activation of cell cycle proliferation by cdk6/cyclin d1 . interleukin ( il)-1 treatment ( 100 units / ml ) had no effect on tunel immunostaining in ad.cdk6/cyclin d1treated islets in the presence of dox ( mean sem , 0.3 0.31% of insulin - positive cells ) vs. controls ( ad.gfp 1.4 0.8% , or ad.cdk6/cyclin d1 islets with no dox 0.95 0.94 ; p = ns , one - way anova ) . similarly , cell death as assessed using cleaved caspase-3 and -9 did not increase in response to il-1 in cdk6/cyclin d1expressing islets ( not shown ) . to mimic normal developmental events , and to avoid oncogenic concerns , we explored whether , and how quickly , the induced cdk6/cyclin d1 might decline after dox withdrawal . six different human islet preparations were transduced with adenoviruses and incubated for 3 days in complete medium containing 1 g / ml dox , at which time they were washed with phosphate - buffered saline and tet - free medium was added . islets were then either harvested immediately ( referred to as day 0 ) or at day 7 . figure 3 shows that 1 ) in six human islet preps , cdk6 and cyclin d1 return to baseline levels observed in control - transduced human islets ; and 2 ) when administered in combination ( as well as when expressed individually as in supplementary fig . 4 further indicates , using an additional six human islet preparations , that 1 ) the decline in human islets is progressive over the course of 1 week , declining by 82 and 94% , respectively ; and 2 ) the decline in cdk6 and cyclin d1 occurs with comparable timing whether administered in combination ( as in fig . a and b : the reversal of cdk6 and cyclin d1 overexpression , with return to endogenous levels observed in control human islets by day 7 , as seen both by cdk6 and cyclin d1 immunoblots as well as by immunoblots of the ha epitope tags . c and d : quantified results of six separate experiments with human islets , demonstrating that after dox withdrawal , cdk6 and cyclin d1 expression are indistinguishable from that seen in control human islets . also , note that in these experiments , in contrast to supplementary fig . 4 in which cdk6 and cyclin d1 adenoviruses were administered separately , here both were administered simultaneously to each human islet preparation . the decline in both cdk6 and cyclin d1 was significant by one - way anova ( p < 0.017 for cdk6 and p values shown over the bars indicate differences between dox - treated groups on day 7 versus day 0 . there were no statistically significant differences between the groups on day 7 . ( a high - quality color representation of this figure is available in the online issue . ) we next queried whether it was possible to reverse the proliferative effect in human -cells by withdrawing dox . 2 , showing that expressing cdk6 and cyclin d1 in -cells for 3 days leads to an increase in brdu incorporation ( top row ) . it further demonstrates that this proliferation continues for at least 10 days in the presence of dox ( second row ) . significantly , 3 days of dox exposure followed by a 7-day washout completely abolished the proliferation ( brdu immunostaining ) in -cells ( third row ) . proliferation in -cells promptly resumed when dox was reintroduced ( bottom row ) . in fig . 4b , the number of brdu and insulin cells over time is quantified , confirming that 3 days of dox induces robust proliferation , which disappears within 7 days of dox withdrawal , and can be reinduced to the full prior extent by re - exposure to dox for 3 days . reversibility of proliferation in human islets transduced with cdk6 and cyclin d1 adenovirus after dox removal . a : confocal microscopy of human islets ( nine different preparations ) dispersed to single cells , plated on coverslips , and transduced with the indicated adenoviruses without dox ( white boxes ) in the media or with 1 g / ml dox ( pink boxes ) for 3 ( top row ) , 10 ( second and third rows ) , or 13 days ( bottom row ) . islet cells were incubated with dox for 3 days followed by a 7-day washout period ( third row ) and then re - exposure to dox for an additional 3 days ( bottom row ) . the dispersed islets were immunostained for insulin ( green ) , brdu ( red ) , and adenovirally delivered cdk6 or cyclin d1 ( indicated by the white ha epitope tag ) . b : quantification of immunohistochemistry in a. the numbers under each bar refer to the numbers of -cells counted per condition . comparisons were made using unpaired student t test ( * p < 0.05 ) or by one - way anova with tukey post hoc analysis ( * * p < 0.05 ) . ( a high - quality digital representation of this figure is available in the online issue . ) first , they sought to determine whether it is possible to reversibly activate , inactivate , and reactivate human -cell replication . and second , they sought to do this in a way that mimics the temporal sequence of human fetal -cell replication , with proliferation being activated for a period of a few weeks or months , with the -cells then allowed to return to quiescence . the results make the novel points that it is indeed possible to inducibly and transiently activate adult human -cell replication , to temporally mimic normal proliferative events in human fetal -cells , and to do so in a way that might navigate away from oncogenic concerns , while also retaining -cell function and survival . we have shown that several members of the g1/s family of molecules , such as cdk6 and cyclin d1 , are able to effectively drive adult human -cell cycle progression ( 811 ) . notably , we have also shown that when transplanted into immunodeficient diabetic mice , cdk6- and cyclin d1overexpressing human -cells continue to proliferate for 68 weeks , and do so in a way that actually improves -cell function / glycemic control ( 9,10 ) . although these prior studies are promising , they are also a cause for worry , because they used adenoviral transduction using the constitutive cmv promoter , which leads to sustained expression . this raises the realistic concern that sustained overexpression of cdks and cyclins , many of which are well - established oncogenes in other contexts , might lead to tumor formation in transplanted islets exposed to these agents . the first current goal , developing a means of reversibly inducing , then reversing , then reactivating brisk human -cell replication , was achieved . these findings make the point that it is indeed possible to reversibly activate as well as arrest human -cell replication , events that provide hope that strategies to allay oncogenic concerns associated with sustained cell cycle activation may be feasible . the second goal , mimicking the transient proliferation that occurs in normal human development , was also achieved . dox was able to activate , its withdrawal inactivate , and its reintroduction reactivate adult human -cell replication . the time span we used ( 3 , 7 , and 10 days ) was briefer than that which occurs in normal development ( weeks to months ) , but further extending proliferation with longer exposure to dox is clearly possible , because we have already demonstrated that prolonged ( 2 months ) induction of human -cell replication is possible using these approaches ( 9,10,15 ) . the proliferation rates herein were comparable to rates achieved by constitutive cmv promoter adenoviruses in prior studies ( 9,10 ) . significantly , we were also able to achieve rates of proliferation that are comparable to , or exceed , those observed in developing human pancreas ( 1214 ) . these observations support the notion that induction of human -cell replication at the 5% range for 23 months may be sufficient to expand endogenous and ex vivo sources of human -cells . one is that they use gene therapy techniques , and as such are unlikely to be directly acceptable for human diabetes therapy . hence , they serve as proof - of - principle studies , demonstrating that therapeutic , reversible human -cell expansion is possible , and provide additional impetus and targets for searches for small molecules , growth factors , nutrients , and signaling pathways that will drive expansion of adult human -cells , whether they are derived from cadaveric or stem cell sources . however , because those experiments are expensive and time consuming , and because we already have shown in two prior studies that transplant of human islets expressing cdk6 and cyclin d1 function superiorly as compared with control human islets ( 9,10 ) , the incremental knowledge gained through such experiments would be marginal . in summary , these studies demonstrate that it is technically feasible to induce robust adult human -cell replication in a regulated manner and to reverse this replication , attenuating oncogenic concerns and mimicking the transient -cell proliferation that normally occurs in the developing human pancreas . these studies document that with the expanding list of -cell cycle targets , and small molecule screens that are in progress , regulated proliferation of adult human -cells is a feasible goal .

induction of proliferation in adult human -cells is challenging . it can be accomplished by introduction of cell cycle molecules such as cyclin - dependent kinase 6 ( cdk6 ) and cyclin d1 , but their continuous overexpression raises oncogenic concerns . we attempted to mimic normal , transient , perinatal human -cell proliferation by delivering these molecules in a regulated and reversible manner . adult cadaveric islets were transduced with doxycycline ( dox)-inducible adenoviruses expressing cdk6 or cyclin d1 . end points were cdk6/cyclin d1 expression and human -cell proliferation , survival , and function . increasing doses of dox led to marked dose- and time - related increases in cdk6 and cyclin d1 , accompanied by a 20-fold increase in -cell proliferation . notably , dox withdrawal resulted in a reversal of both cdk6 and cyclin d1 expression as well as -cell proliferation . re - exposure to dox reinduced both cdk / cyclin expression and proliferation . -cell function and survival were not adversely affected . the adenoviral tetracycline ( tet)-on system has not been used previously to drive human -cell proliferation . human -cells can be induced to proliferate or arrest in a regulated , reversible manner , temporally and quantitatively mimicking the transient perinatal physiological proliferation that occurs in human -cells .

None RESEARCH DESIGN AND METHODS RESULTS Cdk6 and cyclin D Induction of human -cell proliferation by Dox. Expression of cdk6 and cyclin D Removal of Dox results in normalization of cdk6/cyclin D DISCUSSION

it can be accomplished by introduction of cell cycle molecules such as cyclin - dependent kinase 6 ( cdk6 ) and cyclin d1 , but their continuous overexpression raises oncogenic concerns . we attempted to mimic normal , transient , perinatal human -cell proliferation by delivering these molecules in a regulated and reversible manner . adult cadaveric islets were transduced with doxycycline ( dox)-inducible adenoviruses expressing cdk6 or cyclin d1 . end points were cdk6/cyclin d1 expression and human -cell proliferation , survival , and function . increasing doses of dox led to marked dose- and time - related increases in cdk6 and cyclin d1 , accompanied by a 20-fold increase in -cell proliferation . notably , dox withdrawal resulted in a reversal of both cdk6 and cyclin d1 expression as well as -cell proliferation . the adenoviral tetracycline ( tet)-on system has not been used previously to drive human -cell proliferation . human -cells can be induced to proliferate or arrest in a regulated , reversible manner , temporally and quantitatively mimicking the transient perinatal physiological proliferation that occurs in human -cells . adenovirus preparation , immunoblots , glucose - stimulated insulin secretion , cdk and cyclin expression studies , and proliferation and survival studies were performed as described in detail previously ( 811,15,16 ) and in the figure legends . human islets were transduced with an adenovirus delivering the tet transactivator ( ad.tta ) and either a control adenovirus expressing green fluorescent protein ( ad.gfp ) or adenoviruses expressing cdk6 or cyclin d1 under the control of the tet response element ( ad.tre-cdk6 or ad.tre-cyclin d1 ) . increasing amounts of doxycycline ( dox ) ( 01 g / ml ) were added to the medium to define the dose responsiveness of the cdk-6 and cyclin d1 expression . figure 1 shows that cyclin d1 or cdk6 , or both , markedly increased with rising concentrations of dox , plateauing at 0.1 g / ml dox . close examination indicates that both cdk6 and cyclin d1 were expressed at low levels in the absence of dox . dose - related , dox - induced expression of cdk6 or cyclin d1 from ad.tre in human islets . a and b : immunoblots of induced cdk6 or cyclin d1 expression , respectively , in intact human islets 72 h posttransduction with adenoviruses as indicated , and treated with or without increasing doses of dox . in the ad.tre-cdk6 and ad.tre-cyclin d1 viruses , the cmv promoter in the pac - cmvplpa plasmid was replaced with the mfei - ecori promoter fragment of the ptre - shuttle2 from the tet - on system ( clontech ) , now referred to as pac - tre , and the cdk6-ha and ha - cyclin d1 cdnas were ligated into pac - tre . intact islets were transduced with adenovirus at a total of 250 multiplicities of infection ( moi ) ( 150 moi of ad.tre/100 moi of ad.tta ) in rpmi 1640 medium for 1 h. in some experiments , islets were dispersed by trypsinizing for 10 min at 37c . to determine the optimal time course for induction , human islets were transduced , placed in media containing 1 g / ml dox , and then harvested 24 , 48 , or 72 h later . re - cyclin d1 without dox , or ad.gfp with dox ) did not show adenovirus - derived expression of cdk6 or cyclin d1 ( no white hemagglutinin [ ha ] epitope tag staining ) . most importantly , brdu staining can be seen prominently in human -cells expressing cdk6/cyclin d1 . the dispersed islets were immunostained and visualized using laser confocal microscopy for insulin , brdu , and ha ( which is indicative of either adenovirally derived cdk6 or cyclin d1 , both of which contain an ha epitope tag ) . ma ) , anti - insulin antibody 1:1,000 ( dako , carpinteria , ca ) , anti - cdk6 antibody 1:100 ( santa cruz , santa cruz , ca ) , anti - cyclin d1 antibody 1:100 , and anti - ha antibody 1:50 . as can be seen , although very few insulin cells were proliferating ( brdu ) when transduced either with the ad.gfp or with the ad.tta+ad.tre-cyclin d1/cdk6 in the absence of dox , transduction of -cells with ad.tre-cdk6/cyclin d1 in the presence of increasing amounts of dox led to marked , dose - related increases in -cell brdu incorporation , ranging from 2.5 up to 10% . thus , it is possible to activate adult human -cell proliferation by the introduction of dox in a dose - dependent manner . importantly , glucose - stimulated insulin secretion was not adversely affected by induction of proliferation , and insulin and pdx1 mrna expression also were not diminished ( supplementary fig . transferase - mediated dutp nick - end labeling ( tunel ) staining of transduced dispersed islets performed after 3 days of cdk6/cyclin d1 induction revealed no increase in the rare tunel - positive cells ( supplementary fig . cleaved caspase-9 , a product of the intrinsic apoptotic pathway , was not significantly elevated in islets overexpressing cdk6 and cyclin d1 . similarly , cleaved caspase-3 , activated in both the extrinsic and intrinsic pathways , independently confirmed that there was no increase in cell death , despite activation of cell cycle proliferation by cdk6/cyclin d1 . to mimic normal developmental events , and to avoid oncogenic concerns , we explored whether , and how quickly , the induced cdk6/cyclin d1 might decline after dox withdrawal . figure 3 shows that 1 ) in six human islet preps , cdk6 and cyclin d1 return to baseline levels observed in control - transduced human islets ; and 2 ) when administered in combination ( as well as when expressed individually as in supplementary fig . 4 further indicates , using an additional six human islet preparations , that 1 ) the decline in human islets is progressive over the course of 1 week , declining by 82 and 94% , respectively ; and 2 ) the decline in cdk6 and cyclin d1 occurs with comparable timing whether administered in combination ( as in fig . a and b : the reversal of cdk6 and cyclin d1 overexpression , with return to endogenous levels observed in control human islets by day 7 , as seen both by cdk6 and cyclin d1 immunoblots as well as by immunoblots of the ha epitope tags . c and d : quantified results of six separate experiments with human islets , demonstrating that after dox withdrawal , cdk6 and cyclin d1 expression are indistinguishable from that seen in control human islets . 4 in which cdk6 and cyclin d1 adenoviruses were administered separately , here both were administered simultaneously to each human islet preparation . the decline in both cdk6 and cyclin d1 was significant by one - way anova ( p < 0.017 for cdk6 and p values shown over the bars indicate differences between dox - treated groups on day 7 versus day 0 . we next queried whether it was possible to reverse the proliferative effect in human -cells by withdrawing dox . 2 , showing that expressing cdk6 and cyclin d1 in -cells for 3 days leads to an increase in brdu incorporation ( top row ) . 4b , the number of brdu and insulin cells over time is quantified , confirming that 3 days of dox induces robust proliferation , which disappears within 7 days of dox withdrawal , and can be reinduced to the full prior extent by re - exposure to dox for 3 days . reversibility of proliferation in human islets transduced with cdk6 and cyclin d1 adenovirus after dox removal . a : confocal microscopy of human islets ( nine different preparations ) dispersed to single cells , plated on coverslips , and transduced with the indicated adenoviruses without dox ( white boxes ) in the media or with 1 g / ml dox ( pink boxes ) for 3 ( top row ) , 10 ( second and third rows ) , or 13 days ( bottom row ) . islet cells were incubated with dox for 3 days followed by a 7-day washout period ( third row ) and then re - exposure to dox for an additional 3 days ( bottom row ) . the dispersed islets were immunostained for insulin ( green ) , brdu ( red ) , and adenovirally delivered cdk6 or cyclin d1 ( indicated by the white ha epitope tag ) . human islets were transduced with an adenovirus delivering the tet transactivator ( ad.tta ) and either a control adenovirus expressing green fluorescent protein ( ad.gfp ) or adenoviruses expressing cdk6 or cyclin d1 under the control of the tet response element ( ad.tre-cdk6 or ad.tre-cyclin d1 ) . increasing amounts of doxycycline ( dox ) ( 01 g / ml ) were added to the medium to define the dose responsiveness of the cdk-6 and cyclin d1 expression . close examination indicates that both cdk6 and cyclin d1 were expressed at low levels in the absence of dox . dose - related , dox - induced expression of cdk6 or cyclin d1 from ad.tre in human islets . a and b : immunoblots of induced cdk6 or cyclin d1 expression , respectively , in intact human islets 72 h posttransduction with adenoviruses as indicated , and treated with or without increasing doses of dox . islets were maintained in rpmi 1640 medium containing 5.5 mmol / l glucose , 10% tet - free fbs ( clontech , mountain view , ca ) , and 1% penicillin / streptomycin , unless otherwise stated . in the ad.tre-cdk6 and ad.tre-cyclin d1 viruses , the cmv promoter in the pac - cmvplpa plasmid was replaced with the mfei - ecori promoter fragment of the ptre - shuttle2 from the tet - on system ( clontech ) , now referred to as pac - tre , and the cdk6-ha and ha - cyclin d1 cdnas were ligated into pac - tre . intact islets were transduced with adenovirus at a total of 250 multiplicities of infection ( moi ) ( 150 moi of ad.tre/100 moi of ad.tta ) in rpmi 1640 medium for 1 h. in some experiments , islets were dispersed by trypsinizing for 10 min at 37c . to determine the optimal time course for induction , human islets were transduced , placed in media containing 1 g / ml dox , and then harvested 24 , 48 , or 72 h later . re - cyclin d1 without dox , or ad.gfp with dox ) did not show adenovirus - derived expression of cdk6 or cyclin d1 ( no white hemagglutinin [ ha ] epitope tag staining ) . most importantly , brdu staining can be seen prominently in human -cells expressing cdk6/cyclin d1 . the dispersed islets were immunostained and visualized using laser confocal microscopy for insulin , brdu , and ha ( which is indicative of either adenovirally derived cdk6 or cyclin d1 , both of which contain an ha epitope tag ) . as can be seen , although very few insulin cells were proliferating ( brdu ) when transduced either with the ad.gfp or with the ad.tta+ad.tre-cyclin d1/cdk6 in the absence of dox , transduction of -cells with ad.tre-cdk6/cyclin d1 in the presence of increasing amounts of dox led to marked , dose - related increases in -cell brdu incorporation , ranging from 2.5 up to 10% . thus , it is possible to activate adult human -cell proliferation by the introduction of dox in a dose - dependent manner . importantly , glucose - stimulated insulin secretion was not adversely affected by induction of proliferation , and insulin and pdx1 mrna expression also were not diminished ( supplementary fig . cleaved caspase-9 , a product of the intrinsic apoptotic pathway , was not significantly elevated in islets overexpressing cdk6 and cyclin d1 . similarly , cleaved caspase-3 , activated in both the extrinsic and intrinsic pathways , independently confirmed that there was no increase in cell death , despite activation of cell cycle proliferation by cdk6/cyclin d1 . to mimic normal developmental events , and to avoid oncogenic concerns , we explored whether , and how quickly , the induced cdk6/cyclin d1 might decline after dox withdrawal . figure 3 shows that 1 ) in six human islet preps , cdk6 and cyclin d1 return to baseline levels observed in control - transduced human islets ; and 2 ) when administered in combination ( as well as when expressed individually as in supplementary fig . 4 further indicates , using an additional six human islet preparations , that 1 ) the decline in human islets is progressive over the course of 1 week , declining by 82 and 94% , respectively ; and 2 ) the decline in cdk6 and cyclin d1 occurs with comparable timing whether administered in combination ( as in fig . a and b : the reversal of cdk6 and cyclin d1 overexpression , with return to endogenous levels observed in control human islets by day 7 , as seen both by cdk6 and cyclin d1 immunoblots as well as by immunoblots of the ha epitope tags . c and d : quantified results of six separate experiments with human islets , demonstrating that after dox withdrawal , cdk6 and cyclin d1 expression are indistinguishable from that seen in control human islets . 4 in which cdk6 and cyclin d1 adenoviruses were administered separately , here both were administered simultaneously to each human islet preparation . the decline in both cdk6 and cyclin d1 was significant by one - way anova ( p < 0.017 for cdk6 and p values shown over the bars indicate differences between dox - treated groups on day 7 versus day 0 . 2 , showing that expressing cdk6 and cyclin d1 in -cells for 3 days leads to an increase in brdu incorporation ( top row ) . significantly , 3 days of dox exposure followed by a 7-day washout completely abolished the proliferation ( brdu immunostaining ) in -cells ( third row ) . 4b , the number of brdu and insulin cells over time is quantified , confirming that 3 days of dox induces robust proliferation , which disappears within 7 days of dox withdrawal , and can be reinduced to the full prior extent by re - exposure to dox for 3 days . reversibility of proliferation in human islets transduced with cdk6 and cyclin d1 adenovirus after dox removal . islet cells were incubated with dox for 3 days followed by a 7-day washout period ( third row ) and then re - exposure to dox for an additional 3 days ( bottom row ) . the dispersed islets were immunostained for insulin ( green ) , brdu ( red ) , and adenovirally delivered cdk6 or cyclin d1 ( indicated by the white ha epitope tag ) . first , they sought to determine whether it is possible to reversibly activate , inactivate , and reactivate human -cell replication . the results make the novel points that it is indeed possible to inducibly and transiently activate adult human -cell replication , to temporally mimic normal proliferative events in human fetal -cells , and to do so in a way that might navigate away from oncogenic concerns , while also retaining -cell function and survival . we have shown that several members of the g1/s family of molecules , such as cdk6 and cyclin d1 , are able to effectively drive adult human -cell cycle progression ( 811 ) . notably , we have also shown that when transplanted into immunodeficient diabetic mice , cdk6- and cyclin d1overexpressing human -cells continue to proliferate for 68 weeks , and do so in a way that actually improves -cell function / glycemic control ( 9,10 ) . these findings make the point that it is indeed possible to reversibly activate as well as arrest human -cell replication , events that provide hope that strategies to allay oncogenic concerns associated with sustained cell cycle activation may be feasible . the second goal , mimicking the transient proliferation that occurs in normal human development , was also achieved . dox was able to activate , its withdrawal inactivate , and its reintroduction reactivate adult human -cell replication . the time span we used ( 3 , 7 , and 10 days ) was briefer than that which occurs in normal development ( weeks to months ) , but further extending proliferation with longer exposure to dox is clearly possible , because we have already demonstrated that prolonged ( 2 months ) induction of human -cell replication is possible using these approaches ( 9,10,15 ) . significantly , we were also able to achieve rates of proliferation that are comparable to , or exceed , those observed in developing human pancreas ( 1214 ) . these observations support the notion that induction of human -cell replication at the 5% range for 23 months may be sufficient to expand endogenous and ex vivo sources of human -cells . hence , they serve as proof - of - principle studies , demonstrating that therapeutic , reversible human -cell expansion is possible , and provide additional impetus and targets for searches for small molecules , growth factors , nutrients , and signaling pathways that will drive expansion of adult human -cells , whether they are derived from cadaveric or stem cell sources . however , because those experiments are expensive and time consuming , and because we already have shown in two prior studies that transplant of human islets expressing cdk6 and cyclin d1 function superiorly as compared with control human islets ( 9,10 ) , the incremental knowledge gained through such experiments would be marginal . in summary , these studies demonstrate that it is technically feasible to induce robust adult human -cell replication in a regulated manner and to reverse this replication , attenuating oncogenic concerns and mimicking the transient -cell proliferation that normally occurs in the developing human pancreas . these studies document that with the expanding list of -cell cycle targets , and small molecule screens that are in progress , regulated proliferation of adult human -cells is a feasible goal .

resulting from its considerable role in the absorption of nutrients , mainly of short chain fatty acids ( scfas ) and of electrolytes [ 13 ] , the rumen epithelium ranks among the tissues with high metabolic rates [ 4 , 5 ] . a main proportion of the rumen atp utilization is related to activity of a na / k - atpase that has been shown to be expressed at high levels [ 68 ] in the cell membrane of rumen epithelial cells ( rec ) [ 9 , 10 ] . in addition , functional vacuolar - type h pumps ( vh - atpase ) are existent in rec [ 10 , 11 ] . the vh - atpase is well known as being present in intracellular membrane components such as endosomes , lysosomes , clathrin - coated vesicles , and the golgi complex [ 1215 ] . the pump - mediated acidification of such cell compartments is required for a variety of processes , including transcytosis of receptor - ligand complexes and other molecules , for example , nh3/nh4 , coupled transport of neurotransmitters and protein breakdown [ 16 , 17 ] . in addition , a link between electrogenic h secretion by vh - atpases localized on the cell membrane and ion transport and/or the regulation of cytosolic ph has been found in osteoclasts , macrophages , and various epithelia , for example , frog and toad skin , mammalian renal collecting duct , endolymphatic sac of the inner ear , and epididymis [ 2025 ] . the existence of the vh - atpase as an active transport mechanism in addition to the na / k - atpase implies a special functional role of the protein in the rumen . we have shown that the pump plays a considerable role in rec phi regulation being responsible for about 30% of total h release . moreover , indirect evidence for the involvement of vh - atpase in ruminal transport processes comes from experiments showing that mucosal nitrate , known to inhibit vh - atpase activity , reduced propionate and cl absorption markedly [ 26 , 27 ] . foliomycin , a specific vh - atpase blocker , has been found to inhibit the uptake of mg into rec . in our previous study , a variable subcellular distribution of vh - atpase in cell membranes and/or cytosolic pools of the more luminally oriented cell layers ( stratum spinosum , stratum granulosum ) of the rumen epithelium has been observed . we speculate that this flexible location could reflect reversible recycling of ruminal vh - atpase between the plasma membrane and a pool of cytoplasmic vesicles and/or dissociation of v1 catalytic complex from membrane - bound vo domains . in various epithelia and other cell types , such mechanisms are known to be involved in the regulation vh - atpase activity [ 1215 , 3032 ] . regulatory factors in ruminal vh - atpase recycling are unknown but for yeasts [ 3336 ] and renal epithelia ; metabolic control has been demonstrated . physiological signals that modulate vh - localization and activity include phi , hco3 , pco2 , and glucose [ 14 , 15 , 18 , 37 , 38 ] , all related to cell metabolism . the present study was designed to investigate a possible modulation of ruminal vh - atpase activity by substrate / energy availability . to do this , we used fluorescent spectroscopic phi measurements to study the effects of glucose removal and/or reduction of the cellular atp concentration ( [ atp ] ) on vh - atpase functional activity . in addition , western blot and immunocytochemistry are used to analyze if changes of vh - atpase expression and localization play a role in adaptation of the pump activity . medium 199 , trypsin , glutamine , antibiotics ( gentamycin , nystatin , kanamycin , penicillin - streptomycin ) , fetal calf serum ( fcs ) , and dulbecco 's phosphate - buffered saline ( dpbs ) were purchased from pan biotech ( aidenbach , germany ) . foliomycin , amiloride , antimycin a , and 2-deoxyglucose ( 2-dog ) were from sigma aldrich ( munich , germany ) . all chemicals for western blot analysis were purchased from carl roth ( karlsruhe , germany ) . the monoclonal mouse antibodies used in this study were specific for 60-kda subunit of the yeast vh - atpase ( 13d11-b2 , molecular probes ) and the subunit of the sheep na / k - atpase ( m7-pb - e9 , affinity bioreagents ) . both antibodies have been shown to detect the sheep proteins specifically [ 10 , 11 ] . relevant secondary antibodies conjugated to alexa fluor 488 ( invitrogen ) were used for immunocytochemistry . for western blotting , a horseradish - peroxidase ( hrp)-conjugated antibody ( ecl anti - mouse igg ) obtained from amersham bioscience was used . two pieces of rumen tissue , each about 100 cm , were taken from the atrium ruminis , washed at least three times in ice - cold phosphate - buffered saline ( pbs ) containing penicillin - streptomycin , and then transported to the laboratory in the same solution . there , rumen papillae were removed by scissors and washed three times in antibiotic - containing pbs and ones in antibiotic - free pbs . then , primary cultures of ruminal epithelial cells ( rec ) were prepared as described by galfi et al . . briefly , rec were isolated by fractional trypsination and those cell fractions containing mostly cells of the strata spinosum and basale were grown in medium 199 containing 15% fcs , 1.36 mm glutamine , 20 mm hepes , and antibiotics ( 50 mg / l gentamycin , 100 mg / l kanamycin , 2.4 10 u / l nystatin ) in an atmosphere of humidified air-5% co2 at 38c . from day 2 of culture , the medium was nystatin - free and contains 10% fcs only . control experiments were performed in hco3-free , hepes - buffered measuring solution ( in mm ) : 125 nacl , 5 kcl , 1 cacl2 , 2 mgcl2 , 5 glucose , 10 hepes , ph 7.1 . in experiments designed to reduce the energy metabolism of rec ( metabolic inhibition ) : ( 1 ) glucose was removed from the medium and antimycin a ( 5 m ) , a known inhibitor of complex iii of the mitochondrial respiratory chain , was added ; ( 2 ) glucose was replaced with 20 mm 2-dog to inhibit the glycolytic pathway ; ( 3 ) antimycin a ( 5 m ) and 2-dog ( 20 mm ) were added with concomitant glucose removal . the osmolarity of all solutions was adjusted to 280 mosmol / kg using d - mannit . all experiments were performed in the nominal absence of co2/hco3 , to suppress na - hco3 symporter related phi regulation and to enable comparability with results of our previous studies [ 10 , 11 ] . amiloride ( 250 m ) and foliomycin ( 2 m ) , known as specific inhibitors of na / h exchanger ( nhe ) and vh - atpase , respectively , were used to differentiate na- and pump dependent h - secretion . for the determination of phi , cells were loaded with 1 m bcecf - am for 30 minutes and subsequently washed twice in dpbs . rec were incubated for a further 30 minutes to allow complete de - esterification and washed twice before measurement of fluorescence . intracellular ph was detected by measuring the fluorescence of the probe - loaded rec in a spectrofluorometer ( ls-50 b , perkin - elmer ) equipped with a fast - filter accessory that allowed fluorescence to be measured at 20-ms intervals with excitation for bcecf at 440 and 480 nm and emission at 515 nm . all measurements were made at 37c in a 3-ml cuvette containing 2 ml cell suspension ( 10% cytocrit ) under stirring . bcecf signals were calibrated to ph by placing the cells in medium containing 135 mm kcl and the ionophore nigericin ( 10 m ) to equilibrate intra- and extracellular [ h ] . for data evaluation , 10-s data sets were each averaged at the beginning of the measurement and then after 50 , 100 , 150 , 200 , 250 , 300 , 350 , 400 , 450 , 500 , and 550 s. the final phi was determined as the mean phi of the last 10 s of the measurement . thus , for the calculation of any given phi value , 500 data points were used . rec ( 2 10 cells / ml ) were grown on sterile glass cover slips ( neolab , germany ) for 24 to 48 hours . thereafter , a metabolic inhibition ( 20 minutes ) was performed as described above . after being rinsed twice with pbs , if not otherwise stated , all the following steps were carried out at room temperature . after two pbs washes , cells were permeabilized in 0.25% triton x-100 for vh - atpase or in 100 m digitonin for na / k - atpase for 10 minutes and again rinsed three times with pbs . nonspecific binding of igg was suppressed by incubation of specimens with 7% goat serum in pbs for 20 minutes . subsequently , cells were rinsed with pbs ( three times for 5 minutes ) and then incubated overnight at 4c with the primary anti - vh - atpase antibody ( 13 g / ml ) or the primary anti - na / k - atpase antibody ( 15 g / ml ) solved in pbs with 1% bsa ; pbs - bsa . after being rinsed three times with pbs , cells were incubated for 2 hours with the secondary , alexa fluor-488-labeled goat anti - mouse igg1(1 ) antibody ( 1 : 200 in pbs - bsa ) . after three changes of pbs ( 5 minutes each ) , nuclei were counterstained with 300 nm of 4,6-diamidino-2-phenylindole ( dapi ) in s - buffer ( containing : 75 mm kcl , 3 mm mgso4 7h2o , 1 mm egta , 0.2 mm dithiothreitole , 10 mm imidazol , 1 g / ml aprotinin , 0.1 mm phenylmethane sulfonyl - fluoride ) . cover slips were then mounted with 30 l mounting medium ( dianova , hamburg , germany ) . digital images were acquired by using a fluorescence microscope olympus ix50 ( hamburg , germany ) and metamorph version 7.5.2.0 and autodeblur version 1.4.1 software ( visitron systems gmbh , puchheim , germany ) . for western blots , total protein from washed rec was extracted by use of the m - per mammalien protein extraction reagent ( pierce ) , complemented with halt protease inhibitor cocktail ( pierce ) . the protein concentration was determined by using the bradford assay ( bio - rad , munich , germany ) . protein samples ( 10115 g ) were separated by sds ( 12.5%)-polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene fluoride ( pvdf ) membrane ( ge healthcare ) . after transfer , membranes were blocked with 3% non - fat dry milk in pbs ( ph 7.5 ) containing 0.05% tween 20 ( pbs - t ) for 2 hours and washed two times for 5 minutes in pbs - t . thereafter , membranes were incubated at 4c with the primary antibodies ( anti - vh - atpase : 1 : 5,000 dilution ; anti na / k - atpase : 1 : 1,000 dilution ) overnight , washed three times ( 1 15 minutes , 2 5 minutes ) with pbs - t , and incubated for 1 hour with hrp - conjugated secondary antibodies ( 1 : 10,000 dilution ) . then , after three washings ( 1 10 minutes and 2 5 minutes ) in pbs - t , membranes were developed with ecl western blotting substrate ( pierce ) . its ph was measured directly after sampling with a glass electrode ( n 1042a , ph meter cg 841 , schott , mainz , germany ) . then , the rumen fluid was strained through 4 layers of gauze and prepared for scfa analysis . first , a mixture of 5 ml rumen fluid and 2 ml iso - capronic acid ( internal standard ) was centrifuged at 3000 g at 4c for 20 minutes . the filtered ( 0.22 m pore size ) supernatant was used to measure the scfa concentration by gas chromatography ( shimadzu gc-14a , shimadzu corporation , kyoto , japan ) on a capillary column ( free fatty acid phase , 25 m 0.25 mm , machery - nagel gmbh & co. kg , dren , germany ) according to the method of geissler et al . . samples of the rumen wall ( 1 cm surface ) were obtained from identical sites of the atrium ruminis and fixed in 4% neutral formaldehyde solution for morphometric investigations . after rinsing with water , the rumen wall tissues were dehydrated in a graded series of ethanol ( 30% , 50% , 70% , 90% , and absolute ethanol ) , cleared with benzene , saturated with and embedded in paraffin . at each sampling , sections of 5 m thickness were made of 30 papillae and stained with haematoxylin / eosin . the length and width of papillae were determined by the computer - operated image c picture analysis system ( intronic gmbh , berlin , germany ) and the imes analysis program , by using a color video camera ( sony 3 ccd ) and a light microscope ( axiolab , carl zeiss jena , germany ) . the number of papillae per cm mucosa was estimated by using a video camera equipped with a picture analysis system . according to hofmann and schnorr , the total surface of papillae per cm mucosa was determined as length width 2 , multiplied by the number of papillae / cm . if not otherwise stated , data are presented as means standard error ( se ) . significance was determined by student 's t - test or the paired t - test as appropriate . bcecf - loaded rec were suspended in either hepes - buffered na - medium ( control ) or hepes - buffered , glucose - free na - medium with 2-dog and/or antimycin a , and phi was measured over a 10-min period . the appropriate concentrations of 2-dog ( 20 mm ) and antimycin a ( 5 m ) were chosen from dose - response experiments ( figure 1 ) . characteristic original traces showing the time course of the rec phi for all four conditions are shown in figure 2(a ) . data on the phi reduction induced by either antimycin a or 2-dog alone or by a combination of both metabolic inhibitors are summarized in figure 2(b ) . the initial and final phi of control rec incubated in hepes - buffered nacl - medium were 7.2 0.1 and 7.3 0.1 , respectively . the presence of antimycin a and/or 2-dog in the media led to a reduction of phi ( figures 2(a ) and 2(b ) ) . in rec exposed to antimycin a , 2-dog or antimycin a and 2-dog , the phi measured at the beginning of the measuring period was reduced by 0.21 0.03 , 0.35 0.04 , and 0.44 0.04 ph units , respectively . with antimycin a or both inhibitors in the medium , this phi reduction was stable in most cases . however , if 2-dog only was present , the effect was slightly diminished and the observed phi decrease amounted to 0.30 0.03 ph units at the end of the measuring period . next , we investigated whether a decreased vh - atpase activity was responsible for the phi reducing effect of metabolic inhibition ( mi ) . therefore , bcecf - loaded rec were suspended in foliomycin ( 2 m)-containing hepes - buffered na - medium or glucose - free , hepes - buffered na - medium with antimycin a ( 5 m ) , 2-dog ( 20 mm ) , and foliomycin ( 2 m ) . control measurements were performed with cells handled in the same way , but without foliomycin present in the solutions . in comparison to the phi of control cells measured in hepes - buffered na - medium , foliomycin - treated rec showed a decreased phi ( figures 3(a ) and 3(b ) ) . the mean phi reduction observed in foliomycin - treated rec amounted to 0.21 0.05 ph units ( figure 3(b ) ) . as shown in figures 3(a ) and 3(b ) , the foliomycin effect was nearly abolished when rec were incubated in antimycin a- and 2-dog - containing glucose - free media . under such conditions , the phi difference between control ( hepes - buffered , glucose - free na - medium with antimycin a and 2-dog ) and foliomycin - treated rec amounted to 0.03 0.02 ph units only ( figure 3(b ) ) . these results point to a fast deactivation of vh - atpase activity after mi with antimycin a and 2-dog . regulatory mechanisms responsible for such adaptive response could be reversible disassembly of the vo and v1 subunits of the pump [ 30 , 33 ] and/or a translocation between the cell membrane and intracellular compartments . thus , in a next step we tested whether mi affect the distribution of vh - atpase and performed an immunofluorescence study using an antibody directed against the 60-kda subunit of the yeast vh - atpase which has been shown to detect vh - atpase in ovine rumen epithelium . as a control , the na / k - atpase which is constantly found in the cell membrane of rec has been investigated using an subunit - specific anti - na / k - atpase antibody . as expected , the subunit - specific anti - na / k - atpase antibody identified the protein in the cytoplasmic membrane of rec and its localization was not changed by a 20-min incubation in glucose - free , antimycin a- and/or 2-dog - containing media ( figure 4(a ) , a c ) . in comparison with the na / k - atpase , the vh - atpase distribution showed a much higher variability and dependence on the substrate availability . when incubated in glucose - containing standard medium , rec vh - atpase appeared in the cell membrane and/or perimembrane area as well as in the cytoplasma of rec ( figure 4(b ) , a ) . the cytosolic vh - atpase staining is clearly defined and either distributed throughout the cytoplasm or concentrated around the nucleus . after glucose deprivation and application of 2-dog ( figure 4(b ) , b ) , no membrane staining can be found and typically the vh - atpase staining is clustered in the perinuclear area of the cells . compared to control conditions , glucose deprivation and combined application of 2-dog and antimycin a ( figure 4(b ) , c ) induced appearance of diffuse , cytosolic vh - atpase staining . as in our previous investigation with ovine and bovine rec [ 10 , 11 ] , a more or less expressed compensatory phi increase was seen after application of foliomycin . in na - containing media , an nhe is known to be responsible for this effect as application of the nhe inhibitor amiloride reduced rec phi by 65% . therefore , we repeated the above described experiments to investigate the nhe activity before and after mi with 2-dog and antimycin a. experiments were performed as described for the first series , however , in addition to foliomycin ( 2 m ) , the nhe inhibitor amiloride ( 250 m ) was used alone or in combination with the vh - atpase blocker . mi induced a phi decrease of 0.10 0.03 ph units only ( figure 5 ) , which corresponds to a 75% reduction of the effect compared with the results of the first experiment . in addition , the mean inhibitory effect of foliomycin was strongly diminished ( 0.05 0.03 ph units ; figure 5 ) . figure 5 shows that rec responding to substrate deprivation and application of metabolic inhibitors were also foliomycin - sensitive . in such cells phi was reduced by 0.14 0.03 or 0.10 0.05 ph units after mi induction or exposure to foliomycin . in contrast , rec that did not respond to mi were foliomycin - insensitive ( figure 5 ) . in figure 6 , the inhibitory effects of foliomycin and/or amiloride before and after mi are summarized . only results from measurements with rec responding to metabolic inhibition are included and for comparison data from the first series of experiments are also shown . figure 6 shows that compared to series 1 ( a ) the foliomycin effect was strongly reduced ( 0.10 0.02 ph units versus 0.27 ph units ) and b ) the residual foliomycin effect ( 0.08 0.03 ph units ) was not influenced by mi . as in series 1 , foliomycin application led to a compensatory phi increase , an effect that was reduced after mi . a strong amiloride - sensitive component was observed . at the beginning and end of the measurement , the phi of amiloride - treated rec was reduced by 0.83 0.05 and 0.89 0.06 ph - units , respectively . the foliomycin- and amiloride - sensitive components were additive under control conditions and the observed phi decrease amounted to 0.93 0.05 at the start and to 0.95 0.08 at the end of the measuring period . mi led to a reduction of the amiloride - sensitive , nhe - related component . at the end of the measurement , we investigated the expression of vh - atpase b subunit and na / k - atpase subunit protein expression in rec protein extracts from sheep slaughtered during the first or second series of experiments . characteristic examples of immunoblots are given in figure 7 . only with protein samples from series one , a clear 60-kda band representing the vh - atpase b subunit was seen ( figure 7 , lane 1 ) . however , with protein extracts obtained during the second experimental series , the 60-kda band was very weak or completely absent ( figure 7 , lane 2 - 3 ) . in contrast , a distinct 110-kda band was constantly detected showing the presence of na / k - atpase subunit ( figure 7 , lane 46 ) in all the samples . we speculate that varying feeding conditions of sheep could be responsible for the different results seen in both series . to get some information on prior slaughter nutrition , we started rumen fluid analysis ( scfa concentration , ph ) and morphometric measurements of the rumen papillae at the end of the second series of experiments . figure 8 shows measured rumen fluid ph values and the accompanying scfa concentrations determined from 22 sheep . as expected , an inverse relationship ( y = 57,7x + 462.6 ; r = 0.61 ) between ruminal fluid ph and the amount of scfa was observed ( figure 8) . the mean ph value amounted to 7.1 0.1 with minimum and maximum values ranging from 6.4 to 7.9 . mm / l and minimum to maximum levels ranged from 11.7 to 115.7 mm / l . for comparison , data from a preliminary feeding experiment with sheep fed hay ad libitum are also given in figure 8 . although morphometric data showed no significant differences in rumen papillae surface from sheep with ruminal fluid ph values < 6.9 ( n = 6 ) or > 6.9 ( n = 16 ) , there was a tendency to higher surfaces ( 872.0 47.8 mm / cm ) in the low - ph group when compared to the high - ph group ( 797.7 27.1 mm / cm ) . to date , the functional role and the regulation of the recently described ruminal vh - atpase [ 10 , 11 ] is not well understood . based on our own work and that of other investigators [ 15 , 37 , 44 ] showing metabolic regulation of the pump , we assume that the availability of substrates and of energy are main regulatory factors to adapt ruminal vh - atpase activity . to prove this hypothesis , we here investigated the effect of mi induced by glucose removal and application of inhibitors of the glycolytic pathway ( 2-dog ) and/or of mitochondrial atp production ( antimycin a ) on vh - atpase activity . the phi ( 7.2 to 7.3 0.1 ) of rec suspended in control solution ( hepes - buffered , hco3-free na - media with 5 mm glucose ) was in the range of 7.1 to 7.5 reported for rec [ 11 , 40 , 45 ] and other cell types [ 44 , 4648 ] under hco3-free conditions . although oxidative metabolism of scfa , mainly of butyrate and propionate , is the main energy source for metabolically matured rec [ 2 , 4951 ] , glucose has also been shown to be used at a basal rate in vivo and in vitro [ 5 , 5154 ] . the metabolism of scfa has been shown to be supported by glucose and particularly it is known to exert a positive effect on ruminal butyrate metabolism . addition of glucose to butyrate in the incubation medium decreases the rate of butyrate oxidation to co2 and increases the rate of ketogenesis . besides glutamine , glucose is the main metabolic fuel in cultured rec and known to be primarily metabolized through glycolysis . in this study , inhibition of the glycolytic pathway by substitution of glucose with 2-dog induced a significant reduction of phi ( 0.35 0.04 ph units ) in rec showing that their ability to regulate phi , at least partly , depends on this metabolic pathway . like glucose , its analogue 2-dog is transported into the cells and is phosphorylated into 2-dog-6-phosphate . however , then it is not further metabolized and accumulates inside the cells causing energy deprivation . even in the absence of glucose , antimycin a application reduced the rec phi to a lesser extent ( 0.21 0.03 ph units ) than 2-dog showing that an intact glycolytic pathway is more important than mitochondrial atp production for h secretion under our experimental conditions . this is in accordance with our data showing that the combination of glucose substitution with 2-dog and antimycin a led to a stable phi decrease by 0.44 0.04 ph units . in yeast [ 34 , 35 , 56 ] , various mammary cell types [ 18 , 37 , 46 ] , and turtle urinary bladder , a coupling between vh - atpase activity and cellular energetic processes has been demonstrated . to evaluate the role of metabolic regulation on ruminal vh - atpase activity , we investigated the effect of its specific inhibitor foliomycin before and after mi with 2-dog and antimycin a in glucose - free media . under control conditions , the foliomycin - induced phi decrease amounted to 0.21 0.05 ph units at the end of the measurements , which is in agreement with results ( 0.18 0.07 ph units ) seen in a previous study with sheep rec . very similar effects of vh - atpase inhibition with bafilomycin a1 or foliomycin ( 0.16 to 0.26 ph units ) have been reported in studies with cells of the human eccrine sweat duct , cultured rabbit non - pigmented ciliary epithelium and alveolar macrophages . thus , a remarkable part of the rec h secretion resulted from vh - atpase activity . the vh - atpase - related phi component ( 0.21 0.05 ph units ) was nearly abolished ( 0.03 0.02 ph units ) after substitution of glucose by the glycolytic inhibitor 2-dog and treatment with the mitochondrial electron transport inhibitor antimycin a. this result clearly shows that the phi reduction observed after mi mainly results from deactivation of rec vh - atpase . as 2-dog exerts stronger effects on rec phi than treatment with antimycin a , it can be assumed that ruminal vh - atpase activity mainly depends on an intact glycolytic pathway . glucose deprivation and/or inhibition of glycolysis has also been shown to reduce or prevent vh - atpase activity in cells of the medullary collecting duct [ 44 , 60 ] , the porcine kidney proximal tubule cell line llc - pk1 [ 15 , 37 ] , and yeast cells [ 35 , 56 ] . in addition , glucose - induced activation of vh - atpase activity has been shown [ 18 , 37 ] . a coupling between the atp - generating glycolytic pathway and vh - atpase - mediated h secretion is supported by the findings that iodoacetate , an inhibitor of glyceralaldehyde-3-phosphate dehydrogenase ( gapdh ) , reduced vh - atpase activity and that the enzyme coimmunoprecipitates with the pump . also , the rate limiting enzyme of glycolysis , phosphofructokinase-1 interacts with vh - atpase , a subunit which has been suggested to play a role crucial for proton translocation . a direct physical interaction between the vh - atpase e , b and a subunits , and aldolase , an enzyme responsible for cleavage of fructose-6-phosphate in the glycolytic pathway , has been demonstrated [ 34 , 35 ] . with glucose present , this interaction increased dramatically leading to the suggestion that aldolase acts as a glucose sensor and regulates vh - atpase assembly , expression , and activity via direct physical association and by providing atp for h extrusion from cytosol [ 35 , 36 ] . rec conversion of glucose to pyruvate and co2 is rather small in proportion to conversion to lactate and thus , an increased lactate production is indicative of higher rates of glycolysis . interestingly , it was found , that at phe = 7.4 , rec converted 3-fold more d - glucose than n - butyrate and produced about two - fold more lactate than at phe = 6.2 . since glycolysis generates protons , its activation at higher phe will stimulate vh - atpase - mediated h efflux and thereby , will help to maintain scfa absorption under such conditions . provision of extracellular protons is critical for diffusion - mediated uptake of scfa in their undissociated form [ 3 , 50 ] . in this study , deactivation of vh - atpase activity induced by metabolic inhibition has been shown to be a fast process appearing within a few minutes . a general mechanism responsible for such response to energy deprivation could be reversible disassembly of the catalytic vo and the proton - translocating v1 domains of the pump [ 30 , 33 , 56 ] . in yeast cells , glucose deprivation induced a disassembly of 75% of the assembled vh - atpase complexes in as little as 5 minutes . in accordance with our previous study with bovine rumen epithelium , ovine rec vh - atpase b subunit was found in close vicinity of the cell membrane as well as in the cytosolic compartment under control conditions ( glucose - containing nacl - medium ) . however , while na / k - atpase used as control protein was always membrane - bound , we here showed for the first time that the b subunit associated with the rec vh - atpase v1 domain shows diffuse cytosolic distribution after mi . the data provide first evidence that the reduction of vh - atpase - mediated transmembrane proton efflux observed after substrate and energy deprivation of rec could result from a higher proportion of disassembled v1 and vo sectors . since vh - atpases are major cellular proteins that can consume significant amounts of total cellular atp , their graduated disassembly could help to conserve energy under such conditions . however , with the method used in this study , we were not able to exclude endocytosis of vh - atpase - bearing vesicles from the cell membrane into the cytosolic compartment . endo- and exocytotic translocations have been demonstrated in epithelia of kidney , pancreas , and placenta [ 1315 , 64 ] as an additional mechanism for regulating vh - atpase activity . rec belong to cells that have a specific requirement for high levels of proton transport and possess na / h exchangers of subtype 1 to 3 [ 40 , 45 , 65 ] and monocarboxylate transporter 1 ( mct1 ) [ 66 , 67 ] in addition to the vh - atpase . the partial recovery of phi from the acidosis induced by mi or foliomycin application may result from compensatory activation of one or both of these h - secreting transport proteins . the nhe has been shown to be most important for rec phi - regulation under hco3-free conditions responsible for about 70% of proton secretion and the remaining proton secretion was related to vh - atpase . therefore , our second series of experiments was originally designed to investigate the role of nhe before and after mi . unexpectedly however , the effect of mi was decreased by 75% in our secondary series of experiments and this was accompanied by a strongly reduced ( 81% ) foliomycin effect . these results point to an impairment of the glycolytic pathway in those rec and corroborate with tight coupling between glycolysis and/or components of the glycolytic pathway and vh - atpase activity . because rec used in this study were isolated from rumen tissue obtained from a local slaughter house , we can only speculate on the reasons for the very different metabolic properties of the cells . however , the latter has been shown to depend on the level of metabolizable energy ( me ) intake and on the type of nutrition [ 51 , 68 ] . ruminal fluid analysis showed a high proportion of samples ( 16 out of 22 ) showing ph values > 6.9 and [ scfa ] < 60 mm / l ( figure 8) indicating decreased me intake . in a preliminary feeding experiment with sheep fed hay ad libitum ( figure 8) lower ruminal ph values ( 6.7 0.2 ) and higher [ scfa ] ( 81.7 15.5 mm / l ) were observed than in this study ( 7.1 0.1 ; 51.6 5.7 mm / l ) . a tendency for a reduced capacity to oxidize glucose in rec isolated from low - intake sheep had been observed . different feeding conditions are also known to induce changes of the chemical composition of the ruminal fluid . as shown by kauffold et al . , such changes are most strongly expressed between rations consisting of fresh green feed or maize silage and such consisting of concentrate or dried green feeds . the former feeding conditions led to a fast decrease of metabolic activity and protein synthesis in the germinative layers of the rumen epithelium characterized by low o2-consumption and rec nucleus diameter . it seems therefore possible that adaptation to low energy availability that could include reduced proton production from glycolysis [ 69 , 70 ] gives an explanation for the low or absent vh - atpase activity . this hypothesis is supported by our finding that the expression of vh - atpase b subunit protein was drastically reduced or absent in protein extracts from rec used in the second series of experiments . the b subunit is essentially involved in the regulation of normal trafficking , assembly , and activity of vh - atpase [ 36 , 71 ] . interestingly , rec identified to have low metabolic and vh - atpase activity were characterized by a very high amiloride - sensitive component of phi reflecting nhe activity . the amiloride - induced reduction of phi ( 0.89 0.06 ph - units ) was as high as that from butyrate - stimulated rec ( 1.00 0.25 ph units ) . an increased activity of nhe has also been shown to occur during early stages of apoptosis in response to growth factor withdrawal . elevation of nhe activity may compensate for the loss of vh - atpase - related h extrusion thereby improving phi homeostasis in energy - deprived rec . however , after induction of mi , nhe activity decreases slowly resulting in a reduction of the amiloride effect to 0.69 ph units . an explanation is a reduced ability to maintain low intracellular [ na ] by na / k - atpase leading to slow dissipation of the transmembrane na gradient . inhibition of rec na / k - atpase has been shown to reduce phi by lowering nhe activity . . a fast reduction of vh - atpase mediated proton extrusion occurs few minutes after initiation of mi by glucose substitution with 2-dog and application of antimycin a. an intact glycolytic pathway seems to be more important for vh - atpase activity regulation than mitochondrial atp production . the very fast response possibly results from disassembly of the v1 and vo domains of the pump . prolonged energy deficiency may result in a higher level of disassembled , inactive vh - atpase complexes but also led to a reduced expression of the essential b subunit of the pump . this may provide a means to control ruminal epithelial atp consumption in dependence of substrate and energy availability .

in this study , the effect of metabolic inhibition ( mi ) by glucose substitution with 2-deoxyglucose ( 2-dog ) and/or application of antimycin a on ovine rumen epithelial cells ( rec ) vacuolar - type h+-atpase ( vh+-atpase ) activity was investigated . using fluorescent spectroscopy , basal phi of rec was measured to be 7.3 0.1 in hco3-free , glucose - containing nacl medium . mi induced a strong phi reduction ( 0.44 0.04 ph units ) with a more pronounced effect of 2-dog compared to antimycin a ( 0.30 0.03 versus 0.21 0.03 ph units ) . treatment with foliomycin , a specific vh+-atpase inhibitor , decreased rec phi by 0.21 0.05 ph units . after mi induction , this effect was nearly abolished ( 0.03 0.02 ph units ) . in addition , membrane - associated localization of vh+-atpase b subunit disappeared . metabolic control of vh+-atpase involving regulation of its assembly state by elements of the glycolytic pathway could provide a means to adapt rec atp consumption according to energy availability .

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

a main proportion of the rumen atp utilization is related to activity of a na / k - atpase that has been shown to be expressed at high levels [ 68 ] in the cell membrane of rumen epithelial cells ( rec ) [ 9 , 10 ] . in addition , functional vacuolar - type h pumps ( vh - atpase ) are existent in rec [ 10 , 11 ] . in addition , a link between electrogenic h secretion by vh - atpases localized on the cell membrane and ion transport and/or the regulation of cytosolic ph has been found in osteoclasts , macrophages , and various epithelia , for example , frog and toad skin , mammalian renal collecting duct , endolymphatic sac of the inner ear , and epididymis [ 2025 ] . foliomycin , a specific vh - atpase blocker , has been found to inhibit the uptake of mg into rec . in our previous study , a variable subcellular distribution of vh - atpase in cell membranes and/or cytosolic pools of the more luminally oriented cell layers ( stratum spinosum , stratum granulosum ) of the rumen epithelium has been observed . in addition , western blot and immunocytochemistry are used to analyze if changes of vh - atpase expression and localization play a role in adaptation of the pump activity . foliomycin , amiloride , antimycin a , and 2-deoxyglucose ( 2-dog ) were from sigma aldrich ( munich , germany ) . the monoclonal mouse antibodies used in this study were specific for 60-kda subunit of the yeast vh - atpase ( 13d11-b2 , molecular probes ) and the subunit of the sheep na / k - atpase ( m7-pb - e9 , affinity bioreagents ) . then , primary cultures of ruminal epithelial cells ( rec ) were prepared as described by galfi et al . in experiments designed to reduce the energy metabolism of rec ( metabolic inhibition ) : ( 1 ) glucose was removed from the medium and antimycin a ( 5 m ) , a known inhibitor of complex iii of the mitochondrial respiratory chain , was added ; ( 2 ) glucose was replaced with 20 mm 2-dog to inhibit the glycolytic pathway ; ( 3 ) antimycin a ( 5 m ) and 2-dog ( 20 mm ) were added with concomitant glucose removal . intracellular ph was detected by measuring the fluorescence of the probe - loaded rec in a spectrofluorometer ( ls-50 b , perkin - elmer ) equipped with a fast - filter accessory that allowed fluorescence to be measured at 20-ms intervals with excitation for bcecf at 440 and 480 nm and emission at 515 nm . thereafter , a metabolic inhibition ( 20 minutes ) was performed as described above . after three changes of pbs ( 5 minutes each ) , nuclei were counterstained with 300 nm of 4,6-diamidino-2-phenylindole ( dapi ) in s - buffer ( containing : 75 mm kcl , 3 mm mgso4 7h2o , 1 mm egta , 0.2 mm dithiothreitole , 10 mm imidazol , 1 g / ml aprotinin , 0.1 mm phenylmethane sulfonyl - fluoride ) . its ph was measured directly after sampling with a glass electrode ( n 1042a , ph meter cg 841 , schott , mainz , germany ) . bcecf - loaded rec were suspended in either hepes - buffered na - medium ( control ) or hepes - buffered , glucose - free na - medium with 2-dog and/or antimycin a , and phi was measured over a 10-min period . the appropriate concentrations of 2-dog ( 20 mm ) and antimycin a ( 5 m ) were chosen from dose - response experiments ( figure 1 ) . the initial and final phi of control rec incubated in hepes - buffered nacl - medium were 7.2 0.1 and 7.3 0.1 , respectively . the presence of antimycin a and/or 2-dog in the media led to a reduction of phi ( figures 2(a ) and 2(b ) ) . in rec exposed to antimycin a , 2-dog or antimycin a and 2-dog , the phi measured at the beginning of the measuring period was reduced by 0.21 0.03 , 0.35 0.04 , and 0.44 0.04 ph units , respectively . with antimycin a or both inhibitors in the medium , this phi reduction was stable in most cases . however , if 2-dog only was present , the effect was slightly diminished and the observed phi decrease amounted to 0.30 0.03 ph units at the end of the measuring period . next , we investigated whether a decreased vh - atpase activity was responsible for the phi reducing effect of metabolic inhibition ( mi ) . therefore , bcecf - loaded rec were suspended in foliomycin ( 2 m)-containing hepes - buffered na - medium or glucose - free , hepes - buffered na - medium with antimycin a ( 5 m ) , 2-dog ( 20 mm ) , and foliomycin ( 2 m ) . the mean phi reduction observed in foliomycin - treated rec amounted to 0.21 0.05 ph units ( figure 3(b ) ) . as shown in figures 3(a ) and 3(b ) , the foliomycin effect was nearly abolished when rec were incubated in antimycin a- and 2-dog - containing glucose - free media . under such conditions , the phi difference between control ( hepes - buffered , glucose - free na - medium with antimycin a and 2-dog ) and foliomycin - treated rec amounted to 0.03 0.02 ph units only ( figure 3(b ) ) . these results point to a fast deactivation of vh - atpase activity after mi with antimycin a and 2-dog . as a control , the na / k - atpase which is constantly found in the cell membrane of rec has been investigated using an subunit - specific anti - na / k - atpase antibody . as expected , the subunit - specific anti - na / k - atpase antibody identified the protein in the cytoplasmic membrane of rec and its localization was not changed by a 20-min incubation in glucose - free , antimycin a- and/or 2-dog - containing media ( figure 4(a ) , a c ) . when incubated in glucose - containing standard medium , rec vh - atpase appeared in the cell membrane and/or perimembrane area as well as in the cytoplasma of rec ( figure 4(b ) , a ) . after glucose deprivation and application of 2-dog ( figure 4(b ) , b ) , no membrane staining can be found and typically the vh - atpase staining is clustered in the perinuclear area of the cells . compared to control conditions , glucose deprivation and combined application of 2-dog and antimycin a ( figure 4(b ) , c ) induced appearance of diffuse , cytosolic vh - atpase staining . as in our previous investigation with ovine and bovine rec [ 10 , 11 ] , a more or less expressed compensatory phi increase was seen after application of foliomycin . in na - containing media , an nhe is known to be responsible for this effect as application of the nhe inhibitor amiloride reduced rec phi by 65% . therefore , we repeated the above described experiments to investigate the nhe activity before and after mi with 2-dog and antimycin a. experiments were performed as described for the first series , however , in addition to foliomycin ( 2 m ) , the nhe inhibitor amiloride ( 250 m ) was used alone or in combination with the vh - atpase blocker . mi induced a phi decrease of 0.10 0.03 ph units only ( figure 5 ) , which corresponds to a 75% reduction of the effect compared with the results of the first experiment . in addition , the mean inhibitory effect of foliomycin was strongly diminished ( 0.05 0.03 ph units ; figure 5 ) . in such cells phi was reduced by 0.14 0.03 or 0.10 0.05 ph units after mi induction or exposure to foliomycin . in figure 6 , the inhibitory effects of foliomycin and/or amiloride before and after mi are summarized . figure 6 shows that compared to series 1 ( a ) the foliomycin effect was strongly reduced ( 0.10 0.02 ph units versus 0.27 ph units ) and b ) the residual foliomycin effect ( 0.08 0.03 ph units ) was not influenced by mi . at the beginning and end of the measurement , the phi of amiloride - treated rec was reduced by 0.83 0.05 and 0.89 0.06 ph - units , respectively . the foliomycin- and amiloride - sensitive components were additive under control conditions and the observed phi decrease amounted to 0.93 0.05 at the start and to 0.95 0.08 at the end of the measuring period . although morphometric data showed no significant differences in rumen papillae surface from sheep with ruminal fluid ph values < 6.9 ( n = 6 ) or > 6.9 ( n = 16 ) , there was a tendency to higher surfaces ( 872.0 47.8 mm / cm ) in the low - ph group when compared to the high - ph group ( 797.7 27.1 mm / cm ) . to date , the functional role and the regulation of the recently described ruminal vh - atpase [ 10 , 11 ] is not well understood . based on our own work and that of other investigators [ 15 , 37 , 44 ] showing metabolic regulation of the pump , we assume that the availability of substrates and of energy are main regulatory factors to adapt ruminal vh - atpase activity . to prove this hypothesis , we here investigated the effect of mi induced by glucose removal and application of inhibitors of the glycolytic pathway ( 2-dog ) and/or of mitochondrial atp production ( antimycin a ) on vh - atpase activity . the phi ( 7.2 to 7.3 0.1 ) of rec suspended in control solution ( hepes - buffered , hco3-free na - media with 5 mm glucose ) was in the range of 7.1 to 7.5 reported for rec [ 11 , 40 , 45 ] and other cell types [ 44 , 4648 ] under hco3-free conditions . although oxidative metabolism of scfa , mainly of butyrate and propionate , is the main energy source for metabolically matured rec [ 2 , 4951 ] , glucose has also been shown to be used at a basal rate in vivo and in vitro [ 5 , 5154 ] . the metabolism of scfa has been shown to be supported by glucose and particularly it is known to exert a positive effect on ruminal butyrate metabolism . besides glutamine , glucose is the main metabolic fuel in cultured rec and known to be primarily metabolized through glycolysis . in this study , inhibition of the glycolytic pathway by substitution of glucose with 2-dog induced a significant reduction of phi ( 0.35 0.04 ph units ) in rec showing that their ability to regulate phi , at least partly , depends on this metabolic pathway . even in the absence of glucose , antimycin a application reduced the rec phi to a lesser extent ( 0.21 0.03 ph units ) than 2-dog showing that an intact glycolytic pathway is more important than mitochondrial atp production for h secretion under our experimental conditions . this is in accordance with our data showing that the combination of glucose substitution with 2-dog and antimycin a led to a stable phi decrease by 0.44 0.04 ph units . to evaluate the role of metabolic regulation on ruminal vh - atpase activity , we investigated the effect of its specific inhibitor foliomycin before and after mi with 2-dog and antimycin a in glucose - free media . under control conditions , the foliomycin - induced phi decrease amounted to 0.21 0.05 ph units at the end of the measurements , which is in agreement with results ( 0.18 0.07 ph units ) seen in a previous study with sheep rec . very similar effects of vh - atpase inhibition with bafilomycin a1 or foliomycin ( 0.16 to 0.26 ph units ) have been reported in studies with cells of the human eccrine sweat duct , cultured rabbit non - pigmented ciliary epithelium and alveolar macrophages . thus , a remarkable part of the rec h secretion resulted from vh - atpase activity . the vh - atpase - related phi component ( 0.21 0.05 ph units ) was nearly abolished ( 0.03 0.02 ph units ) after substitution of glucose by the glycolytic inhibitor 2-dog and treatment with the mitochondrial electron transport inhibitor antimycin a. this result clearly shows that the phi reduction observed after mi mainly results from deactivation of rec vh - atpase . as 2-dog exerts stronger effects on rec phi than treatment with antimycin a , it can be assumed that ruminal vh - atpase activity mainly depends on an intact glycolytic pathway . glucose deprivation and/or inhibition of glycolysis has also been shown to reduce or prevent vh - atpase activity in cells of the medullary collecting duct [ 44 , 60 ] , the porcine kidney proximal tubule cell line llc - pk1 [ 15 , 37 ] , and yeast cells [ 35 , 56 ] . in addition , glucose - induced activation of vh - atpase activity has been shown [ 18 , 37 ] . also , the rate limiting enzyme of glycolysis , phosphofructokinase-1 interacts with vh - atpase , a subunit which has been suggested to play a role crucial for proton translocation . a direct physical interaction between the vh - atpase e , b and a subunits , and aldolase , an enzyme responsible for cleavage of fructose-6-phosphate in the glycolytic pathway , has been demonstrated [ 34 , 35 ] . in this study , deactivation of vh - atpase activity induced by metabolic inhibition has been shown to be a fast process appearing within a few minutes . a general mechanism responsible for such response to energy deprivation could be reversible disassembly of the catalytic vo and the proton - translocating v1 domains of the pump [ 30 , 33 , 56 ] . in yeast cells , glucose deprivation induced a disassembly of 75% of the assembled vh - atpase complexes in as little as 5 minutes . in accordance with our previous study with bovine rumen epithelium , ovine rec vh - atpase b subunit was found in close vicinity of the cell membrane as well as in the cytosolic compartment under control conditions ( glucose - containing nacl - medium ) . however , while na / k - atpase used as control protein was always membrane - bound , we here showed for the first time that the b subunit associated with the rec vh - atpase v1 domain shows diffuse cytosolic distribution after mi . however , with the method used in this study , we were not able to exclude endocytosis of vh - atpase - bearing vesicles from the cell membrane into the cytosolic compartment . rec belong to cells that have a specific requirement for high levels of proton transport and possess na / h exchangers of subtype 1 to 3 [ 40 , 45 , 65 ] and monocarboxylate transporter 1 ( mct1 ) [ 66 , 67 ] in addition to the vh - atpase . the nhe has been shown to be most important for rec phi - regulation under hco3-free conditions responsible for about 70% of proton secretion and the remaining proton secretion was related to vh - atpase . unexpectedly however , the effect of mi was decreased by 75% in our secondary series of experiments and this was accompanied by a strongly reduced ( 81% ) foliomycin effect . these results point to an impairment of the glycolytic pathway in those rec and corroborate with tight coupling between glycolysis and/or components of the glycolytic pathway and vh - atpase activity . because rec used in this study were isolated from rumen tissue obtained from a local slaughter house , we can only speculate on the reasons for the very different metabolic properties of the cells . in a preliminary feeding experiment with sheep fed hay ad libitum ( figure 8) lower ruminal ph values ( 6.7 0.2 ) and higher [ scfa ] ( 81.7 15.5 mm / l ) were observed than in this study ( 7.1 0.1 ; 51.6 5.7 mm / l ) . the former feeding conditions led to a fast decrease of metabolic activity and protein synthesis in the germinative layers of the rumen epithelium characterized by low o2-consumption and rec nucleus diameter . the b subunit is essentially involved in the regulation of normal trafficking , assembly , and activity of vh - atpase [ 36 , 71 ] . the amiloride - induced reduction of phi ( 0.89 0.06 ph - units ) was as high as that from butyrate - stimulated rec ( 1.00 0.25 ph units ) . a fast reduction of vh - atpase mediated proton extrusion occurs few minutes after initiation of mi by glucose substitution with 2-dog and application of antimycin a. an intact glycolytic pathway seems to be more important for vh - atpase activity regulation than mitochondrial atp production . this may provide a means to control ruminal epithelial atp consumption in dependence of substrate and energy availability .

this paper presents two materials that originate from the same synthetic reagents but differ in second - harmonic - generation ( shg ) phase matchability . materials for shg applications require a noncentrosymmetric ( ncs ) arrangement of their crystal structure . these compounds are often characterized by acentric basic building units ( bbus ) such as polar early - transition - metal ( etm ) polyhedra or lone - pair cations ; the synthesis of ncs materials is an ongoing challenge with contributions toward shg - active materials , piezoelectrics , multiferroics , and other functional solids . as per chen s anionic group theory , to design efficient shg - active crystals , not only should the bbus display strong individual polar moments , but also the polar moments should be aligned along one direction ( specifically along one crystallographic axis ) . various works have described the use of polar anions with different polyhedral geometries to construct polar solids . we , and others , have described the use of multiple ions ( such as an octahedral ion and a tetrahedral ion ) to generate polar materials because the disparate ions would not be relatable via symmetry operations . in the materials of rb5nb3of18 ( ions of [ nbf7 ] and [ nbo2/2f4 ] ) and ag3(moo3f3)(moo4)cl ( ions of [ moo3f3 ] and [ moo4 ] ) , the crystal structures show polar alignment of [ moo3f3]/[moo4 ] or [ nbo2/2f4 ] ions . the vanadate family of compounds offers interesting properties of recorded ncs arrangements , multiple ionic environments ( strong polar distortions that exist in both tetrahedral and octahedral arrangements ) , and magnetic activity of vanadium(3+/4 + ) ions that can contribute to the possibility of multiferroics . fluoride vanadates with mixed ionic content because vanadium cations can exist as [ vo4 ] tetrahedra and [ voxf6x ] octahedra . in this manner , a mixed - valence vanadium(4+/5 + ) material could have isolated [ vo4 ] tetrahedra and [ voxf6x ] octahedra . hydrochloric acid is a known reducing agent of vanadium ; an in situ reduction process would create vanadium(4 + ) , while vanadium(5 + ) would also be present under nonequilibrium conditions . more recently , we employed bromide ions of kbr in acidic hydrothermal conditions to reduce vanadium(5 + ) to form the fully vanadium(4 + ) kvof3 . the reaction of sodium chloride in hydrofluoric acid with vanadium oxide produces two products in similar synthetic conditions : navof4(h2o ) ( i ) and navo2xf2+x ( ii ) , where x = /3 . notably , the structures are both ncs : compound i is type 1 non - phase - matchable ( npm ) , while compound ii is type 1 phase - matchable ( pm ) . analyses of similar synthetic conditions could afford similar ncs materials that allow for a better understanding of tailoring phase matchability in solid - state , purely inorganic materials . caution ! hydrofluoric acid is toxic and corrosive ! it must be handled with extreme caution and the appropriate protective gear and training . the dissolution of compounds in aqueous hydrofluoric acid can be exothermic and may cause volatilization of hydrofluoric acid . the use of teflon at elevated temperatures allows hfaq to permeate the film ; caution should be used in handling the backfill of hydrothermal reactions . vanadium oxide has noted toxicity , particularly when in small , particulate form ; vanadium oxide should be handled cautiously and in well - ventilated areas . hydrofluoric acid ( 49% hf in water by weight ) was obtained from sigma - aldrich . vanadium oxide ( v2o5 , 99.6% min ) and sodium chloride ( nacl , 99% ) were obtained from alfa - aesar . deionized water was used as backfill in the pressure vessel and as boiling water . the compound navof4(h2o ) ( i ) was synthesized by combining 0.5475 g of v2o5 ( 6.020 mmol v ) , 0.3514 g of nacl ( 6.013 mmol na ) , and 1.00 ml of hfaq ( 24.5 mmol hf ) in a teflon pouch that was made and sealed as previously described . the pouch was then placed in a beaker of boiling water , and the reaction mixture was refluxed within the pouch for 30 min . the contents were allowed to crystallize for an additional 48 h , and the pouch was opened and filtered in air to afford a mixture of clear platelets of i and nacl . the ph of the reaction mixture was about 1 before and after the reaction . to monitor reaction conditions , two reactions with identical contents were performed simultaneously with the reaction described above , but the contents were removed after reaction for total periods of 1 and 2 h. powder x - ray diffraction ( pxrd ) scans showed the presence of nacl in the remaining product mixtures . we were unable to avoid the presence of nacl either by alteration of the reaction conditions or by rinsing with water after the synthesis because i is water - soluble . the compound navo2xf2+x ( ii ; x = /3 ) was synthesized by the combination of the same amounts of reagents as those for i [ 0.5330 g of v2o5 ( 5.861 mmol v ) , 0.3520 g of nacl ( 6.023 mmol na ) , and 1.00 ml of hfaq ( 24.5 mmol hf ) ] in a teflon pouch , but the pouch was placed in a parr acid digestion vessel , individually , heated to 150 c , held at that temperature for 24 h , cooled at a rate of 0.1 c / min , removed , allowed to sit at ambient conditions for 48 h , filtered in air , rinsed with deionized water three times to remove unreacted nacl , and allowed to dry to obtain 0.3441 g of clear , dark - blue platelets of compound ii ( 2.374 mmol , 40.51% yield with respect to v2o5 ) . it was found that if a pouch of i ( before the pouch was opened ) was subsequently placed in the hydrothermal conditions described above , compound ii was formed . pxrd scans with laboratory radiation did not show other phases in the obtained compound ii ; see the supporting information ( si ) . compound i was unable to be examined for elemental content because we were unable to obtain the compound phase - pure . to confirm the metallic content of ii , standard solutions were prepared with millipore - filtered water with a solution of 997 g / ml vanadium in 1.3% nitric acid in water ( ricca ) and a solution of 1000 g / ml sodium in 2% nitric acid in water ( fluka ) . three samples of ii were dissolved in 10 ml of nitric acid , separately , and diluted to the appropriate concentrations with millipore - filtered water . the spectra were acquired and analyzed with varian icp - expert ii software . the concentrations of vanadium and sodium for each sample were obtained with the spectral emission peak of the icp - aes spectrum that was the most gaussian in shape . each concentration was obtained by the average of five measurements on the same sample . a sample of compound ii was examined via ion - selective - electrode analysis by midwest microlab , llc . the analysis was performed on three separate samples to reveal an elemental content of 30.88(23)% by mass . this corresponds to a formula of navo2xf2+x [ x = 0.3234(167 ) ] or approximately na(vv2)xo2xf2+x ( x = /3 ) . this material shows increased fluoride content compared to the previously published material navo2f2 and results in mixed oxide fluoride occupancy on crystallographic sites of the structure . the fluoride content increased as a result of charge balance to account for vanadium(4 + ) that was observed via magnetic susceptibility . single crystals of compounds i and ii were mounted on a glass fiber in paratone oil and cooled to 100 k under a flow of nitrogen . the data were obtained with a bruker kappa apex 2 ccd diffractometer using monochromated mo k radiation ( = 0.71073 ) . absorption corrections ( multiscan , sadabs ) were applied to the data in the program apex2 . the structures were solved with the use of xs to determine the atomic coordinates of the metallic cations ; the resulting anions and parameters were refined with fourier difference maps with the shelxl algorithm within the olex2 suite . the unit cell of ii is a commensurate modulation of a previous structure , navo2f2 . full crystallographic data sets were obtained on the same crystal at 100 and 298 k , and the q vector was invariant ; this indicates a commensurate modulation , and thus a supercell was used to describe the system . the unit cell and space group of ii was ambiguous : the structure could be described in six different unit cell / space group combinations . the structure could additionally be described in two centrosymmetric ( cs ) settings , but these were disregarded because the material exhibited an shg response ( characteristic of ncs structures ) and poor refinement parameters ( see the si ) . the structure was solved in each possibility with the same weighting scheme , and significant disorder was present in ii as a result of valence disorder [ disorder of vanadium(4+)/vanadium(5 + ) on the same site ] . under single - crystal x - ray crystallography , it was determined that the vanadium cations were split along bonds to specific anions x ( x = o , f ) ; this indicates disorder of the x site . the crystal was solved in each of six possible descriptions , and hamilton s r test was applied to determine the best description . the model that showed the greatest correspondence to the data , as determined from the r factors , was used to describe the crystal . the cifs for compounds i and ii are provided in the si . a summary of the other unit cell / space group descriptions and refinement is provided in the si . the obtained crystal structure of compound ii is a model of the obtained data : further investigation into the disorder may show long - range order of the oxide the elemental occupancies at these sites were defined with the data acquired from elemental analyses to arrive at the formula of ii . refinement of the commensurate structure with the subcell and q vector ( 0 , 0 , /2 ) in the program jana2006 showed that the vanadium positional disorder was present if the commensurate symmetry was accounted for ; subsequently , the supercell was used for further analysis for simplicity . symmetry analysis with platon suggested halving of the unit cell of ii with maintenance of the space group ( e.g. , returning to the unit cell of navo2f2 ) , but this was disregarded owing to the satellite peaks observed . samples of i and ii were ground with a mortar and pestle and sieved into discrete size ranges . synthesis of i resulted in unreacted nacl that could not be avoided or separated from i. the presence of nacl dilutes the shg - active phase . the shg response was measured with the kurtz perry method and compared to standards of -sio2 . the average nonlinear - optical susceptibilities of i and ii are approximately 0.55 and 1.15 pm / v , respectively . shg responses as a function of the particle sizes for ( a ) compound i ( type 1 npm ) and ( b ) compound ii ( type 1 pm ) . the lines are drawn to guide the eye and are not fits to the data . the sample was placed in a magnetic property measurement system ( mpms)-xl magnetometer from quantum design . the sample was measured under a 1000 oe field in a zero - field - cooled ( zfc ) scan , a 5000 oe field in zfc and field - cooled scans , and a 1 t field in a zfc scan . the resulting magnetic susceptibilities were corrected for the diamagnetic contributions of polyethylene ( 50/ 10 emu / kg ) and the diamagnetism of the compound ( 53.033 10 emu / mol ) . the spectra showed temperature - independent paramagnetism , and a term was included to account for this phenomenon . the resulting fits showed eff of 0.303 b / mol , which is consistent with a material with /3 the ftir spectra of i and ii were obtained on a bruker 37 tensor ftir instrument equipped with an atr accessory with a germanium crystal . the spectra were obtained from 5504000 cm with 256 scans at 2 cm resolution , and a background spectrum was subtracted . the data of the v x stretching region for compound ii ( 5501200 cm ) was then fit to a summation of gaussian functions . the resulting spectra and fitting parameters are provided in the si . for compound ii , no absorptions that could be attributed to x h ( x = f , o ) stretches are observed , which indicates that no protons are present within the structure . compound i contained impurities so its ftir spectrum was not fit , but the spectrum displays a broad peak that corresponds to water moieties present in the compound . the structure contains 0d bbus of [ vof4(h2o ) ] anions that are separated from other vanadium moieties . the sodium cations exist in octahedra with fluoride anions and a coordinated water molecule . the octahedral environments of these species are shown in figure 2b : the vanadium octahedron coordinates solely to the sodium cations via fluoride anions and the water molecule ; the oxide anion coordinates solely to the vanadium ion to fulfill its valence . the unit cell of navof4(h2o ) is shown in figure 2c : the polar moments of the vanadium octahedra partially align along one axis ( the polar c axis ) . representation of the structure of compound i. ( a ) ordered , 0d anionic octahedra of [ vo1/1f4/1(h2o)1/1 ] and [ naf2/2f3/1(h2o)1/2 ] . the fluoride and oxide anions and water moieties are ordered among the cations . the polar moments of the vanadium octahedral anions are shown with blue arrows ; the polar moments are partially oriented along the c axis . the structure of ii is commensurately modulated and presents very interesting vanadium octahedra in 1d chains , namely , in the split sites of vanadium that are shown pictorially in figure 3 . the site occupancies show two vanadium sites in approximately 1:2 ratios , similar to the 1:2 vanadium(4+)/vanadium(5 + ) ratio of the chemical formula indicated by elemental analysis . the increase of the dimensionality ( from 0d to 1d ) for i and ii is a result of increased temperature , which increases the condensation of vanadium oxide fluoride bbus . general structure analysis of ii is difficult in light of the disorder present , even under consideration of the supercell . the vanadium cations are each disordered among two positions ; the occupancies of these sites were refined so as to add to 1 ; this is consistent with the finding of the 1:1 sodium / vanadium ratio ( it is possible that the sodium / vanadium ratio is 1:1 and the structure is equally sodium- and vanadium - deficient , but this is very unlikely ) . free refinement of the sodium or vanadium cation occupancies in the crystallographic data resulted in full occupancy on each site . this indicates that the anions along these bonds are disordered , mixed oxide fluoride sites . the v o bonds are typically shorter than the v f bonds , and subsequently o / f disorder causes the ellipsoid of the anionic site to be elongated along the bonding axis . during refinement , fluoride ( 2o:1f ) site so as to maintain the formula found with elemental analyses . the vanadium cations are disordered along the bonding axis of one symmetrically distinct anion ; this anion is therefore disordered and was refined so as to account for the content of navo2xf2+x ( x = /3 ) . certain oxide and fluoride sites could be split and have their occupancies refined ; these occupancies are relatively consistent with a parity of vanadium valence identity of 1:2 vanadium(4+)/vanadium(5 + ) . there may be some correlation of the site occupancies with the 1:2 vanadium(4+)/vanadium(5 + ) ratio : the ions v1b , v2b , o4a , and f5a versus , respectively , v1a , v2a , o4b , and f5b refined in roughly 1:21:3 ratios . the individual octahedra of figure 3a can not be analyzed with bond - valence - sum calculations to determine if one site corresponds to vanadium(4 + ) or vanadium(5 + ) owing to the high degree of site identity and occupancy disorder . the representations of the environments of the vanadium anions are shown in figure 3b . . these data may not be completely accurate owing to nacl impurities present with i and absorptions of ii in the near - ir ( nir ) and visible regions . perry experiment to examine shg responses , the laser source ( first harmonic ) was 1064 nm and the measured output ( second harmonic ) was 532 nm . compound ii absorbs strongly in both regions ( see the si ) , so its shg response is likely greater . icp - aes and fluoride elemental analysis indicate the formula of compound ii to be na(vv2)xo2xf2+x where x = /3 . the crystallographic structure indicated disorder along bonds to two anions ( see figure 3c ) . the decreased cationic charge by reduction of vanadium(5 + ) to vanadium(4 + ) results in a decreased anionic charge by the partial replacement of oxygen(2 ) with fluorine(1 ) . fluoride site vacancies , but this is not observed in the single - crystal data : free refinement of the disordered anion as a fluoride ion results in a site occupancy of about 1 , and refinement as an oxide site results in a site occupancy of about 1.1 ( consistent with the presence of fluoride ions with a greater z value than oxide ions ) . compound i is ordered in terms of oxide fluoride anions or water molecules . notably , the sodium ion packs in such a way that it predominantly coordinates to five fluoride anions and one water molecule ; it does not coordinate to the softer oxide anion ( softer relative to the fluoride anion ) . this is consistent with hard / soft acid / base interactions that have been previously observed and discussed . the mixed valence of vanadium in ii occurs on account of the presence of chloride ions in solution of hydrothermal synthesis . we infer that reduction occurs on account of generated hydrochloric acid ( generated by chloride ions in highly acidic hf ) . when the reaction is performed with naf as a sodium source in place of nacl , a small amount of clear crystals are produced . the pxrd and single - crystal diffraction data show the crystals to have the same unit cell of ii ; that is , the clear material also shows a supercell ( 1 1 2 ) of navo2f2 . we were unable to reproduce the reported synthesis of navo2f2 because the synthesis required high temperatures ( 552 c ) with hf . mixed vanadate species generally have magnetic moments that correspond to noninteger numbers of electrons . thus , magnetic measurements showed ii to have an effective magnetic moment of 0.303 b / mol . the band gap was determined with uv vis nir spectroscopy to be approximately 0.632 ev . the mixed vanadium(4+)/vanadium(5 + ) valences occur in many compounds , and this property is of interest for battery and magnetic materials . however , mixed vanadium(3+)/vanadium(5 + ) valences are rare . these two species could be identified in previous structures owing to different environments : while vanadium(5 + ) cations may crystallize in a tetrahedral environment , vanadium(3 + ) cations do not . thus , vanadium(3 + ) has been identified in the center of octahedra , while vanadium(5 + ) adopts a tetrahedral environment for k3v(vo4)2 , and ba3v2o3s4 . other interesting structures such as ba8v7o22 and fe6.5v11.5o35 combine vanadium in three oxidation states : 3 + , 4 + , and 5 + . these multivalent materials are usually identifiable by their distinct environment for the vanadium with different oxidation states . we conjecture , but can not decisively prove , that the vanadium of ii is present in the vanadium(4 + ) and vanadium(5 + ) states owing to the low magnetic moment and paramagnetic behavior at low temperatures . if vanadium(3 + ) ions were present , we could expect to observe inner - orbital interactions between the unpaired electrons of vanadium(3 + ) . we do not observe tetrahedra in the crystal structure of ii , so we are unable to decisively identify vanadium(5 + ) species . bond - valence - sum calculations return noninteger values for the valence , indicating valence - site disorder . the vanadium oxide fluoride anions have a polar moment that is partially aligned along one axis ( the polar c axis ) . the polar ions are separated by sodium ions into 0d bbus ( i.e. , the vanadium octahedra do not share ions / water molecules with other vanadium octahedra ) . these sodium ions the polar moments of the overall structure from other polar moments ; such an arrangement can be favorable because the polar moments do not have an adjacent neighbor . a vanadium bbu could situate itself antiparallel to a neighboring vanadium bbu to cancel out the polarity and maximize the local electroneutrality . the presence of such mediating ions that charge balance but still allow polar moments could be a viable path to develop ncs structures . this was found to be a factor in the polarity of the highly efficient shg material k3b6o10cl ; the potassium counterions contributed to the overall polarity of the structure . in the structures of i and ii , the sodium octahedra , via geometric calculations , are found to be very weakly polar . from this analysis , the polar contribution to the shg response of compounds i and ii originates from the vanadium octahedra ; however , more extensive density functional theory calculations should be performed to examine whether this is the case . it should also be noted that , although the sodium cations are necessary for charge balance , they do dilute the polar contributions of individual ions in the structure . if the sodium ions could contribute to the polarity of the structure , a greater shg response may be observed . in i , the vanadium polar moments pack in a 1d bbu of vanadium cations separated by a 1d ribbon of sodium cations . figure 4a shows these coordination environments : the 21 screw axis of i describes the geometry of these ribbons , with oxide anions partially oriented in one direction . figure 4b shows that these ribbons pack so that the terminal oxides of the vanadium bbus are situated toward the channels . this type of environment minimizes the number of contacts to the terminal oxide ; such an environment has been noted to correlate with ncs crystal classes ; however , this may be a correlation and not an inherent cause . the cs polymorph of knanbof5 only has one contact to the oxide : the linear nb = o k bond . it is likely that a reduced number of contacts to the terminal oxide of an etm still can facilitate low - symmetry environments [ such as in the ncs knanbof5 and cuvof4(h2o)7 systems ] . these low coordination numbers avoid oh and td environments around the oxide anion but do not necessarily avoid cv . this linear point group does not contain an inversion center but does allow a number of coordination environments : such variability can allow cs environments around the terminal oxide . ( a ) 1d unit of the individual vanadium oxide fluoride bbus as described by the 21 screw axis along the c axis . void space exists , and the terminal oxide anions are situated toward these voids . initially , the structure of ii looks nonpolar based on the unit cells shown in figure 3 . the disorder convolutes structural interpretation , but visualization of the structure of ii in figure 5 shows the preferential presence of a terminal oxide and a vanadium ion in one location . this concept of partial disorder within an inorganic phase could allow ( i ) polar moments and ( ii ) stabilization afforded by increased entropy . this disorder could be introduced by in situ reduction / oxidation to modify known phases ( such as , in this case , navo2f2 ) . partial occupancies illustrated with pie chart atoms : the unit cell of compound ii with only the polyhedra of v2 shown . a polar moment exists along the b axis owing to the preference of one oxide anion to situate in one location ; vanadium distortion is predominantly directed down the b axis . compound i is colorless owing to the presence of fluoride anions ; these anions blue - shift the absorption of the metal into the uv region . such transparency in the uv region is necessary for shg materials to provide high - energy lasers in the uv region for the potential use of lasers for nanolithography . the material navof40.7h2o has been previously reported but not crystallographically described ; the synthesis of the material is similar to the synthesis of i. the structure of i is chemically and structurally similar to [ c2n2h10][vof4(h2o ) ] . the zero dimensionality of the vanadium bbus refers to coordination of the vanadium bbus with other vanadium bbus : each vanadium octahedron is isolated from other vanadium octahedra . the hybrid material of [ c2n2h10][vof4(h2o ) ] contains vanadium(4 + ) ions , while ii contains vanadium(5 + ) ions . the former phase has greater polar alignment [ at an angle of 25.241(63) with respect to the polar b axis ] compared to the latter phase [ at an angle of 55.266(37) with respect to the polar b axis ] . the hybrid material s polar alignment is attributed to hydrogen bonds . although hydrogen bonds are , in fact , present for i , the stronger interactions with the sodium ions are more influential within the phase . the shg response of [ c2n2h10][vof4(h2o ) ] is about an order of magnitude greater than the shg response of i. this may be a consequence of the presence of nacl impurities within the sample of i but also may indicate a greater shg response for [ c2n2h10][vof4(h2o ) ] on account of the polar alignment and chen s anionic group theory . as noted , ii is a modulated variation of navo2f2 . the modulation is relatively complex to analyze because , from the solution of the supercell , the structure presents disorder in ( i ) the vanadium valence , ( ii ) vanadium site positions / occupancies , and ( iii ) oxide the ftir spectrum of compound ii shows numerous , broad peaks from 1020 to 500 cm . the number and broadness indicates several ( disordered ) v = o and v x ( x = f , o ) bonding environments . the relatively high energy ( 1018 cm ) of one peak indicates that a strong v = o bond is present . the single - crystal data do not indicate other long - range order to be present ; e.g. , there is no evidence of separate chains that consist of different geometries , nor are there signs of ordering of cations along the chain [ such as every third vanadium in a chain being vanadium(4 + ) ] . the shg responses obtained for i and ii are about the same as that of navo2f2 ( on the order of -sio2 ) . compound ii absorbs strongly at the fundamental frequency that was used in the shg measurement ( 1064 nm ) and the second harmonic ( 532 nm ) of light that was produced via shg ( see figure 1 ) . therefore , the actual shg efficiency of ii is likely greater than that of navo2f2 . despite chemical similarity , ii is pm and i is npm . phase matchability has been extensively described for organic molecular crystals ; future studies of inorganic systems are needed to rationalize the phase matchability in inorganic , extended solids . the material navof4(h2o ) is a 0d ncs material that was synthesized under reflux conditions . by increasing the synthetic temperature to hydrothermal conditions , the higher - dimensional ( 1d ) material navo2xf2+x ( x = /3 ) was synthesized ; it is a commensurately modulated variation of navo2f2 . the modulated material is a multivalent compound that was obtained by in situ reduction with a mild reducing agent of nacl in hf at 150 c . such materials could be of interest to multiferroics in addition to studies of shg - active materials and fundamental solid - state synthetic chemistry . navof4(h2o ) is type 1 npm , while navo2xf2+x ( x = /3 ) is type 1 pm . future theoretical work into the effects of dispersion and electronic configurations of ncs materials should address the topic of phase matchability of extended , inorganic solids . an understanding of how materials may or may not be pm for industrial purposes would greatly enhance future targeted efforts to generate suitable materials for shg purposes .

the syntheses of two noncentrosymmetric ( ncs ) vanadium oxide fluoride compounds that originate from the same synthetic reagent concentrations are presented . hydrothermal and low - temperature syntheses allow the isolation of metastable products that may form new phases ( or decompose ) upon heating and allow creation of chemically similar but structurally different materials . ncs materials synthesis has been a long - standing goal in inorganic chemistry : in this article , we compare two chemically similar ncs inorganic materials , navof4(h2o ) ( i ) and navo2xf2+x ( ii ; x = 1/3 ) . these materials originate from the same , identical reagent mixtures but are synthesized at different temperatures : 100 c and 150 c , respectively . compound i crystallizes in pna21 : a = 9.9595(4 ) , b = 9.4423(3 ) , and c = 4.8186(2 ) . compound ii crystallizes in p21 : a = 6.3742(3 ) , b = 3.5963(2 ) , c = 14.3641(7 ) , and = 110.787(3). both materials display second - harmonic - generation activity ; however , compound i is type 1 non - phase - matchable , whereas compound ii is type 1 phase - matchable .

Introduction Experimental Methods Results Discussion Conclusion

this paper presents two materials that originate from the same synthetic reagents but differ in second - harmonic - generation ( shg ) phase matchability . materials for shg applications require a noncentrosymmetric ( ncs ) arrangement of their crystal structure . these compounds are often characterized by acentric basic building units ( bbus ) such as polar early - transition - metal ( etm ) polyhedra or lone - pair cations ; the synthesis of ncs materials is an ongoing challenge with contributions toward shg - active materials , piezoelectrics , multiferroics , and other functional solids . we , and others , have described the use of multiple ions ( such as an octahedral ion and a tetrahedral ion ) to generate polar materials because the disparate ions would not be relatable via symmetry operations . in the materials of rb5nb3of18 ( ions of [ nbf7 ] and [ nbo2/2f4 ] ) and ag3(moo3f3)(moo4)cl ( ions of [ moo3f3 ] and [ moo4 ] ) , the crystal structures show polar alignment of [ moo3f3]/[moo4 ] or [ nbo2/2f4 ] ions . the vanadate family of compounds offers interesting properties of recorded ncs arrangements , multiple ionic environments ( strong polar distortions that exist in both tetrahedral and octahedral arrangements ) , and magnetic activity of vanadium(3+/4 + ) ions that can contribute to the possibility of multiferroics . in this manner , a mixed - valence vanadium(4+/5 + ) material could have isolated [ vo4 ] tetrahedra and [ voxf6x ] octahedra . hydrochloric acid is a known reducing agent of vanadium ; an in situ reduction process would create vanadium(4 + ) , while vanadium(5 + ) would also be present under nonequilibrium conditions . more recently , we employed bromide ions of kbr in acidic hydrothermal conditions to reduce vanadium(5 + ) to form the fully vanadium(4 + ) kvof3 . the reaction of sodium chloride in hydrofluoric acid with vanadium oxide produces two products in similar synthetic conditions : navof4(h2o ) ( i ) and navo2xf2+x ( ii ) , where x = /3 . notably , the structures are both ncs : compound i is type 1 non - phase - matchable ( npm ) , while compound ii is type 1 phase - matchable ( pm ) . analyses of similar synthetic conditions could afford similar ncs materials that allow for a better understanding of tailoring phase matchability in solid - state , purely inorganic materials . vanadium oxide has noted toxicity , particularly when in small , particulate form ; vanadium oxide should be handled cautiously and in well - ventilated areas . vanadium oxide ( v2o5 , 99.6% min ) and sodium chloride ( nacl , 99% ) were obtained from alfa - aesar . the compound navof4(h2o ) ( i ) was synthesized by combining 0.5475 g of v2o5 ( 6.020 mmol v ) , 0.3514 g of nacl ( 6.013 mmol na ) , and 1.00 ml of hfaq ( 24.5 mmol hf ) in a teflon pouch that was made and sealed as previously described . the pouch was then placed in a beaker of boiling water , and the reaction mixture was refluxed within the pouch for 30 min . the contents were allowed to crystallize for an additional 48 h , and the pouch was opened and filtered in air to afford a mixture of clear platelets of i and nacl . we were unable to avoid the presence of nacl either by alteration of the reaction conditions or by rinsing with water after the synthesis because i is water - soluble . the compound navo2xf2+x ( ii ; x = /3 ) was synthesized by the combination of the same amounts of reagents as those for i [ 0.5330 g of v2o5 ( 5.861 mmol v ) , 0.3520 g of nacl ( 6.023 mmol na ) , and 1.00 ml of hfaq ( 24.5 mmol hf ) ] in a teflon pouch , but the pouch was placed in a parr acid digestion vessel , individually , heated to 150 c , held at that temperature for 24 h , cooled at a rate of 0.1 c / min , removed , allowed to sit at ambient conditions for 48 h , filtered in air , rinsed with deionized water three times to remove unreacted nacl , and allowed to dry to obtain 0.3441 g of clear , dark - blue platelets of compound ii ( 2.374 mmol , 40.51% yield with respect to v2o5 ) . it was found that if a pouch of i ( before the pouch was opened ) was subsequently placed in the hydrothermal conditions described above , compound ii was formed . pxrd scans with laboratory radiation did not show other phases in the obtained compound ii ; see the supporting information ( si ) . compound i was unable to be examined for elemental content because we were unable to obtain the compound phase - pure . to confirm the metallic content of ii , standard solutions were prepared with millipore - filtered water with a solution of 997 g / ml vanadium in 1.3% nitric acid in water ( ricca ) and a solution of 1000 g / ml sodium in 2% nitric acid in water ( fluka ) . three samples of ii were dissolved in 10 ml of nitric acid , separately , and diluted to the appropriate concentrations with millipore - filtered water . each concentration was obtained by the average of five measurements on the same sample . a sample of compound ii was examined via ion - selective - electrode analysis by midwest microlab , llc . this corresponds to a formula of navo2xf2+x [ x = 0.3234(167 ) ] or approximately na(vv2)xo2xf2+x ( x = /3 ) . this material shows increased fluoride content compared to the previously published material navo2f2 and results in mixed oxide fluoride occupancy on crystallographic sites of the structure . the unit cell of ii is a commensurate modulation of a previous structure , navo2f2 . full crystallographic data sets were obtained on the same crystal at 100 and 298 k , and the q vector was invariant ; this indicates a commensurate modulation , and thus a supercell was used to describe the system . the structure could additionally be described in two centrosymmetric ( cs ) settings , but these were disregarded because the material exhibited an shg response ( characteristic of ncs structures ) and poor refinement parameters ( see the si ) . the structure was solved in each possibility with the same weighting scheme , and significant disorder was present in ii as a result of valence disorder [ disorder of vanadium(4+)/vanadium(5 + ) on the same site ] . under single - crystal x - ray crystallography , it was determined that the vanadium cations were split along bonds to specific anions x ( x = o , f ) ; this indicates disorder of the x site . the crystal was solved in each of six possible descriptions , and hamilton s r test was applied to determine the best description . the model that showed the greatest correspondence to the data , as determined from the r factors , was used to describe the crystal . the obtained crystal structure of compound ii is a model of the obtained data : further investigation into the disorder may show long - range order of the oxide the elemental occupancies at these sites were defined with the data acquired from elemental analyses to arrive at the formula of ii . , returning to the unit cell of navo2f2 ) , but this was disregarded owing to the satellite peaks observed . the average nonlinear - optical susceptibilities of i and ii are approximately 0.55 and 1.15 pm / v , respectively . shg responses as a function of the particle sizes for ( a ) compound i ( type 1 npm ) and ( b ) compound ii ( type 1 pm ) . the sample was measured under a 1000 oe field in a zero - field - cooled ( zfc ) scan , a 5000 oe field in zfc and field - cooled scans , and a 1 t field in a zfc scan . the resulting magnetic susceptibilities were corrected for the diamagnetic contributions of polyethylene ( 50/ 10 emu / kg ) and the diamagnetism of the compound ( 53.033 10 emu / mol ) . the spectra showed temperature - independent paramagnetism , and a term was included to account for this phenomenon . the spectra were obtained from 5504000 cm with 256 scans at 2 cm resolution , and a background spectrum was subtracted . the data of the v x stretching region for compound ii ( 5501200 cm ) was then fit to a summation of gaussian functions . for compound ii , no absorptions that could be attributed to x h ( x = f , o ) stretches are observed , which indicates that no protons are present within the structure . compound i contained impurities so its ftir spectrum was not fit , but the spectrum displays a broad peak that corresponds to water moieties present in the compound . the unit cell of navof4(h2o ) is shown in figure 2c : the polar moments of the vanadium octahedra partially align along one axis ( the polar c axis ) . representation of the structure of compound i. the structure of ii is commensurately modulated and presents very interesting vanadium octahedra in 1d chains , namely , in the split sites of vanadium that are shown pictorially in figure 3 . the increase of the dimensionality ( from 0d to 1d ) for i and ii is a result of increased temperature , which increases the condensation of vanadium oxide fluoride bbus . general structure analysis of ii is difficult in light of the disorder present , even under consideration of the supercell . this indicates that the anions along these bonds are disordered , mixed oxide fluoride sites . the v o bonds are typically shorter than the v f bonds , and subsequently o / f disorder causes the ellipsoid of the anionic site to be elongated along the bonding axis . the vanadium cations are disordered along the bonding axis of one symmetrically distinct anion ; this anion is therefore disordered and was refined so as to account for the content of navo2xf2+x ( x = /3 ) . certain oxide and fluoride sites could be split and have their occupancies refined ; these occupancies are relatively consistent with a parity of vanadium valence identity of 1:2 vanadium(4+)/vanadium(5 + ) . there may be some correlation of the site occupancies with the 1:2 vanadium(4+)/vanadium(5 + ) ratio : the ions v1b , v2b , o4a , and f5a versus , respectively , v1a , v2a , o4b , and f5b refined in roughly 1:21:3 ratios . these data may not be completely accurate owing to nacl impurities present with i and absorptions of ii in the near - ir ( nir ) and visible regions . compound ii absorbs strongly in both regions ( see the si ) , so its shg response is likely greater . icp - aes and fluoride elemental analysis indicate the formula of compound ii to be na(vv2)xo2xf2+x where x = /3 . fluoride site vacancies , but this is not observed in the single - crystal data : free refinement of the disordered anion as a fluoride ion results in a site occupancy of about 1 , and refinement as an oxide site results in a site occupancy of about 1.1 ( consistent with the presence of fluoride ions with a greater z value than oxide ions ) . compound i is ordered in terms of oxide fluoride anions or water molecules . the pxrd and single - crystal diffraction data show the crystals to have the same unit cell of ii ; that is , the clear material also shows a supercell ( 1 1 2 ) of navo2f2 . the mixed vanadium(4+)/vanadium(5 + ) valences occur in many compounds , and this property is of interest for battery and magnetic materials . however , mixed vanadium(3+)/vanadium(5 + ) valences are rare . thus , vanadium(3 + ) has been identified in the center of octahedra , while vanadium(5 + ) adopts a tetrahedral environment for k3v(vo4)2 , and ba3v2o3s4 . other interesting structures such as ba8v7o22 and fe6.5v11.5o35 combine vanadium in three oxidation states : 3 + , 4 + , and 5 + . we conjecture , but can not decisively prove , that the vanadium of ii is present in the vanadium(4 + ) and vanadium(5 + ) states owing to the low magnetic moment and paramagnetic behavior at low temperatures . if vanadium(3 + ) ions were present , we could expect to observe inner - orbital interactions between the unpaired electrons of vanadium(3 + ) . the vanadium oxide fluoride anions have a polar moment that is partially aligned along one axis ( the polar c axis ) . the polar ions are separated by sodium ions into 0d bbus ( i.e. from this analysis , the polar contribution to the shg response of compounds i and ii originates from the vanadium octahedra ; however , more extensive density functional theory calculations should be performed to examine whether this is the case . this type of environment minimizes the number of contacts to the terminal oxide ; such an environment has been noted to correlate with ncs crystal classes ; however , this may be a correlation and not an inherent cause . it is likely that a reduced number of contacts to the terminal oxide of an etm still can facilitate low - symmetry environments [ such as in the ncs knanbof5 and cuvof4(h2o)7 systems ] . ( a ) 1d unit of the individual vanadium oxide fluoride bbus as described by the 21 screw axis along the c axis . void space exists , and the terminal oxide anions are situated toward these voids . this concept of partial disorder within an inorganic phase could allow ( i ) polar moments and ( ii ) stabilization afforded by increased entropy . this disorder could be introduced by in situ reduction / oxidation to modify known phases ( such as , in this case , navo2f2 ) . partial occupancies illustrated with pie chart atoms : the unit cell of compound ii with only the polyhedra of v2 shown . compound i is colorless owing to the presence of fluoride anions ; these anions blue - shift the absorption of the metal into the uv region . the material navof40.7h2o has been previously reported but not crystallographically described ; the synthesis of the material is similar to the synthesis of i. the structure of i is chemically and structurally similar to [ c2n2h10][vof4(h2o ) ] . the zero dimensionality of the vanadium bbus refers to coordination of the vanadium bbus with other vanadium bbus : each vanadium octahedron is isolated from other vanadium octahedra . the hybrid material s polar alignment is attributed to hydrogen bonds . as noted , ii is a modulated variation of navo2f2 . the modulation is relatively complex to analyze because , from the solution of the supercell , the structure presents disorder in ( i ) the vanadium valence , ( ii ) vanadium site positions / occupancies , and ( iii ) oxide the ftir spectrum of compound ii shows numerous , broad peaks from 1020 to 500 cm . the number and broadness indicates several ( disordered ) v = o and v x ( x = f , o ) bonding environments . the single - crystal data do not indicate other long - range order to be present ; e.g. the shg responses obtained for i and ii are about the same as that of navo2f2 ( on the order of -sio2 ) . compound ii absorbs strongly at the fundamental frequency that was used in the shg measurement ( 1064 nm ) and the second harmonic ( 532 nm ) of light that was produced via shg ( see figure 1 ) . therefore , the actual shg efficiency of ii is likely greater than that of navo2f2 . despite chemical similarity , ii is pm and i is npm . phase matchability has been extensively described for organic molecular crystals ; future studies of inorganic systems are needed to rationalize the phase matchability in inorganic , extended solids . the material navof4(h2o ) is a 0d ncs material that was synthesized under reflux conditions . by increasing the synthetic temperature to hydrothermal conditions , the higher - dimensional ( 1d ) material navo2xf2+x ( x = /3 ) was synthesized ; it is a commensurately modulated variation of navo2f2 . the modulated material is a multivalent compound that was obtained by in situ reduction with a mild reducing agent of nacl in hf at 150 c . navof4(h2o ) is type 1 npm , while navo2xf2+x ( x = /3 ) is type 1 pm . future theoretical work into the effects of dispersion and electronic configurations of ncs materials should address the topic of phase matchability of extended , inorganic solids .

chronic obstructive pulmonary disease ( copd ) is primarily a smoking - related lung disease and afflicts more than 200 million people worldwide . , airway inflammation leads to increased mucus production and reduced mucociliary clearance , causing bronchoconstriction and airflow limitation . patients with copd frequently suffer disease exacerbations often induced by bacterial or viral respiratory infections , suggestive of impaired innate immunity . during disease exacerbation , although there is clear benefit to continuous low - level oxygen therapy in chronic , stable copd disease [ 1 , 2 ] , excessive amounts of nontitrated oxygen may in fact be harmful during copd exacerbation [ 3 , 4 ] . therefore , how oxygen modulates the effects of cigarette smoke on lung immunity may be relevant for patients with copd exacerbation and other smoke - induced lung diseases . the immunomodulatory effects of moderate and high levels of oxygen exposure ( fio2 0.61.0 ) have been well described in experimental models . mice exposed to four days of 95% oxygen had increased mortality with impaired macrophage phagocytosis in a klebsiella pneumoniae model . 60% oxygen exposure shortly following lps - induced lung injury markedly exacerbated lung inflammation in part mediated by macrophage - induced recruitment of alveolar neutrophils . although higher levels of oxygen appear to impair lung immunity , there is limited data as to how lower levels of oxygen supplementation ( fio2 0.300.4 ) can modulate lung immunity , particularly with coexisting cigarette smoke exposure . macrophages are prominent resident cells of the alveolar space and are critical for regulation of immune responses in the lung [ 79 ] . with smoke - induced copd , macrophage numbers can increase 5- to 10-fold in the lungs and alveolar space [ 10 , 11 ] and can correlate with disease severity , although they do not conform to the classic m1/m2 dichotomy . alveolar macrophage dysfunction induced by cigarette smoke exposure leads to excess oxidative stress and may contribute to higher bacterial colonization and increased susceptibility to exacerbations . alveolar macrophage phenotype and function can be modulated by exposure to varying levels of supplemental oxygen [ 5 , 14 ] . as a model to help understanding oxygen effects on the lung immune response to bacteria subsequent to cigarette smoke exposure , we challenged mice or macrophages in isolation with subacute durations of cigarette smoke ( cs ) followed by exposure to low - dose oxygen and pseudomonas aeruginosa bacteria . we chose a shorter cigarette smoke exposure to focus on the effects cs and oxygen - induced changes in inflammatory cell populations , similar to bronchitis , and without emphysema - like changes in lung architecture . c57bl/6 mice were purchased ( jackson labs , bar harbor , me ) and housed at the johns hopkins university asthma and allergy center . experiments were conducted under a protocol approved by the johns hopkins animal care and use committee . mice were exposed to cigarette smoke 5 hours / day , 5 days / week for 5 weeks , by burning 3r4f reference cigarettes ( 2.45 mg nicotine / cigarette ; tobacco research institute , university of kentucky ) using a smoking machine ( model te-10 , teague enterprises ) . for specified groups , the morning after a 5-week cigarette smoke ( cs ) exposure , we instilled pseudomonas aeruginosa ( pao1 , atcc , manassas , va ) ( 3 10 cfus , in 50 l pbs ) or vehicle control ( pbs ) via an intratracheal ( i.t . ) route through a 20-gauge endotracheal catheter as before . mice were anesthetized with intraperitoneal ketamine / acetylpromazine ( 100/2.5 g / g ) prior to exposure of the trachea . within 2 hours of instillation of i.t . pao1 or vehicle control , mice were exposed to 3540% oxygen or 21% oxygen for up to five days . for oxygen exposure , mice were placed in customized and sealed cages with ad libitum food and water . 3540% oxygen was achieved with a mixture of air and medical grade oxygen ( roberts oxygen , rockville , md ) at adjustable flow rates and constant pressure , with continuous measurements via an oxygen analyzer with a feedback loop to automatically adjust oxygen concentrations ( model 65 , advanced micro instruments , huntington beach , ca ) . oxygen exposure was uninterrupted except for 5 min every other day for cage cleaning . mice were harvested after 5 weeks of cigarette smoke exposure ( or air exposure as a control ) and 3 days of 3540% oxygen ( or 21% oxygen as a control ) . in addition , following 5 weeks of cs exposure ( or air exposure ) , designated mice were exposed to i.t . pao1 ( or vehicle control ) and 2 days of 3540% oxygen ( or 21% oxygen as control ) until sacrifice and harvest of lungs for assessment of cfus and inflammatory parameters or up to 5 days for assessment of mortality . mice were anesthetized with intraperitoneal ketamine / acetylpromazine ( 150/13.5 mg / kg ) prior to harvest and killed by exsanguination from the inferior vena cava . the lungs were perfused free of blood with 1 ml of phosphate - buffered saline ( pbs ) . the right lung was lavaged with two aliquots of 0.7 ml calcium - free pbs . for quantitative measures of bacteria , whole lungs were homogenized without prior lavage , and the lysates were diluted in pbs and streaked on agar plates . bal fluid was centrifuged ( 700 g , 10 min at 4c ) , and cell - free supernatants were stored at 80c . the cell pellet was diluted in pbs , and the total cell number was counted with a hemocytometer after staining with trypan blue . cell populations were determined by counting 300 cells / sample , and a percentage was calculated based on a minimum of three mice per group . mh - s alveolar mouse macrophage cells were obtained from atcc ( manassas , va ) . cells from passages 410 were maintained in dulbecco 's modified eagle 's media with 10% fetal bovine serum , 1% l - glutamine , and 1% penicillin / streptomycin mixture at 37c in 5% co2 . prior to experiments , cells were scraped from culture , collected , and centrifuged at 300 rcf for 5 minutes and then counted via trypan blue exclusion . mh - s cells were added in media to 0.4 nm - tissue culture pet - membrane inserts ( falcon ) and placed in media and in six - well plates ( falcon ) . cells were maintained at 37c in 5% co2 between 2 and 24 hours to allow adherence to tissue culture inserts prior to experiments , which was verified by light microscopy . in experiments utilizing serum - free media , mh - s alveolar macrophage cells cultured on inserts were placed into a vitrocell chamber ( waldkirch , germany ) for exposure to whole cigarette smoke ( 2 cigarettes , 7 min / cigarette ) or room air sham for 2 exposures over 24 hours . cells were treated with 40% o2/5% co2 or 21% o2/5% co2 ( control ) between cs exposures . bacteria was harvested from agar plates following 24-hour incubation at 37c , inoculated in lb broth , and then incubated at 37c and 200 rpm until in log phase of growth . bacteria were then diluted to od = 0.1 at 600 nm , approximating 1 10 cfu / ml of live bacteria . mh - s cells were incubated with gfp - labeled pseudomonas aeruginosa ( pao1-gfp ) in phosphate - buffered saline ( pbs ) for 3 hours , during which time exposure to 40% oxygen/5% co2 ( or control , 21% o2/5% co2 ) was continued . after 3 hours of incubation , the media were then removed and plated to determine extracellular pao1-gfp cfu counts or collected for quantification of cytokines . after removal of pao1-gfp inoculum , the mh - s cells on inserts were washed in warm pbs , treated with gentamicin ( 3060 minutes ) to kill adherent bacteria , and then washed twice in warm pbs and scraped from the inserts into detergent solution ( 1% triton - x ) that was plated for intracellular cfu counts using serial dilution . using cell - free bal fluid from mice or supernatants from cell culture , cytokine analysis of tnf- and il-6 was performed by standard elisa kits following the manufacturer 's recommendations ( r&d systems , minneapolis , mn ) . all samples were run in duplicate . for surface staining , primary lung cells or mh - s cells were incubated with fc block-2.4g2 ( bd pharmingen ) ab to block fcg iii / iirs before staining with a specific ab . the following antibodies were purchased from bd pharmingen ( san diego , ca ) and biolegend ( san diego , ca ) : anti - gr1-bv570 , anti - cd11b - petr , anti - cd86-bv421 , anti - mmr - ax647 , anti - dectin-1-ax700 , and anti - f4/80-allophycocyanin - cy7 , along with relevant isotype antibodies . the fitc / ax488 channel was left open for pao1-gfp . for intracellular staining of cytokines , cells were isolated and resuspended ( 0.5 10 cells / ml ) in rpmi 1640/fcs / penicillin / streptomycin / golgi plug ( unstimulated ) or with additional leukocyte activation mixture ( bd biosciences , san jose , ca ; pma + ionomycin + brefeldin a ; 2 ml / ml , stimulated , to enhance intracellular cytokine signal ) for 4 hrs . live - dead discrimination was performed with fixable uv - excitable blue dead cell stain ( invitrogen ) . cells were fc blocked ; surface stained for macrophage , neutrophil , and lymphocyte markers ; and fixed / permeabilized ( cytofix / cytoperm , bd pharmingen , san jose , ca ) and intracellularly stained 30 min for cytokines including anti - tnf--percp . monocytes , alveolar macrophages , neutrophils , and lymphocytes were gated with characteristic forward scatter / side scatter using a facsaria instrument , celldiva for data acquisition ( bd biosciences , san jose , ca ) , and flowjo for analysis ( tree star , san carlos , ca ) . student 's t - test was used for comparisons between two variables with significance determined using the holm - sidak method . multiple comparisons were performed using ordinary one - way anova with bonferroni or tukey 's correction for multiple comparisons . the in vivo model consisted of wild - type c57bl/6 mice exposure to 5 weeks of cigarette smoke via chamber as before controls were age - matched and exposed to room air also via chamber . we have shown that a subacute duration ( 46 weeks ) of cs exposure was associated with increased alveolar epithelial permeability and increased accumulation of inflammatory cells in the alveolar space but did not induce changes in lung architecture . in the current study , we observed a twofold increase in alveolar macrophages recovered by bronchoalveolar lavage ( bal ) from cigarette - smoke - exposed mice compared to sham - exposed controls ( figure 1(a ) ) . the addition of 3 days of continuous 3540% oxygen exposure did not change the bal macrophage count . pao1 , 3 10 cfus ) followed by low - dose oxygen ( 3540% o2 ) or control ( 21% o2 , room air ) for 2 days . compared to air - exposed controls , smoke - exposed mice had increased pao1 cfus recovered from the lung , as others have also shown . however , exposure to 3540% oxygen ( cs + o2 ) resulted in a significant decrease in pao1 cfus recovered from the whole lung compared to smoke - exposed mice exposed to room air ( cs + room air ) ( figure 1(b ) ) . low - dose supplemental oxygen also appeared to reduce bacteremia in cs - exposed mice as the cs + o2 mice had no evidence of bacteremia compared to ~500 pao1 cfus recovered from the blood of mice exposed to room air . furthermore , between groups of cs - exposed mice , mice that received 3540% oxygen for up to 5 days after pao1 exposure had significantly reduced mortality compared to control ( room air ) exposure for the same period ( figure 1(c ) , p = 0.0357 by mantel - cox ) . pao1 in either non - cs - exposed group of mice ( not shown ) . to determine whether the benefits on bacterial clearance translated to other relevant endpoints , we also quantified lung injury parameters . at day 2 after i.t . pao1 , bal protein was significantly increased in cs - exposed mice treated with 21% oxygen ( cs + room air ) compared to non - cs - exposed mice ( figure 1(d ) ) pao1 ( cs + o2 ) reduced bal protein to levels observed in non - cs - exposed mice . in contrast , the bal total cell count at day 2 was not different between groups irrespective of cs or oxygen exposure ( figure 1(e ) ) . therefore , these data suggest that exposing mice to 3540% oxygen following cigarette smoke and pao1 exposure markedly improves bacterial clearance to improve survival , with some associated changes in lung injury parameters . with the significant mortality benefit in o2-exposed mice following cs and i.t . pao1 exposure , we measured bal cytokine and cellular profiles to assess for other phenotypic differences ( figure 2 ) . the addition of 3540% o2 to cs and pao1-exposed mice did not change bal il-6 levels but appeared to reduce bal tnf- at day 2 after i.t . in addition , 3540% oxygen exposure did not change the percentage of alveolar neutrophils ( gr1 ) among cs- and pao1-exposed mice at day 2 after i.t . pao1 ( not shown ) . to further investigate how 3540% oxygen exposure may induce macrophage - specific effects in cs- and bacteria - exposed mice , we used facs analysis to measure tnf- expression in macrophages as well as expression of macrophage surface receptors . after gating on a similar percentage and number of macrophages ( f4 - 80 ) between groups , mice exposed to low - dose oxygen had significantly decreased expression of tnf- by facs compared to room air exposure ( figure 2(b ) ) . cd86 , a proinflammatory m1 macrophage marker involved in cellular cosignaling , was significantly increased on macrophages derived from oxygen - exposed mice following cs and i.t . pao1 exposure significantly increased expression of dectin-1 and trended towards increased expression of mannose receptor , both m2 , anti - inflammatory markers involved in phagocytosis . collectively , these data suggest that low - dose oxygen modulates proinflammatory cytokine production and upregulates expression of macrophage receptors that may be important for bacterial clearance . to further understand macrophage - specific effects induced by oxygen exposure after 24 hours of exposure of mh - s alveolar macrophages to cs ( or control ) and 40% o2 ( or 21% o2 ) , il-6 and tnf- cytokines were quantified in the cell - free media , and macrophage marker expression was evaluated by facs . cs exposure significantly increased il-6 in the cell - free media compared to control ( air ) exposure ( figure 3(a ) ) but did not induce a difference in tnf- at 24 hours , consistent with prior work . cs exposure significantly increased cd86 expression compared to air ( control ) ( figure 3(b ) ) , but 40% oxygen did not further augment cd86 expression . in addition , cs also appeared to increase mmr expression on mh - s cells . following 24 hours of cs ( or control ) exposure , mh - s cells were incubated with p. aeruginosa ( pao1 ) with concurrent exposure to 40% oxygen ( or 21% oxygen ) . after 3 hours , we quantified intracellular pao1 cfus . exposure to 40% oxygen after cs and pao1 resulted in a significant , greater than twofold increase in intracellular pao1 by cfu counts when compared to air or cs - exposed groups that did not receive 40% oxygen ( figure 3(c ) ) . because pao1 was gfp - tagged , we were also able to quantify the association of bacteria with macrophages using facs ( figure 3(d ) ) . similar to the pattern observed with intracellular cfu counts , the addition of 40% oxygen exposure appeared to increase the gfp signal associated with mh - s macrophages both by mean fluorescence intensity ( mfi ) among gfp - positive macrophages , as well as the phagocytosis index ( gfp mfi % pao1-gfp ) . as a potential confounding factor , oxygen levels , we quantified cfus among extracellular bacteria not adherent to or phagocytosed by macrophages . among all 4 exposure groups , we observed a strong inverse correlation ( r = 0.085 ) of extracellular cfus with intracellular cfus from the same well of mh - s cells ( figure 3(e ) ) , suggesting that differences in intracellular bacteria were not attributable to oxygen - induced differences in bacterial growth in the media . collectively , these data demonstrate that isolated macrophages exposed to cigarette smoke can augment bacterial clearance when treated with low - dose supplemental oxygen . we also examined whether oxygen - enhanced bacterial phagocytosis by mh - s cells was associated with changes in its inflammatory profile by measuring selected proinflammatory cytokines and macrophage m1/m2 marker expression following pao1 exposure . although il-6 was not different between groups ( not shown ) , the addition of 40% oxygen to either cs or control - exposed mh - s cells significantly reduced tnf- levels ( figure 4(a ) ) . expression of the m1 marker cd86 was increased on mh - s following cs and pao1 exposure ; the addition of 40% oxygen did not further augment cd86 expression ( figure 4(b ) ) . among the scavenger , m2 receptors , dectin-1 showed a possible oxygen - mediated effect following pao1 exposure , as the fold change of mfi expression was highest on mh - s cells exposed to cs and 40% oxygen ; in contrast , mmr was not different between the groups . using facs to assess pao1-gfp association with mh - s cells , we observed a strong correlation between dectin-1 mfi and pao1-gfp mfi ( r = 0.7905 ) ( figure 4(c ) ) . this data suggests that cells expressing higher levels of dectin-1 also had higher bacterial association and also supports that 40% oxygen was an important modifier of both markers . in contrast , the correlation between cd86 mfi and pao1-gfp mfi or mmr mfi and pao1-gfp mfi was not nearly as strong . we also measured oxygen - induced effects on other macrophage surface receptors including marco and cd200r . cs - exposed mh - s cells demonstrated increased expression of marco ; 40% oxygen did not further regulate marco expression ( figure 5(a ) ) . following subsequent incubation with pao1-gfp , marco expression was not statistically different between groups , although trended towards an increase on macrophages exposed to cs + 40% oxygen . in contrast , the combination of cigarette smoke and 40% oxygen exposure significantly increased cd200r expression ( figure 5(b ) ) compared to control and 21% oxygen . following incubation with pao1-gfp , however , macrophages exposed to cs + 40% oxygen did not significantly increase cd200 expression but did trend in that direction . collectively , this data suggests that low - dose oxygen can regulate expression of multiple scavenger or inhibitory receptors that may be important for clearance of bacteria and other immune - mediated functions . in this study , we sought to understand how oxygen therapy may modulate cigarette smoke - induced immune dysfunction by using an experimental model of cigarette smoke exposure and intrapulmonary inoculation with pseudomonas aeruginosa , followed by room air or low - dose oxygen . pseudomonas aeruginosa is an important respiratory pathogen in patients with lung disease , including copd . the addition of low - dose oxygen ( fio2 = 0.350.40 ) to cigarette smoke - exposed mice improved clearance of pseudomonas aeruginosa leading to a reduction in mortality and measureable differences in lung injury . phenotypically , macrophages derived from these mice upregulated pattern recognition and scavenger receptors . in cell culture , short - term exposure to 40% oxygen following cigarette smoke enhanced macrophage clearance of pao1 . along with supportive data regarding macrophage phenotyping and cytokine production , our findings in cell culture solidify evidence for a direct effect of low - dose oxygen to improve macrophage function . however , the variable effects of 40% oxygen on macrophage surface receptors highlight potentially diverse regulation of phagocytosis . our study identifies a novel , protective role for low - dose oxygen to enhance macrophage phagocytosis of bacteria and mitigate lung inflammation following a subacute duration of cs exposure . although the murine model of subacute cs exposure followed by bacterial challenge may be a reasonable model for some of the inflammatory changes seen with human bronchitis , it does not account for changes related to mucus production or for structural changes seen with emphysema . if similar benefits of low - dose oxygen on promoting bacterial clearance were found in humans with copd or other smoking - related lung diseases , these findings may provide some basis on the benefit of oxygen therapy for patients with smoking - related lung disease during pathogen - induced disease exacerbation [ 1 , 2 ] . in that context , low - dose oxygen therapy may also limit disease progression by enhancing macrophage phagocytosis and thereby limiting the severity of bacteria - induced disease exacerbations . most prior studies demonstrate a detrimental effect of high oxygen levels ( 60100% ) on lung immunity . our findings somewhat contrast the work of baleeiro and colleagues who demonstrated a detrimental effect of sublethal oxygen exposure on host defense against gram - negative pneumonia . however , important differences in study design include the level of oxygen exposure ( 95% versus 40% ) and the use of cigarette smoke . their work identified a reduction in macrophage toll - like receptor 4 ( tlr4 ) expression following 95% oxygen exposure resulting in impaired recognition of gram - negative bacteria . however , in our study , with the addition of 40% oxygen following cs exposure , we did not observe differences in macrophage surface tlr4 expression by flow cytometry ( unpublished observations ) . we have also demonstrated that moderate levels of oxygen exposure ( 60% ) ~12 hours after lps - induced lung inflammation were sufficient to significantly exacerbate lung damage . in one of the few studies of host defense at lower levels of supplemental oxygen , knighton and colleagues showed that 45% oxygen decreased tissue necrosis and increased bacterial clearance compared to 12% oxygen following skin infection with e. coli in guinea pigs . therefore , in addition to the impact of cigarette smoke , these studies also suggest that oxygen - induced effects on lung immunity may be dependent on the level of oxygen exposure . we noted marked changes in macrophage cell surface phagocytic and scavenger receptor expression in response to cigarette smoke and 40% oxygen . interestingly , dectin-1 expression was increased following low - dose oxygen exposure in cs - exposed macrophages , and this increase strongly correlated with increased pao1 uptake by macrophages . dectin-1 is a type ii transmembrane receptor involved in -glucan - derived fungal pathogen immune responses [ 25 , 26 ] . macrophage dectin-1 expression has been shown to be regulated by leukotriene b4 as a part of the gm - csf / pu1.1 axis , and leukotriene b4 levels were elevated in human bal fluid following exposure to 50% oxygen . dectin-1 is critical for clearance of fungal pathogens via recognition of beta - glucans [ 26 , 29 , 30 ] . however , beta - glucans are present in the cell walls of other nonfungal pathogens including p. aeruginosa [ 31 , 32 ] and h. influenza ; for the latter , beta - glucan recognition by epithelial dectin-1 was critical to generate an inflammatory response [ 33 , 34 ] . with the strong correlation between macrophage dectin-1 expression and pao1 phagocytosis among mh - s macrophages in culture , oxygen - induced upregulation of dectin-1 may be contributory towards clearance of pao1 via beta - glucan recognition and binding . we suggest that oxygen improves bacterial phagocytosis through upregulation of cell surface phagocytic and scavenger receptors . one consideration is that room - air - exposed mice were more hypoxic following bacteria exposure and supplemental oxygen may have protected the mice from hypoxia - induced complications . we did not assess for hypoxia in vivo , but we did demonstrate a significant increase in macrophage phagocytosis of bacteria with low - dose oxygen exposure in our cell culture system where hypoxia was not a factor . another potential mechanism by which low - dose supplemental oxygen may improve bacterial phagocytosis is through enhanced production of reactive oxygen species ( ros ) . nadph oxidase , a key enzyme in macrophages for generation of superoxide that requires molecular oxygen , modulated bacterial overgrowth . however , unregulated ros can also be detrimental to bacterial control , as a loss of macrophage - generated extracellular superoxide dismutase ( ec - sod ) , an antioxidant enzyme , was found to impair phagocytosis of e. coli . interestingly , hyperoxia ( 100% oxygen ) in ec - sod knockout mice leads to increased lung edema and diminished survival , a further evidence that unopposed ros may augment lung damage . overall , our findings suggest that oxygen - induced modulation of macrophage function is complex and likely associated with both the underlying lung substrate and the level of supplemental oxygen that is administered . our study also highlights the complex regulation of cell surface signaling receptors that is influenced by supplemental oxygen . to our knowledge , this is the first report of macrophage cd200r expression in response to oxygen and cigarette smoke exposure . cd200r demonstrates an inhibitory effect on inflammatory signaling when engaged by the cd200 ligand that is expressed by the respiratory epithelium and other immunomodulating cells ( e.g. , t cells ) . prior studies have shown decreased expression of cd200r in human monocytes after treatment with diesel emission particles . in contrast , we showed that the combination of cigarette smoke and 40% oxygen exposure increased alveolar macrophage cd200r expression . cd200r may protect against excess cs- and bacteria - induced damage , as it did in a murine influenza model . in addition , due to the important immunomodulatory effects of macrophage cd200r tethering to epithelial cd200 , we would anticipate a synergistic effect in terms of limiting inflammation in a macrophage - epithelial coculture system . one , because we did not directly measure bacterial killing by macrophages , it is conceivable that low - dose oxygen exposure did not enhance phagocytosis , but instead impaired bacterial killing . if true , we would not anticipate a benefit of low - dose oxygen on murine mortality and lung inflammation . two , we did not evaluate for an oxygen - mediated effect on neutrophil clearance of bacteria . although neutrophil numbers in the alveolar space did not appear to be influenced by oxygen following cs and pao1 exposure , we did not directly assess neutrophil function . macrophage cd86 and dectin-1 , both modulated by cs and oxygen exposure in our studies , can promote neutrophil costimulation and pathogen clearance . however , since low - dose oxygen - enhanced macrophage phagocytosis of bacteria in isolated macrophages in culture , any oxygen - mediated effects on neutrophils would likely enhance , not diminish , our findings . three , although the in vitro model for cs delivery induced il-6 secretion by alveolar macrophages , we did not observe a reduction in pao1 phagocytosis amongst cs - exposed mh - s cells as compared to control exposure . however , prior study of cell culture systems investigating bacteria phagocytosis have primarily used cigarette smoke extract ( cse ) and not direct cigarette smoke exposure [ 20 , 41 , 42 ] ; thus little is known about what duration and intensity of direct cs exposure to macrophages are required to induce a change in phagocytosis . four , because we have not proven that upregulation of one or several scavenger receptors is critical for low - dose oxygen to enhance macrophage phagocytosis , it is possible that receptor upregulation is correlative and not causal for oxygen - enhanced phagocytosis . given the concomitant use of diverse pattern recognition receptors including dectin-1 , mmr , and the tlrs by immune cells , we would anticipate synergy between these receptors towards antimicrobial immunity . our results are similar to that of hodge and colleagues who demonstrated a clear benefit of azithromycin exposure on human alveolar macrophage phagocytosis with a correlative increase in mmr expression , yet specific cellular mechanisms were not ascertained . our findings demonstrate a novel , protective role for low - dose oxygen in cigarette smoke and bacteria - exposed mice that appears to be mediated by enhanced macrophage phagocytosis of bacteria . upregulation of scavenger and other pattern recognition receptors also denotes a unique cellular phenotype with coinduction of m1 and m2 macrophage markers . the individual role of these receptors is not yet clear , but our work has identified additional potential therapeutic targets to support enhanced bacterial clearance and decreased inflammation seen in response to 3540% oxygen treatment in the lungs of cigarette - smoke - exposed mice .

background . chronic obstructive pulmonary disease ( copd ) is a common , smoking - related lung disease . patients with copd frequently suffer disease exacerbations induced by bacterial respiratory infections , suggestive of impaired innate immunity . low - dose oxygen is a mainstay of therapy during copd exacerbations ; yet we understand little about whether oxygen can modulate the effects of cigarette smoke on lung immunity . methods . wild - type mice were exposed to cigarette smoke for 5 weeks , followed by intratracheal instillation of pseudomonas aeruginosa ( pao1 ) and 21% or 3540% oxygen . after two days , lungs were harvested for pao1 cfus , and bronchoalveolar fluid was sampled for inflammatory markers . in culture , macrophages were exposed to cigarette smoke and oxygen ( 40% ) for 24 hours and then incubated with pao1 , followed by quantification of bacterial phagocytosis and inflammatory markers . results . mice exposed to 3540% oxygen after cigarette smoke and pao1 had improved survival and reduced lung cfus and inflammation . macrophages from these mice expressed less tnf- and more scavenger receptors . in culture , macrophages exposed to cigarette smoke and oxygen also demonstrated decreased tnf- secretion and enhanced phagocytosis of pao1 bacteria . conclusions . our findings demonstrate a novel , protective role for low - dose oxygen following cigarette smoke and bacteria exposure that may be mediated by enhanced macrophage phagocytosis .

1. Background 2. Methods 3. Results 4. Discussion 5. Conclusion

chronic obstructive pulmonary disease ( copd ) is primarily a smoking - related lung disease and afflicts more than 200 million people worldwide . , airway inflammation leads to increased mucus production and reduced mucociliary clearance , causing bronchoconstriction and airflow limitation . patients with copd frequently suffer disease exacerbations often induced by bacterial or viral respiratory infections , suggestive of impaired innate immunity . during disease exacerbation , although there is clear benefit to continuous low - level oxygen therapy in chronic , stable copd disease [ 1 , 2 ] , excessive amounts of nontitrated oxygen may in fact be harmful during copd exacerbation [ 3 , 4 ] . therefore , how oxygen modulates the effects of cigarette smoke on lung immunity may be relevant for patients with copd exacerbation and other smoke - induced lung diseases . the immunomodulatory effects of moderate and high levels of oxygen exposure ( fio2 0.61.0 ) have been well described in experimental models . mice exposed to four days of 95% oxygen had increased mortality with impaired macrophage phagocytosis in a klebsiella pneumoniae model . although higher levels of oxygen appear to impair lung immunity , there is limited data as to how lower levels of oxygen supplementation ( fio2 0.300.4 ) can modulate lung immunity , particularly with coexisting cigarette smoke exposure . alveolar macrophage dysfunction induced by cigarette smoke exposure leads to excess oxidative stress and may contribute to higher bacterial colonization and increased susceptibility to exacerbations . as a model to help understanding oxygen effects on the lung immune response to bacteria subsequent to cigarette smoke exposure , we challenged mice or macrophages in isolation with subacute durations of cigarette smoke ( cs ) followed by exposure to low - dose oxygen and pseudomonas aeruginosa bacteria . we chose a shorter cigarette smoke exposure to focus on the effects cs and oxygen - induced changes in inflammatory cell populations , similar to bronchitis , and without emphysema - like changes in lung architecture . c57bl/6 mice were purchased ( jackson labs , bar harbor , me ) and housed at the johns hopkins university asthma and allergy center . mice were exposed to cigarette smoke 5 hours / day , 5 days / week for 5 weeks , by burning 3r4f reference cigarettes ( 2.45 mg nicotine / cigarette ; tobacco research institute , university of kentucky ) using a smoking machine ( model te-10 , teague enterprises ) . for specified groups , the morning after a 5-week cigarette smoke ( cs ) exposure , we instilled pseudomonas aeruginosa ( pao1 , atcc , manassas , va ) ( 3 10 cfus , in 50 l pbs ) or vehicle control ( pbs ) via an intratracheal ( i.t . ) within 2 hours of instillation of i.t . pao1 or vehicle control , mice were exposed to 3540% oxygen or 21% oxygen for up to five days . 3540% oxygen was achieved with a mixture of air and medical grade oxygen ( roberts oxygen , rockville , md ) at adjustable flow rates and constant pressure , with continuous measurements via an oxygen analyzer with a feedback loop to automatically adjust oxygen concentrations ( model 65 , advanced micro instruments , huntington beach , ca ) . mice were harvested after 5 weeks of cigarette smoke exposure ( or air exposure as a control ) and 3 days of 3540% oxygen ( or 21% oxygen as a control ) . in addition , following 5 weeks of cs exposure ( or air exposure ) , designated mice were exposed to i.t . pao1 ( or vehicle control ) and 2 days of 3540% oxygen ( or 21% oxygen as control ) until sacrifice and harvest of lungs for assessment of cfus and inflammatory parameters or up to 5 days for assessment of mortality . the lungs were perfused free of blood with 1 ml of phosphate - buffered saline ( pbs ) . for quantitative measures of bacteria , whole lungs were homogenized without prior lavage , and the lysates were diluted in pbs and streaked on agar plates . bal fluid was centrifuged ( 700 g , 10 min at 4c ) , and cell - free supernatants were stored at 80c . prior to experiments , cells were scraped from culture , collected , and centrifuged at 300 rcf for 5 minutes and then counted via trypan blue exclusion . mh - s cells were added in media to 0.4 nm - tissue culture pet - membrane inserts ( falcon ) and placed in media and in six - well plates ( falcon ) . in experiments utilizing serum - free media , mh - s alveolar macrophage cells cultured on inserts were placed into a vitrocell chamber ( waldkirch , germany ) for exposure to whole cigarette smoke ( 2 cigarettes , 7 min / cigarette ) or room air sham for 2 exposures over 24 hours . bacteria was harvested from agar plates following 24-hour incubation at 37c , inoculated in lb broth , and then incubated at 37c and 200 rpm until in log phase of growth . mh - s cells were incubated with gfp - labeled pseudomonas aeruginosa ( pao1-gfp ) in phosphate - buffered saline ( pbs ) for 3 hours , during which time exposure to 40% oxygen/5% co2 ( or control , 21% o2/5% co2 ) was continued . after 3 hours of incubation , the media were then removed and plated to determine extracellular pao1-gfp cfu counts or collected for quantification of cytokines . after removal of pao1-gfp inoculum , the mh - s cells on inserts were washed in warm pbs , treated with gentamicin ( 3060 minutes ) to kill adherent bacteria , and then washed twice in warm pbs and scraped from the inserts into detergent solution ( 1% triton - x ) that was plated for intracellular cfu counts using serial dilution . using cell - free bal fluid from mice or supernatants from cell culture , cytokine analysis of tnf- and il-6 was performed by standard elisa kits following the manufacturer 's recommendations ( r&d systems , minneapolis , mn ) . the following antibodies were purchased from bd pharmingen ( san diego , ca ) and biolegend ( san diego , ca ) : anti - gr1-bv570 , anti - cd11b - petr , anti - cd86-bv421 , anti - mmr - ax647 , anti - dectin-1-ax700 , and anti - f4/80-allophycocyanin - cy7 , along with relevant isotype antibodies . cells were fc blocked ; surface stained for macrophage , neutrophil , and lymphocyte markers ; and fixed / permeabilized ( cytofix / cytoperm , bd pharmingen , san jose , ca ) and intracellularly stained 30 min for cytokines including anti - tnf--percp . monocytes , alveolar macrophages , neutrophils , and lymphocytes were gated with characteristic forward scatter / side scatter using a facsaria instrument , celldiva for data acquisition ( bd biosciences , san jose , ca ) , and flowjo for analysis ( tree star , san carlos , ca ) . the in vivo model consisted of wild - type c57bl/6 mice exposure to 5 weeks of cigarette smoke via chamber as before controls were age - matched and exposed to room air also via chamber . pao1 , 3 10 cfus ) followed by low - dose oxygen ( 3540% o2 ) or control ( 21% o2 , room air ) for 2 days . compared to air - exposed controls , smoke - exposed mice had increased pao1 cfus recovered from the lung , as others have also shown . however , exposure to 3540% oxygen ( cs + o2 ) resulted in a significant decrease in pao1 cfus recovered from the whole lung compared to smoke - exposed mice exposed to room air ( cs + room air ) ( figure 1(b ) ) . low - dose supplemental oxygen also appeared to reduce bacteremia in cs - exposed mice as the cs + o2 mice had no evidence of bacteremia compared to ~500 pao1 cfus recovered from the blood of mice exposed to room air . furthermore , between groups of cs - exposed mice , mice that received 3540% oxygen for up to 5 days after pao1 exposure had significantly reduced mortality compared to control ( room air ) exposure for the same period ( figure 1(c ) , p = 0.0357 by mantel - cox ) . pao1 , bal protein was significantly increased in cs - exposed mice treated with 21% oxygen ( cs + room air ) compared to non - cs - exposed mice ( figure 1(d ) ) pao1 ( cs + o2 ) reduced bal protein to levels observed in non - cs - exposed mice . therefore , these data suggest that exposing mice to 3540% oxygen following cigarette smoke and pao1 exposure markedly improves bacterial clearance to improve survival , with some associated changes in lung injury parameters . in addition , 3540% oxygen exposure did not change the percentage of alveolar neutrophils ( gr1 ) among cs- and pao1-exposed mice at day 2 after i.t . to further investigate how 3540% oxygen exposure may induce macrophage - specific effects in cs- and bacteria - exposed mice , we used facs analysis to measure tnf- expression in macrophages as well as expression of macrophage surface receptors . after gating on a similar percentage and number of macrophages ( f4 - 80 ) between groups , mice exposed to low - dose oxygen had significantly decreased expression of tnf- by facs compared to room air exposure ( figure 2(b ) ) . pao1 exposure significantly increased expression of dectin-1 and trended towards increased expression of mannose receptor , both m2 , anti - inflammatory markers involved in phagocytosis . collectively , these data suggest that low - dose oxygen modulates proinflammatory cytokine production and upregulates expression of macrophage receptors that may be important for bacterial clearance . to further understand macrophage - specific effects induced by oxygen exposure after 24 hours of exposure of mh - s alveolar macrophages to cs ( or control ) and 40% o2 ( or 21% o2 ) , il-6 and tnf- cytokines were quantified in the cell - free media , and macrophage marker expression was evaluated by facs . following 24 hours of cs ( or control ) exposure , mh - s cells were incubated with p. aeruginosa ( pao1 ) with concurrent exposure to 40% oxygen ( or 21% oxygen ) . exposure to 40% oxygen after cs and pao1 resulted in a significant , greater than twofold increase in intracellular pao1 by cfu counts when compared to air or cs - exposed groups that did not receive 40% oxygen ( figure 3(c ) ) . collectively , these data demonstrate that isolated macrophages exposed to cigarette smoke can augment bacterial clearance when treated with low - dose supplemental oxygen . we also examined whether oxygen - enhanced bacterial phagocytosis by mh - s cells was associated with changes in its inflammatory profile by measuring selected proinflammatory cytokines and macrophage m1/m2 marker expression following pao1 exposure . expression of the m1 marker cd86 was increased on mh - s following cs and pao1 exposure ; the addition of 40% oxygen did not further augment cd86 expression ( figure 4(b ) ) . among the scavenger , m2 receptors , dectin-1 showed a possible oxygen - mediated effect following pao1 exposure , as the fold change of mfi expression was highest on mh - s cells exposed to cs and 40% oxygen ; in contrast , mmr was not different between the groups . following subsequent incubation with pao1-gfp , marco expression was not statistically different between groups , although trended towards an increase on macrophages exposed to cs + 40% oxygen . in contrast , the combination of cigarette smoke and 40% oxygen exposure significantly increased cd200r expression ( figure 5(b ) ) compared to control and 21% oxygen . following incubation with pao1-gfp , however , macrophages exposed to cs + 40% oxygen did not significantly increase cd200 expression but did trend in that direction . collectively , this data suggests that low - dose oxygen can regulate expression of multiple scavenger or inhibitory receptors that may be important for clearance of bacteria and other immune - mediated functions . in this study , we sought to understand how oxygen therapy may modulate cigarette smoke - induced immune dysfunction by using an experimental model of cigarette smoke exposure and intrapulmonary inoculation with pseudomonas aeruginosa , followed by room air or low - dose oxygen . pseudomonas aeruginosa is an important respiratory pathogen in patients with lung disease , including copd . the addition of low - dose oxygen ( fio2 = 0.350.40 ) to cigarette smoke - exposed mice improved clearance of pseudomonas aeruginosa leading to a reduction in mortality and measureable differences in lung injury . phenotypically , macrophages derived from these mice upregulated pattern recognition and scavenger receptors . in cell culture , short - term exposure to 40% oxygen following cigarette smoke enhanced macrophage clearance of pao1 . along with supportive data regarding macrophage phenotyping and cytokine production , our findings in cell culture solidify evidence for a direct effect of low - dose oxygen to improve macrophage function . however , the variable effects of 40% oxygen on macrophage surface receptors highlight potentially diverse regulation of phagocytosis . our study identifies a novel , protective role for low - dose oxygen to enhance macrophage phagocytosis of bacteria and mitigate lung inflammation following a subacute duration of cs exposure . although the murine model of subacute cs exposure followed by bacterial challenge may be a reasonable model for some of the inflammatory changes seen with human bronchitis , it does not account for changes related to mucus production or for structural changes seen with emphysema . if similar benefits of low - dose oxygen on promoting bacterial clearance were found in humans with copd or other smoking - related lung diseases , these findings may provide some basis on the benefit of oxygen therapy for patients with smoking - related lung disease during pathogen - induced disease exacerbation [ 1 , 2 ] . in that context , low - dose oxygen therapy may also limit disease progression by enhancing macrophage phagocytosis and thereby limiting the severity of bacteria - induced disease exacerbations . most prior studies demonstrate a detrimental effect of high oxygen levels ( 60100% ) on lung immunity . our findings somewhat contrast the work of baleeiro and colleagues who demonstrated a detrimental effect of sublethal oxygen exposure on host defense against gram - negative pneumonia . however , important differences in study design include the level of oxygen exposure ( 95% versus 40% ) and the use of cigarette smoke . however , in our study , with the addition of 40% oxygen following cs exposure , we did not observe differences in macrophage surface tlr4 expression by flow cytometry ( unpublished observations ) . in one of the few studies of host defense at lower levels of supplemental oxygen , knighton and colleagues showed that 45% oxygen decreased tissue necrosis and increased bacterial clearance compared to 12% oxygen following skin infection with e. coli in guinea pigs . therefore , in addition to the impact of cigarette smoke , these studies also suggest that oxygen - induced effects on lung immunity may be dependent on the level of oxygen exposure . we noted marked changes in macrophage cell surface phagocytic and scavenger receptor expression in response to cigarette smoke and 40% oxygen . interestingly , dectin-1 expression was increased following low - dose oxygen exposure in cs - exposed macrophages , and this increase strongly correlated with increased pao1 uptake by macrophages . with the strong correlation between macrophage dectin-1 expression and pao1 phagocytosis among mh - s macrophages in culture , oxygen - induced upregulation of dectin-1 may be contributory towards clearance of pao1 via beta - glucan recognition and binding . we suggest that oxygen improves bacterial phagocytosis through upregulation of cell surface phagocytic and scavenger receptors . one consideration is that room - air - exposed mice were more hypoxic following bacteria exposure and supplemental oxygen may have protected the mice from hypoxia - induced complications . we did not assess for hypoxia in vivo , but we did demonstrate a significant increase in macrophage phagocytosis of bacteria with low - dose oxygen exposure in our cell culture system where hypoxia was not a factor . another potential mechanism by which low - dose supplemental oxygen may improve bacterial phagocytosis is through enhanced production of reactive oxygen species ( ros ) . overall , our findings suggest that oxygen - induced modulation of macrophage function is complex and likely associated with both the underlying lung substrate and the level of supplemental oxygen that is administered . in contrast , we showed that the combination of cigarette smoke and 40% oxygen exposure increased alveolar macrophage cd200r expression . cd200r may protect against excess cs- and bacteria - induced damage , as it did in a murine influenza model . one , because we did not directly measure bacterial killing by macrophages , it is conceivable that low - dose oxygen exposure did not enhance phagocytosis , but instead impaired bacterial killing . if true , we would not anticipate a benefit of low - dose oxygen on murine mortality and lung inflammation . although neutrophil numbers in the alveolar space did not appear to be influenced by oxygen following cs and pao1 exposure , we did not directly assess neutrophil function . however , since low - dose oxygen - enhanced macrophage phagocytosis of bacteria in isolated macrophages in culture , any oxygen - mediated effects on neutrophils would likely enhance , not diminish , our findings . however , prior study of cell culture systems investigating bacteria phagocytosis have primarily used cigarette smoke extract ( cse ) and not direct cigarette smoke exposure [ 20 , 41 , 42 ] ; thus little is known about what duration and intensity of direct cs exposure to macrophages are required to induce a change in phagocytosis . four , because we have not proven that upregulation of one or several scavenger receptors is critical for low - dose oxygen to enhance macrophage phagocytosis , it is possible that receptor upregulation is correlative and not causal for oxygen - enhanced phagocytosis . given the concomitant use of diverse pattern recognition receptors including dectin-1 , mmr , and the tlrs by immune cells , we would anticipate synergy between these receptors towards antimicrobial immunity . our results are similar to that of hodge and colleagues who demonstrated a clear benefit of azithromycin exposure on human alveolar macrophage phagocytosis with a correlative increase in mmr expression , yet specific cellular mechanisms were not ascertained . our findings demonstrate a novel , protective role for low - dose oxygen in cigarette smoke and bacteria - exposed mice that appears to be mediated by enhanced macrophage phagocytosis of bacteria . the individual role of these receptors is not yet clear , but our work has identified additional potential therapeutic targets to support enhanced bacterial clearance and decreased inflammation seen in response to 3540% oxygen treatment in the lungs of cigarette - smoke - exposed mice .

halogen bonding ( xb ) refers to the attractive noncovalent interaction arising between the electron - deficient -hole of a polarized halogen atom , such as bromine or iodine , and an electron - rich lewis base . while xb has been well - developed in the context of solid - state crystal engineering and in the design of functional materials , the utilization of xb in solution is relatively underexploited with seminal applications in the areas of reactivity , catalysis , self - assembly , transport , and medicinal chemistry only recently being reported . anion supramolecular chemistry has experienced rapid growth over the past few decades stimulated by the crucial roles that anions play in many chemical , biological , and environmental processes . while supramolecular interactions such as hydrogen bonding ( hb ) have dominated the field of anion coordination , recognition , and transport , halogen bonding ( xb ) has only recently begun to emerge as a powerful alternative interaction capable of coordinating anions in competitive solvent media . the strength of xb in solution has been demonstrated to be comparable to hb , while the stringent linear directionality of xb and ph - independence is advantageous for the design of selective receptors for anions with particular geometries . it is noteworthy that the relatively few examples of acyclic and macrocyclic xb anion receptors reported to date all display significantly contrasting anion recognition behavior compared to hb analogues . furthermore , rotaxane interlocked host structural frameworks containing convergent xb triazolium donor groups have been demonstrated to selectively recognize and sense halide anions in highly competitive aqueous media . herein we report a bis - iodotriazole - pyridinium motif , incorporated within both an acyclic and a novel catenane - based receptor , capable of binding anions via two convergent r ix halogen bonds . the catenane host structural framework , prepared via chloride anion templation , is demonstrated to bind halide anions with impressive selectivity in competitive organic in addition , we report for the first time cl and br k - edge xas analysis of the interactions observed in these receptors with anions , revealing significant charge transfer from the halides to the iodotriazole xb donors , and the first measure of covalency in these systems . these data allow for the first quantitative evaluation of the degree of covalency in halogen - bonded species . pre - edge features in the xas spectra are modeled and supplemented with dft calculations and tddft simulations of the xas data which reproduce the experimentally observed pre - edge features . the synthesis of the acyclic bis - triazole - pyridinium receptors is shown in scheme 1 . 3,5-diethynylpyridine 1 was prepared by deprotecting 3,5-bis(trimethylsilylethynyl ) pyridine with koh in methanol , while azides 2a c were prepared via a modification of a literature procedure . the bidentate bis - triazole - pyridinium acyclic precursor , 3a , was prepared via a cuaac click reaction between 1 and 2.2 equiv of hexylazide , prepared in situ from the corresponding bromide . the bidentate bis - iodotriazole - pyridinium acyclic receptor precursors , 3b and 3c , were prepared using a modification of a cuaac click ci which were characterized by high - resolution esi mass spectrometry , h , c nmr , and f as well as p nmr spectroscopy where appropriate . acyclic receptors 4a and , for solubility reasons , 4b were anion exchanged to their corresponding noncoordinating hexafluorophosphate salts in preparation for anion titration experiments , while 4cx was used in x - ray crystallographic structure analysis ( x = cl ) and x - ray absorption spectroscopic analysis ( x = cl , br , pf6 ) . reagents and conditions : ( i ) x = h : cuso45h2o , ascorbic acid , na2co3 , nan3 , hexyl - bromide , 3a : 79% ; x = i : nai , cu(clo4)26h2o , tbta , dbu , thf , 3b : 76% , 3c : 79% ; ( ii ) ch3i , dcm , 4ai : 81% , 4ci : 80% ; ( iii ) nh4pf6(aq ) , 4apf6 : 89% , 4bpf6 : 53% . the anion recognition properties of acyclic receptors 4apf6 and 4bpf6 were investigated using h nmr anion titration experiments by adding aliquots of anions as their tetrabutylammonium salts to solutions of the receptor . initial titrations with tbacl in acetone - d6 determined 1:1 stoichiometric association constants of > 10 m for both receptors . consequently , titrations were repeated in the more competitive cd3cn affording the data in table 1 for hb receptor 4apf6 ; however , association constants for xb receptor 4bpf6 were still > 10 m. as a result , titrations for 4bpf6 were performed in the even more competitive solvent dmso - d6 indicating the superior anion - binding capability of the xb receptor . addition of anions typically caused downfield perturbations of 4apf6 triazole proton d , directly involved in anion coordination , and the 4bpf6 pyridinium proton c incumbent on anion binding ( figure 1 ) . wineqnmr2 analysis of the titration data determined the 1:1 stoichiometric association constants shown in table 1 . anion binding titrations with 4bpf6 following proton c in dmso - d6 : experimental titration data ( circles ) with fitted binding isotherms indicated as lines ( 298 k ) . calculated using wineqnmr2 monitoring the chemical shift data of 4apf6 proton d and 4bpf6 proton c ; estimated standard errors given in parentheses ( 298 k ) . the anion binding selectivity of prototriazole receptor 4apf6 follows anion basicity trends , as would be expected for an anion receptor in an aprotic solvent , with the larger halides complexed more weakly than chloride . similarly , for the xb acyclic receptor 4bpf6 , selectivity follows anion basicity trends for the halides , with oac being the most strongly associated anion ; the association constant , ka , is 2.5 times larger in magnitude than chloride . interestingly , no binding was observed for h2po4 and no3 , while so4 was bound at > 10 m , presumably due to the greater negative charge . to the best of our knowledge , this is only the second example of an xb oxoanion - selective anion receptor . crystals suitable for x - ray diffraction structural analysis were obtained from the chloride salt of the octyl - appended acyclic receptor 4ccl . iodotriazole heterocyclic motif , despite the large size of the iodine substituents ( figure 2 ) . the chelating xbs vary in length from 3.121(2 ) to 3.195(2 ) corresponding to 8486% of the sum of the van der waals halide radii , which is indicative of strong xb interactions to the anion . perspective ( left ) and space - filling representation ( right ) views of the crystal structure of 4ccl . gray = carbon , blue = nitrogen , purple = iodine , green = chlorine . as a result of the enhanced anion recognition properties of the acyclic xb receptor 4bpf6 , in comparison to the hb analogue , 4apf6 , the bis - iodotriazole - pyridinium motif was integrated into a catenane host structural framework . to this end , macrocycle precursor 6 was prepared initially , by reaction of 1 with 2.2 equiv of vinyl - appended azide 5 , via the same modified cuaac methodology ( scheme 2 ) . methylation was achieved using ch3i , and thereafter , the vinyl - appended bis - iodotriazole - pyridinium - containing macrocycle precursor was anion exchanged to the corresponding chloride salt by repeated washing with nh4cl(aq ) to afford 7cl . reagents and conditions : ( i ) nai , cu(clo4)26h2o , tbta , dbu , thf , 56% ; ( ii ) ch3i , dcm , 99% ; ( iii ) nh4cl(aq ) , 66% . synthesis of catenane 9cl was accomplished using chloride templation via a ring - closing metathesis strategy . an initial interpenetrated assembly was prepared by mixing equimolar amounts of macrocycle 8(71 ) with 7cl in dry ch2cl2 . addition of grubbs ii ring - closing metathesis catalyst afforded the catenane in 41% yield following purification by preparative thin layer chromatography . catenane 9cl was characterized by h , c and 2d cosy nmr experiments and high - resolution mass spectrometry . notable shifts in the pyridinium and isophthalamide protons c and 3 , respectively , were observed in addition to the splitting and upfield perturbation of the hydroquinone protons 5 and 6 consistent with inter - ring donor acceptor interactions between the electron - rich hydroquinone groups and the electron - deficient pyridinium group , which confirms the interlocked nature of the catenane ( figure 3 ) . further evidence for the interlocking of the two macrocyclic components was obtained by 2d roesy nmr which revealed through - space interactions including those between hydroquinone and pyridinium protons of the two macrocycles ( see supporting information ( si ) ) . truncated h nmr spectra of 7cl ( top ) , 9cl ( middle ) , and 8 ( bottom ) ( 300 mhz , 293 k , solvent : cdcl3 ) . the chloride anion template was removed by repeated washing with nh4pf6(aq ) to afford 9pf6 for anion recognition studies and was similarly fully characterized ( scheme 3 ) . reagents and conditions : ( i ) grubbs ii ( 10 wt % ) , dcm , 41% ; ( ii ) nh4pf6(aq ) , 86% . association constants for the formation of 1:1 stoichiometric host / guest complexes of catenane 9pf6 with various anions were determined using h nmr titration experiments , monitoring shifts in the internal isophthalamide proton 3 upon addition of aliquots of anions added as their tetrabutylammonium salts ( figure 4 ) . a preliminary titration of 9pf6 with tbacl in 1:1 cdcl3/cd3od resulted in an association constant ka > 10 m , much greater than the value obtained for the previously reported analogous prototriazole - containing hb catenane ( table 2 ) . consequently , all subsequent titrations were conducted in the aqueous solvent mixture 10:45:45 d2o / cdcl3/cd3od , which is more competitive . the association constants shown in table 2 were determined by wineqnmr2 analysis of the titration data . d2o / cdcl3/cd3od : experimental titration data ( circles ) with calculated fit indicated as the line ( 298 k ) . calculated using wineqnmr2 monitoring the internal pyridinium proton ; estimated standard errors given in parentheses ( 293 k , solvent : 10:45:45 d2o / cdcl / cd3od ) . 1:1 cdcl3/cd3od , 298 k. nb : no binding : < 0.07 ppm . very strong binding of the halides indeed , association constants determined for br and i are both > 10 m which highlights the fact that binding is dramatically enhanced in the xb catenane when compared to the hb catenane analogue . furthermore , in contrast , the xb host demonstrates a preference for the larger halides despite the voluminous iodine xb donors . indeed , a reversal in selectivity for the halides is observed for 9pf6 in comparison to the acyclic receptor 4bpf6 due the interlocked host s unique three - dimensional binding cavity . despite the oac selectivity observed for the acyclic xb receptor 4bpf6 , no evidence for oxoanion binding was discerned for oac or h2po4 for the xb catenane 9pf6 . this may be attributed to the oxoanions large size and unfavorable geometry to bind and be encapsulated by the unique interlocked catenane host cavity ( vide infra ) . crystals suitable for x - ray diffraction structural analysis were obtained for catenane 9 with chloride , bromide , iodide , dihydrogen phosphate , and sulfate counteranions . all structures crystallize in p1 with 1:1 complexes obtained for chloride ( figure 5 ) , bromide , and iodide , which are isomorphous , and 2:1 complexes were obtained for dihydrogen phosphate and sulfate , which in turn are isomorphous . while both hydrogen and halogen bonds are observed between the host 9 and the anionic guest , the catenane curiously adopts an open conformation in the solid state rather than completely encapsulating the anion ; presumably this is due to the large iodo - substituted triazole and the most energetically favorable conformation for crystallization . while a varying degree of disorder for the polyether ends of the macrocycles is observed in each complex , it does not affect the anion - binding cavity of the catenane and is omitted for clarity here . perspective ( left ) and space - filling representation ( right ) views of the crystal structure of 9cl . triazole icl halogen bonds ( 3.110(2)3.300(3 ) ) are indicated as red dashed lines . gray = carbon , blue = nitrogen , red = oxygen , purple = iodine , green = chlorine , white = hydrogen . for further information regarding the isomorphous 9br and 9i structures , see si . examining the halide anion structures , strong halogen bonds are observed indicated by bond lengths significantly shorter than the sum of the van der waals radii and are summarized in table 3 . calculated errors given in parentheses . the structure of ( 9)2so4 ( figure 6 ) clearly shows the so4 anion situated between two hosts rather than being encapsulated in the cavity , as it is too large to penetrate the interlocked binding pocket . furthermore , the anion is on the inversion center in the asymmetric unit allowing it to adopt one of two symmetry - related orientations ( see supporting information ) . halogen bonds are formed rather than bifurcated halogen bonds from one o to each of the iodine xb donors . the dihydrogen phosphate structure ( see supporting information ) , while of lower quality , shows a very similar structure ; i.e. , the large oxoanion binds outside of the cavity . crystal structure of ( 9)2so4 showing a 2:1 catenane / anion complex with the anion ( so4 ) positioned between the two catenane hosts rather than penetrating the binding cavity . gray = carbon , blue = nitrogen , red = oxygen , purple = iodine , white = hydrogen , orange = phosphorus , yellow = sulfur . to establish the nature of the interactions between the halide ( cl / br ) donor and the iodine acceptor , we have exploited cl and br k - edge x - ray absorption spectroscopy ( xas ) . it has been clearly established that cl k - edge xas can be used to evaluate the degree of delocalization in chloro - metal bonds . in such cases , a pre - edge feature is observed in the spectrum that formally corresponds to excitation of a cl1s electron to empty valence d - orbitals on the transition metal acceptor . the intensity of such transitions is directly proportional to the amount of cl3p character mixed into these empty metal acceptor orbitals , providing a convenient method of obtaining a quantitative measure of the degree of mixing between the chloro donor and the metal acceptor . this methodology is equally applicable in cases where nonmetal acceptors are under investigation , although to our knowledge it has not been applied to other types of donor acceptor systems . within the context of this work , one would expect a halide ion ( cl and/or br ) to exhibit simple k - edge xanes spectra with no pre - edge features . electric - dipole allowed cl3p cl1s ( or br4p br1s ) transitions would not be observed since these valence p - states are filled ( nsnp ) . covalent delocalization of the filled cl3p orbital with an empty acceptor orbital ( e.g. , via hydrogen or halogen bonding ) would result in the possibility of a new allowed transition corresponding to charge transfer from the chloride to its bonding partner ( axcl * cl1s ) . given that the intensity of such transitions is directly proportional to the amount of cl3p in the final state wave function , axcl * , the intensity of any observable pre - edge feature provides us a direct measure of the degree of covalency in the halogen bond . chlorine k - edge xas data were obtained for a series of donor acceptor complexes at beamline 4 - 3 at the stanford synchrotron radiation lightsource . the near - edge spectra for 4cl and 9cl ( see figure 7 ) each exhibit an intense pre - edge feature that is not present in ionic chloride salts . the presence of this intense feature can only result from charge transfer between the chloro donor and its partners . fitting of the pre - edge and edge features allows experimental quantification of the degree of charge donation in each of these species ( table 4 ) . these results clearly demonstrate charge transfer from the donor via halogen bonding in 4cl , and a combination of xb and hb interactions in 9cl . for comparison , data were also collected on the h - bonding analogue of 4cl ( 10cl ) as well as a simple x - bonded adduct 5-iodo-3-methyl-1,4-dioctyl-1h-1,2,3-triazol-3-ium chloride ( 11cl ) ( figure 7 ; see si for the synthesis of 10cl and 11cl ) . these data provide a measure of the total donation from the chloride anion to all of its bonding partners , and thus the number of interactions must be considered when interpreting these results . normalized and background subtracted : ( a ) cl k- and ( b ) br k - edge xas data for 11cl / br , 4cl / br , 10cl / br , and 9cl / br . calculated errors ( parentheses ) are obtained from statistical distribution of fit results for > 50 fits for each data set . total donation is calculated in comparison with cl k - edge xas data on a cucl4 reference , errors include an estimate of the error in charge transfer of this reference compound . it is apparent that , with a chloride donor , x - bonding interactions are significantly more covalent in character than comparable h - bonding interactions . these data are consistent with previous reports that charge transfer is an important factor in xb bonds . these xas data , however , allow us to quantify the degree of covalency in the xb bond and thus compare the degree of charge transfer in different systems , which to our knowledge is unprecedented . in 11cl , where only a single halogen bonding interaction is possible , we find that the degree of donation is consistent with 6% charge transfer to the iodinated triazole acceptor . the magnitude of charge donation / covalent character in the xb bond is notable in that it is a similar magnitude to that which is commonly observed in transition metal complexes , where covalent contributions are considered to be of great importance in defining chemical properties . for example , in simple divalent metal chlorides ( [ mcl4 ] , where m = cu , ni , co , fe ) , bond covalencies have been determined to range from 6% to 9% . in the bis - triazole 4cl , the total charge donation from the halide ion almost doubles to 11% , which implies that each of the halogen bonds are independent from each other and additive . by contrast , replacement of the iodine acceptors for protons in the bis - triazole ( 10cl ) leads to an almost complete loss of intensity in the cl k - edge xas pre - edge feature , reflecting very poor charge donation through h - bonding in this system . data for the catenane 9cl , where both h- and x - bonds are present , indicate that charge donation from the chloride anion decreases substantially ( as compared to 4cl ) . this presumably reflects weakened halogen bonding due to competition with amide h - bond interactions . qualitatively similar data were obtained for the bromide donor complexes 11br , 4br , 10br , and 9br ( see figure 7 ) . unfortunately , lifetime broadening at the higher energy br k - edge , as well as smaller energy separation between features , leads to poorer resolution of the pre - edge features of interest . the data indicate the presence of a low - energy shoulder , indicating a pre - edge feature to that observed in the cl k - edge data . we note that the pre - edge shoulder is larger for the x - bonded systems as compared to the h - bonded system ( 10br ) . however , quantitative comparisons between the different x - bonded systems are inconclusive due to large errors in the associated fits ( see section s6 ) . to further explore the details of the covalent nature of x - bonding in these systems , we performed a series of density functional calculations , and tddft simulations of the xas data . i in 4cl are calculated to be 3.18(3 ) , which compare favorably with that observed crystallographically ( 3.120(2)3.195(2 ) ) . tddft simulations of the cl k- and br k - edge xas data reproduce the low energy pre - edge feature assigned to the axx * x1s transition in each case . the relevant axx * orbitals for 11cl and 4cl are shown in figure 8 . in each case , these antibonding acceptor orbitals reflect the covalent nature of the x i bond(s ) . two relevant acceptor orbitals are present in 4cl since two x i interactions are possible ; the splitting of these two orbitals in 4cl is small ( 0.3 ev ) , and thus the resulting transitions can not be resolved experimentally . all calculated values are for fully optimized structures at the b3lyp / def2-tzvp+zora level of theory . visual representation of the important axx * kohn sham acceptor orbitals for 11 , 4 , and 9 . tddft - based simulations of the cl k- and br k - edge xas data are in good qualitative agreement with the experimental data ( figure 9 ) . as anticipated , the intensities of the axx * x1s pre - edge transitions correlate with the degree of charge transfer from the halide to the acceptor . the trends observed in the tddft - calculated intensities of the transitions are in reasonable agreement with those observed in the experimental data . however , the calculated pre - edge intensities ( and calculated xb charge donation ) are in good agreement with those obtained from the cl k - edge xas data . trends observed in pre - edge transition energies are also reproduced in the dft results . a small shift to higher energy ( 0.4 ev ) on going from a single xb acceptor ( 11cl ) to two acceptors ( in 4cl ) is consistent with a similar shift observed in the experimental data ( see table 4 ) . this shift correlates with decreased electron density on the chloride , which leads to stabilization of the cl1s donor orbital . tddft simulated cl k - edge ( left ) and br k - edge ( right ) xas spectra for selected xb adducts . a constant linear shift was applied to the calculated energies to better match experimental k - edge energies . the bromide adducts show similar trends as those observed in the chlorides ; the tddft simulations are also in reasonable agreement with the br k - edge experimental data . the calculated degree of charge donation is very similar between the two halides , suggesting that the degree of covalent mixing is similar in both cases . investigations are currently underway to further evaluate the specific factors that affect the covalency of these xb interactions and the resultant binding affinities in the catenanes . in conclusion , the xb bis - iodotriazole - pyridinium motif is shown to be a potent anion - coordinating motif in competitive aqueous media , especially when incorporated into a catenane host structural framework . the acyclic xb receptor displays a marked enhancement in anion recognition over the hb analogue and the first instance of acetate selectivity . moreover , the xb catenane notably exhibits further augmentation of anion recognition ability over its hb analogue , selectively binding halides very strongly in competitive aqueous media with no binding of oxoanions which is the reverse acetate selectivity trend of the acyclic xb receptor . this suggests the xb catenane s unique interlocked host binding cavity is of complementary size and shape for halides , whereas the oxoanions are too large and are of the wrong geometry for encapsulation . single crystal x - ray structural analysis provided solid - state evidence for the association of anions with the catenane host framework where halide anions are able to penetrate the cavity of the catenane . by contrast oxoanions ( h2po4 and so4 ) are found outside the three - dimensional binding pocket in support of the solution - state anion recognition binding observations . cl and br k - edge xas revealed the presence of intense pre - edge features characteristic of charge transfer between the halide donor and the xb acceptor . quantitative fitting of these pre - edge features provided a direct measure of the degree of covalency in the halogen bonding interaction , which is comparable to that observed in transition metal complexes . furthermore , we confirm that perpendicular xb interactions are independent and additive , but that the degree of xb covalency can be mitigated through the presence of hb donors . these conclusions are well supported and substantiated by tddft simulations of the xas data , which indicate that the degree of covalency is essentially the same for both cl and br donors . most importantly , these results offer the first evidence of this kind for covalency in halogen bonds of anion receptors with halide anions . furthermore , the presence of both xb and hb donors in the catenane host framework provides interesting insight into the interplay between these two competing interactions in a single anion receptor system .

the synthesis and anion binding properties of novel halogen - bonding ( xb ) bis - iodotriazole - pyridinium - containing acyclic and [ 2]catenane anion host systems are described . the xb acyclic receptor displays selectivity for acetate over halides with enhanced anion recognition properties compared to the analogous hydrogen - bonding ( hb ) acyclic receptor . a reversal in halide selectivity is observed in the xb [ 2]catenane , in comparison to the acyclic xb receptor , due to the interlocked host s unique three - dimensional binding cavity , and no binding is observed for oxoanions . notable halide anion association constant values determined for the [ 2]catenane in competitive organic aqueous solvent mixtures demonstrate considerable enhancement of anion recognition as compared to the hb catenane analogue . x - ray crystallographic analysis of a series of halide catenane complexes reveal strong xb interactions in the solid state . these interactions were studied using cl and br k - edge x - ray absorption spectroscopy ( xas ) indicating intense pre - edge features characteristic of charge transfer from the halide to its bonding partner ( axx * x1s ) , and providing a direct measure of the degree of covalency in the halogen bond(s ) . the data reveal that the degree of covalency is similar to that which is observed in transition metal coordinate covalent bonds . these results are supported by dft results , which correlate well with the experimental data .

Introduction Results and Discussion Conclusion

halogen bonding ( xb ) refers to the attractive noncovalent interaction arising between the electron - deficient -hole of a polarized halogen atom , such as bromine or iodine , and an electron - rich lewis base . while supramolecular interactions such as hydrogen bonding ( hb ) have dominated the field of anion coordination , recognition , and transport , halogen bonding ( xb ) has only recently begun to emerge as a powerful alternative interaction capable of coordinating anions in competitive solvent media . it is noteworthy that the relatively few examples of acyclic and macrocyclic xb anion receptors reported to date all display significantly contrasting anion recognition behavior compared to hb analogues . herein we report a bis - iodotriazole - pyridinium motif , incorporated within both an acyclic and a novel catenane - based receptor , capable of binding anions via two convergent r ix halogen bonds . the catenane host structural framework , prepared via chloride anion templation , is demonstrated to bind halide anions with impressive selectivity in competitive organic in addition , we report for the first time cl and br k - edge xas analysis of the interactions observed in these receptors with anions , revealing significant charge transfer from the halides to the iodotriazole xb donors , and the first measure of covalency in these systems . these data allow for the first quantitative evaluation of the degree of covalency in halogen - bonded species . pre - edge features in the xas spectra are modeled and supplemented with dft calculations and tddft simulations of the xas data which reproduce the experimentally observed pre - edge features . the synthesis of the acyclic bis - triazole - pyridinium receptors is shown in scheme 1 . the bidentate bis - triazole - pyridinium acyclic precursor , 3a , was prepared via a cuaac click reaction between 1 and 2.2 equiv of hexylazide , prepared in situ from the corresponding bromide . the bidentate bis - iodotriazole - pyridinium acyclic receptor precursors , 3b and 3c , were prepared using a modification of a cuaac click ci which were characterized by high - resolution esi mass spectrometry , h , c nmr , and f as well as p nmr spectroscopy where appropriate . acyclic receptors 4a and , for solubility reasons , 4b were anion exchanged to their corresponding noncoordinating hexafluorophosphate salts in preparation for anion titration experiments , while 4cx was used in x - ray crystallographic structure analysis ( x = cl ) and x - ray absorption spectroscopic analysis ( x = cl , br , pf6 ) . the anion recognition properties of acyclic receptors 4apf6 and 4bpf6 were investigated using h nmr anion titration experiments by adding aliquots of anions as their tetrabutylammonium salts to solutions of the receptor . consequently , titrations were repeated in the more competitive cd3cn affording the data in table 1 for hb receptor 4apf6 ; however , association constants for xb receptor 4bpf6 were still > 10 m. as a result , titrations for 4bpf6 were performed in the even more competitive solvent dmso - d6 indicating the superior anion - binding capability of the xb receptor . similarly , for the xb acyclic receptor 4bpf6 , selectivity follows anion basicity trends for the halides , with oac being the most strongly associated anion ; the association constant , ka , is 2.5 times larger in magnitude than chloride . interestingly , no binding was observed for h2po4 and no3 , while so4 was bound at > 10 m , presumably due to the greater negative charge . crystals suitable for x - ray diffraction structural analysis were obtained from the chloride salt of the octyl - appended acyclic receptor 4ccl . the chelating xbs vary in length from 3.121(2 ) to 3.195(2 ) corresponding to 8486% of the sum of the van der waals halide radii , which is indicative of strong xb interactions to the anion . as a result of the enhanced anion recognition properties of the acyclic xb receptor 4bpf6 , in comparison to the hb analogue , 4apf6 , the bis - iodotriazole - pyridinium motif was integrated into a catenane host structural framework . methylation was achieved using ch3i , and thereafter , the vinyl - appended bis - iodotriazole - pyridinium - containing macrocycle precursor was anion exchanged to the corresponding chloride salt by repeated washing with nh4cl(aq ) to afford 7cl . notable shifts in the pyridinium and isophthalamide protons c and 3 , respectively , were observed in addition to the splitting and upfield perturbation of the hydroquinone protons 5 and 6 consistent with inter - ring donor acceptor interactions between the electron - rich hydroquinone groups and the electron - deficient pyridinium group , which confirms the interlocked nature of the catenane ( figure 3 ) . truncated h nmr spectra of 7cl ( top ) , 9cl ( middle ) , and 8 ( bottom ) ( 300 mhz , 293 k , solvent : cdcl3 ) . a preliminary titration of 9pf6 with tbacl in 1:1 cdcl3/cd3od resulted in an association constant ka > 10 m , much greater than the value obtained for the previously reported analogous prototriazole - containing hb catenane ( table 2 ) . consequently , all subsequent titrations were conducted in the aqueous solvent mixture 10:45:45 d2o / cdcl3/cd3od , which is more competitive . very strong binding of the halides indeed , association constants determined for br and i are both > 10 m which highlights the fact that binding is dramatically enhanced in the xb catenane when compared to the hb catenane analogue . furthermore , in contrast , the xb host demonstrates a preference for the larger halides despite the voluminous iodine xb donors . indeed , a reversal in selectivity for the halides is observed for 9pf6 in comparison to the acyclic receptor 4bpf6 due the interlocked host s unique three - dimensional binding cavity . despite the oac selectivity observed for the acyclic xb receptor 4bpf6 , no evidence for oxoanion binding was discerned for oac or h2po4 for the xb catenane 9pf6 . crystals suitable for x - ray diffraction structural analysis were obtained for catenane 9 with chloride , bromide , iodide , dihydrogen phosphate , and sulfate counteranions . all structures crystallize in p1 with 1:1 complexes obtained for chloride ( figure 5 ) , bromide , and iodide , which are isomorphous , and 2:1 complexes were obtained for dihydrogen phosphate and sulfate , which in turn are isomorphous . while both hydrogen and halogen bonds are observed between the host 9 and the anionic guest , the catenane curiously adopts an open conformation in the solid state rather than completely encapsulating the anion ; presumably this is due to the large iodo - substituted triazole and the most energetically favorable conformation for crystallization . while a varying degree of disorder for the polyether ends of the macrocycles is observed in each complex , it does not affect the anion - binding cavity of the catenane and is omitted for clarity here . examining the halide anion structures , strong halogen bonds are observed indicated by bond lengths significantly shorter than the sum of the van der waals radii and are summarized in table 3 . the structure of ( 9)2so4 ( figure 6 ) clearly shows the so4 anion situated between two hosts rather than being encapsulated in the cavity , as it is too large to penetrate the interlocked binding pocket . to establish the nature of the interactions between the halide ( cl / br ) donor and the iodine acceptor , we have exploited cl and br k - edge x - ray absorption spectroscopy ( xas ) . it has been clearly established that cl k - edge xas can be used to evaluate the degree of delocalization in chloro - metal bonds . in such cases , a pre - edge feature is observed in the spectrum that formally corresponds to excitation of a cl1s electron to empty valence d - orbitals on the transition metal acceptor . the intensity of such transitions is directly proportional to the amount of cl3p character mixed into these empty metal acceptor orbitals , providing a convenient method of obtaining a quantitative measure of the degree of mixing between the chloro donor and the metal acceptor . within the context of this work , one would expect a halide ion ( cl and/or br ) to exhibit simple k - edge xanes spectra with no pre - edge features . , via hydrogen or halogen bonding ) would result in the possibility of a new allowed transition corresponding to charge transfer from the chloride to its bonding partner ( axcl * cl1s ) . given that the intensity of such transitions is directly proportional to the amount of cl3p in the final state wave function , axcl * , the intensity of any observable pre - edge feature provides us a direct measure of the degree of covalency in the halogen bond . chlorine k - edge xas data were obtained for a series of donor acceptor complexes at beamline 4 - 3 at the stanford synchrotron radiation lightsource . the near - edge spectra for 4cl and 9cl ( see figure 7 ) each exhibit an intense pre - edge feature that is not present in ionic chloride salts . fitting of the pre - edge and edge features allows experimental quantification of the degree of charge donation in each of these species ( table 4 ) . these results clearly demonstrate charge transfer from the donor via halogen bonding in 4cl , and a combination of xb and hb interactions in 9cl . for comparison , data were also collected on the h - bonding analogue of 4cl ( 10cl ) as well as a simple x - bonded adduct 5-iodo-3-methyl-1,4-dioctyl-1h-1,2,3-triazol-3-ium chloride ( 11cl ) ( figure 7 ; see si for the synthesis of 10cl and 11cl ) . these data provide a measure of the total donation from the chloride anion to all of its bonding partners , and thus the number of interactions must be considered when interpreting these results . normalized and background subtracted : ( a ) cl k- and ( b ) br k - edge xas data for 11cl / br , 4cl / br , 10cl / br , and 9cl / br . total donation is calculated in comparison with cl k - edge xas data on a cucl4 reference , errors include an estimate of the error in charge transfer of this reference compound . it is apparent that , with a chloride donor , x - bonding interactions are significantly more covalent in character than comparable h - bonding interactions . these xas data , however , allow us to quantify the degree of covalency in the xb bond and thus compare the degree of charge transfer in different systems , which to our knowledge is unprecedented . in 11cl , where only a single halogen bonding interaction is possible , we find that the degree of donation is consistent with 6% charge transfer to the iodinated triazole acceptor . the magnitude of charge donation / covalent character in the xb bond is notable in that it is a similar magnitude to that which is commonly observed in transition metal complexes , where covalent contributions are considered to be of great importance in defining chemical properties . in the bis - triazole 4cl , the total charge donation from the halide ion almost doubles to 11% , which implies that each of the halogen bonds are independent from each other and additive . by contrast , replacement of the iodine acceptors for protons in the bis - triazole ( 10cl ) leads to an almost complete loss of intensity in the cl k - edge xas pre - edge feature , reflecting very poor charge donation through h - bonding in this system . data for the catenane 9cl , where both h- and x - bonds are present , indicate that charge donation from the chloride anion decreases substantially ( as compared to 4cl ) . unfortunately , lifetime broadening at the higher energy br k - edge , as well as smaller energy separation between features , leads to poorer resolution of the pre - edge features of interest . the data indicate the presence of a low - energy shoulder , indicating a pre - edge feature to that observed in the cl k - edge data . we note that the pre - edge shoulder is larger for the x - bonded systems as compared to the h - bonded system ( 10br ) . however , quantitative comparisons between the different x - bonded systems are inconclusive due to large errors in the associated fits ( see section s6 ) . to further explore the details of the covalent nature of x - bonding in these systems , we performed a series of density functional calculations , and tddft simulations of the xas data . tddft simulations of the cl k- and br k - edge xas data reproduce the low energy pre - edge feature assigned to the axx * x1s transition in each case . visual representation of the important axx * kohn sham acceptor orbitals for 11 , 4 , and 9 . tddft - based simulations of the cl k- and br k - edge xas data are in good qualitative agreement with the experimental data ( figure 9 ) . as anticipated , the intensities of the axx * x1s pre - edge transitions correlate with the degree of charge transfer from the halide to the acceptor . the trends observed in the tddft - calculated intensities of the transitions are in reasonable agreement with those observed in the experimental data . however , the calculated pre - edge intensities ( and calculated xb charge donation ) are in good agreement with those obtained from the cl k - edge xas data . trends observed in pre - edge transition energies are also reproduced in the dft results . a small shift to higher energy ( 0.4 ev ) on going from a single xb acceptor ( 11cl ) to two acceptors ( in 4cl ) is consistent with a similar shift observed in the experimental data ( see table 4 ) . tddft simulated cl k - edge ( left ) and br k - edge ( right ) xas spectra for selected xb adducts . a constant linear shift was applied to the calculated energies to better match experimental k - edge energies . the bromide adducts show similar trends as those observed in the chlorides ; the tddft simulations are also in reasonable agreement with the br k - edge experimental data . the calculated degree of charge donation is very similar between the two halides , suggesting that the degree of covalent mixing is similar in both cases . investigations are currently underway to further evaluate the specific factors that affect the covalency of these xb interactions and the resultant binding affinities in the catenanes . in conclusion , the xb bis - iodotriazole - pyridinium motif is shown to be a potent anion - coordinating motif in competitive aqueous media , especially when incorporated into a catenane host structural framework . the acyclic xb receptor displays a marked enhancement in anion recognition over the hb analogue and the first instance of acetate selectivity . moreover , the xb catenane notably exhibits further augmentation of anion recognition ability over its hb analogue , selectively binding halides very strongly in competitive aqueous media with no binding of oxoanions which is the reverse acetate selectivity trend of the acyclic xb receptor . this suggests the xb catenane s unique interlocked host binding cavity is of complementary size and shape for halides , whereas the oxoanions are too large and are of the wrong geometry for encapsulation . single crystal x - ray structural analysis provided solid - state evidence for the association of anions with the catenane host framework where halide anions are able to penetrate the cavity of the catenane . by contrast oxoanions ( h2po4 and so4 ) are found outside the three - dimensional binding pocket in support of the solution - state anion recognition binding observations . cl and br k - edge xas revealed the presence of intense pre - edge features characteristic of charge transfer between the halide donor and the xb acceptor . quantitative fitting of these pre - edge features provided a direct measure of the degree of covalency in the halogen bonding interaction , which is comparable to that observed in transition metal complexes . furthermore , we confirm that perpendicular xb interactions are independent and additive , but that the degree of xb covalency can be mitigated through the presence of hb donors . these conclusions are well supported and substantiated by tddft simulations of the xas data , which indicate that the degree of covalency is essentially the same for both cl and br donors . most importantly , these results offer the first evidence of this kind for covalency in halogen bonds of anion receptors with halide anions .

bronchiectasis is a multidimensional disease , and hence , its severity or prognosis could not be defined with any precision using a single variable.13 accordingly , some recently published multidimensional scores combine clinical , functional , microbiological , and radiological variables to evaluate the prognosis and severity of bronchiectasis more comprehensively.2,3 one of these scores is the faced score,3 an acronym derived from five dichotomized variables ( f : forced expiratory volume in 1 s [ fev1 ] ; a : age ; c : chronic colonization by pseudomonas aeruginosa [ pa ] , e : radiological extension [ number of pulmonary lobes affected ] , and d : dyspnea ) . the faced score is easy to remember and to apply in clinical practice ; it presents a range of values of growing severity spanning 07 points , and it has demonstrated an excellent prognostic capacity for mortality ( both all - cause and respiratory ) in both medium and long term , and an excellent capacity to discriminate different degrees of severity.4 it also had a good external validation in europe as well as in various latin american countries in bronchiectasis patients with wide - ranging etiology and severity and with characteristics different from those of the initial cohort.46 nevertheless , some authors have found that the faced score does not predict exacerbations accurately.6 one of the key clinical aspects in the prognosis and severity of airway diseases is exacerbations , particularly serious ones that require hospitalization . in bronchiectasis , exacerbations have been associated with higher mortality2 and a greater impairment of lung function,7 as well as more severe forms of the disease.8,9 furthermore , another important aspect of exacerbations in these patients is the substantial concomitant health costs.10 as exacerbations are potentially preventable , exacerbations and hospitalizations are main outcomes in most of the clinical studies that seek to evaluate the efficacy and , above all , cost - efficiency of different treatments for bronchiectasis.1116 hence , tools that can predict future exacerbations and particularly those patients ar risk of presenting multiple exacerbations ( especially severe ones requiring hospitalizations ) are required . since the faced score was not constructed with the variable exacerbations , the present study aimed to analyze , in a wide - ranging series of patients different from the original faced cohort , the prognostic capacity for exacerbations ( and more particularly for patients with multiple exacerbations ) of a new score called e - faced and compare it with that of faced . it also sought to evaluate whether e - faced also maintains the faced score s excellent predictive capacity for all - cause and respiratory mortality , as well as its capacity to discriminate different degrees of severity of bronchiectasis . this is an observational , multicenter study of two large historical cohorts involving seven teaching centers in spain ( construction series ) and six teaching centers in latin america ( validation series ) , all with multidisciplinary and protocolized monographic bronchiectasis outpatient clinics . the spanish series corresponds with the one used for the construction and internal validation of the faced score . it comprised a total of 819 patients with bronchiectasis whose characteristics have previously been published.3 this series will be used as the construction cohort for the e - faced . meanwhile , 651 patients with bronchiectasis from six latin american centers ( four in brazil , one in argentina , and one in chile ) who were the subjects for the external validation of the faced score will also be used for the external validation of the e - faced score . all the patients were diagnosed as having bronchiectasis by means of a primary diagnosis with hrct , and they presented a wide range of severity , etiologies , and clinical and functional alterations , following the criteria described by naidich et al.17 patients aged < 18 years were excluded , along with those whose vital state was unknown at the end of the follow - up . this study was approved by the ethics and research committee of each participating center ( table s1 ) . the variables recorded in both the construction cohort ( n=819 ) and the validation cohort ( n=651 ) were the same as those used in the original studies for the construction , internal validation , and external validation of the faced score . in other words , the recorded variables corresponded as closely as possible to a radiological diagnosis of bronchiectasis . the exacerbations , hospitalizations , and microbiological data of the construction cohort pertained to the year prior to the patients inclusion in the study , while these data were also available longitudinally for the validation cohort during the 5 years of the follow - up . since there is no standard definition of an exacerbation , this study defined an exacerbation as a worsening of the respiratory symptoms concomitant with an increase in the volume or purulence of sputum that requires antibiotic treatment . this enabled identification of the number of exacerbations more objectively , according to the number of antibiotic treatments prescribed for changes in the volume or purulence of sputum or symptoms . similarly , a severe exacerbation was defined as one that was considered by the physician to require hospitalization . patients from both cohorts were followed up for 5 years after the radiological diagnosis of bronchiectasis . the number of deaths and their causes were identified via the hospitals computerized records or the corresponding death certificates . the main variable was the prognostic capacity for exacerbations in a year , according to different cut - off points as regards number and severity . the secondary outcomes were established as the predictive capacity for death from any cause or from respiratory causes , and the capacity to discriminate different degrees of severity . the spss 20.0 ( ibm corporation , armonk , ny , usa ) statistical package was used for all the calculations . the quantitative variables were evaluated as mean ( standard deviation ) in cases of normal distribution or as median ( inter - quartile range ) in other cases . the qualitative or dichotomic variables were evaluated as an absolute value ( percentage of the total ) . various logistic regressions were made to determine the optimal cut - off point for the number of exacerbations and hospitalizations that would add the best independent prognostic value to the faced score . the independent values in the construction series ( n=819 ) , apart from the value of the faced itself , the cut - off points of at least one , at least two , or more than two exacerbations or hospitalizations in the year prior to inclusion in the study , including a composite of at least two exacerbations or one hospitalization in the previous year . the dependent variable was the number of deaths from any cause in the spanish construction cohort ( 819 patients ) . the prognostic capacity of the best score for the number or severity of the exacerbations was quantified by means of the odds ratio ( or ; 95% confidence interval [ 95% ci ] ) , while the value of the or , rounded up to its highest whole value , was used as a relative weight for the variable in the new score that was formed ( e - faced ) . in order to simplify the score , only different cut - off points were used for the number or severity of exacerbations , to make the variable dichotomic , as in the original faced score . once the range of the e - faced score had been calculated and obtained , its prognostic capacity for future exacerbations and hospitalizations over the course of a year ( according to different cut - off points for number and severity ) was determined by using the external validation database ( n=651 ) and calculating the corresponding areas under the receiver operating characteristic curve ( auc - roc ) and 95% ci . a prognostic capacity was considered excellent if the auc - roc is > 80% of the total area.18 the prognostic capacity of the e - faced for all - cause and respiratory mortality was then calculated , again by using the auc - roc . all the results were compared with those obtained from the application of the faced score to the same series of patients . the roc curves obtained from the e - faced and faced scores were compared by means of c - statistics . finally , the kaplan meier curves and the log - rank test for comparing the curves two by two were used to determine the capacity of the new index ( e - faced ) to discriminate the degree of severity , in order to divide bronchiectasis into mild ( first tertile ) , moderate ( second tertile ) , or severe ( third tertile ) . in all cases , this is an observational , multicenter study of two large historical cohorts involving seven teaching centers in spain ( construction series ) and six teaching centers in latin america ( validation series ) , all with multidisciplinary and protocolized monographic bronchiectasis outpatient clinics . the spanish series corresponds with the one used for the construction and internal validation of the faced score . it comprised a total of 819 patients with bronchiectasis whose characteristics have previously been published.3 this series will be used as the construction cohort for the e - faced . meanwhile , 651 patients with bronchiectasis from six latin american centers ( four in brazil , one in argentina , and one in chile ) who were the subjects for the external validation of the faced score will also be used for the external validation of the e - faced score . all the patients were diagnosed as having bronchiectasis by means of a primary diagnosis with hrct , and they presented a wide range of severity , etiologies , and clinical and functional alterations , following the criteria described by naidich et al.17 patients aged < 18 years were excluded , along with those whose vital state was unknown at the end of the follow - up . this study was approved by the ethics and research committee of each participating center ( table s1 ) . the variables recorded in both the construction cohort ( n=819 ) and the validation cohort ( n=651 ) were the same as those used in the original studies for the construction , internal validation , and external validation of the faced score . in other words , the recorded variables corresponded as closely as possible to a radiological diagnosis of bronchiectasis . the exacerbations , hospitalizations , and microbiological data of the construction cohort pertained to the year prior to the patients inclusion in the study , while these data were also available longitudinally for the validation cohort during the 5 years of the follow - up . since there is no standard definition of an exacerbation , this study defined an exacerbation as a worsening of the respiratory symptoms concomitant with an increase in the volume or purulence of sputum that requires antibiotic treatment . this enabled identification of the number of exacerbations more objectively , according to the number of antibiotic treatments prescribed for changes in the volume or purulence of sputum or symptoms . similarly , a severe exacerbation was defined as one that was considered by the physician to require hospitalization . patients from both cohorts were followed up for 5 years after the radiological diagnosis of bronchiectasis . the number of deaths and their causes were identified via the hospitals computerized records or the corresponding death certificates . the main variable was the prognostic capacity for exacerbations in a year , according to different cut - off points as regards number and severity . the secondary outcomes were established as the predictive capacity for death from any cause or from respiratory causes , and the capacity to discriminate different degrees of severity . the spss 20.0 ( ibm corporation , armonk , ny , usa ) statistical package was used for all the calculations . the quantitative variables were evaluated as mean ( standard deviation ) in cases of normal distribution or as median ( inter - quartile range ) in other cases . the qualitative or dichotomic variables were evaluated as an absolute value ( percentage of the total ) . various logistic regressions were made to determine the optimal cut - off point for the number of exacerbations and hospitalizations that would add the best independent prognostic value to the faced score . the independent values in the construction series ( n=819 ) , apart from the value of the faced itself , the cut - off points of at least one , at least two , or more than two exacerbations or hospitalizations in the year prior to inclusion in the study , including a composite of at least two exacerbations or one hospitalization in the previous year . the dependent variable was the number of deaths from any cause in the spanish construction cohort ( 819 patients ) . the prognostic capacity of the best score for the number or severity of the exacerbations was quantified by means of the odds ratio ( or ; 95% confidence interval [ 95% ci ] ) , while the value of the or , rounded up to its highest whole value , was used as a relative weight for the variable in the new score that was formed ( e - faced ) . in order to simplify the score , only different cut - off points were used for the number or severity of exacerbations , to make the variable dichotomic , as in the original faced score . once the range of the e - faced score had been calculated and obtained , its prognostic capacity for future exacerbations and hospitalizations over the course of a year ( according to different cut - off points for number and severity ) was determined by using the external validation database ( n=651 ) and calculating the corresponding areas under the receiver operating characteristic curve ( auc - roc ) and 95% ci . a prognostic capacity was considered excellent if the auc - roc is > 80% of the total area.18 the prognostic capacity of the e - faced for all - cause and respiratory mortality was then calculated , again by using the auc - roc . all the results were compared with those obtained from the application of the faced score to the same series of patients . the roc curves obtained from the e - faced and faced scores were compared by means of c - statistics . finally , the kaplan meier curves and the log - rank test for comparing the curves two by two were used to determine the capacity of the new index ( e - faced ) to discriminate the degree of severity , in order to divide bronchiectasis into mild ( first tertile ) , moderate ( second tertile ) , or severe ( third tertile ) . in all cases , the construction cohort comprised 819 patients with bronchiectasis from the initial spanish cohort for the construction and internal validation of the faced , whereas the external validation cohort ( n=651 ) comprised the latin american cohort for the external validation of the faced . data from 20 patients from the initial spanish cohort ( 2.4% ) and 21 patients from the latin american cohort ( 3.1% ) were not obtained , mainly because of lack of information on their vital status at the end of the study ( figure 1 ) . the differential characteristics of both cohorts are shown in table 1 . as can be seen , patients in the external validation cohort are younger , and they present greater clinical and functional severity , a greater radiological extension of bronchiectasis , a lower percentage of etiologies of unknown origin , and a significantly lower number of exacerbations . table 2 shows the results of the logistical regressions , which included different cut - off points for the number of exacerbations and hospitalizations in the previous year ( at least one , at least two , or more than two , as well as a composite of two exacerbations or at least one hospitalization ) to evaluate the faced score s independent prognostic capacity for all - cause mortality . as can be seen , the cut - off point that added the most statistically significant prognostic value to the faced score was at least one hospitalization in the previous year , with an or of 1.8 , and so this was the variable chosen to be added to the faced . this process therefore added two extra points to the faced score in cases of at least one hospitalization in the previous year , or none in cases of no hospitalizations . table 3 shows that the e - faced score has a range of values of 09 points . the three tertiles running from least to greatest severity are therefore 03 points , 46 points , and 79 points . table 4 shows how the prognostic capacity of the e - faced score was significantly greater than that of the faced , in both the number and severity ( hospitalizations ) of the future exacerbations over the course of a year . the greatest benefit from e - faced was found in the identification of patients with future multiple exacerbations ( at least two exacerbations per year [ figure 2 ] , or at least two exacerbations or one hospitalization per year ) . figure 3 shows the auc - roc of both the e - faced and faced scores for all - cause mortality in the initial construction cohort ( n=819 ) . these are 0.87 ( 0.830.91 ) and 0.87 ( 0.820.91 ) , respectively , with no significant differences between them ; they are always > 0.80 , which means that they both present an excellent prognostic capacity . furthermore , in the case of prognostic capacity for respiratory deaths , the results were again similarly excellent : the e - faced score presented an auc - roc of 0.86 ( 0.810.9 ) , whereas the faced score presented one of 0.82 ( 0.780.87 ) . similarly , when both the e - faced and the faced scores were applied to the series of 651 patients from the latin american cohorts , the auc - rocs were 0.84 ( 0.800.88 ) and 0.81 ( 0.770.86 ) , respectively , which are as excellent as those of the construction cohort , with no statistically significant differences between the two . furthermore , in the case of the prognostic capacity for respiratory mortality , the e - faced presented an auc - roc of 0.87 ( 0.830.91 ) and the faced one of 0.84 ( 0.800.88 ) ( figure 4 ) . it was also demonstrated ( figure 5 ) that the three tertiles of both the faced and the e - faced scores accurately differentiated patients into three groups of increasing severity , with no significant differences between the classifications created by the faced and e - faced scores . similarly , the comparative division of this validation cohort into three tertiles in both the e - faced and the faced scores also presented an excellent discriminatory capacity in three groups of increasing severity , with no significant differences from the construction cohort ( figure 6 ) . table 2 shows the results of the logistical regressions , which included different cut - off points for the number of exacerbations and hospitalizations in the previous year ( at least one , at least two , or more than two , as well as a composite of two exacerbations or at least one hospitalization ) to evaluate the faced score s independent prognostic capacity for all - cause mortality . as can be seen , the cut - off point that added the most statistically significant prognostic value to the faced score was at least one hospitalization in the previous year , with an or of 1.8 , and so this was the variable chosen to be added to the faced . this process therefore added two extra points to the faced score in cases of at least one hospitalization in the previous year , or none in cases of no hospitalizations . table 3 shows that the e - faced score has a range of values of 09 points . the three tertiles running from least to greatest severity are therefore 03 points , 46 points , and 79 points . table 4 shows how the prognostic capacity of the e - faced score was significantly greater than that of the faced , in both the number and severity ( hospitalizations ) of the future exacerbations over the course of a year . the greatest benefit from e - faced was found in the identification of patients with future multiple exacerbations ( at least two exacerbations per year [ figure 2 ] , or at least two exacerbations or one hospitalization per year ) . figure 3 shows the auc - roc of both the e - faced and faced scores for all - cause mortality in the initial construction cohort ( n=819 ) . these are 0.87 ( 0.830.91 ) and 0.87 ( 0.820.91 ) , respectively , with no significant differences between them ; they are always > 0.80 , which means that they both present an excellent prognostic capacity . furthermore , in the case of prognostic capacity for respiratory deaths , the results were again similarly excellent : the e - faced score presented an auc - roc of 0.86 ( 0.810.9 ) , whereas the faced score presented one of 0.82 ( 0.780.87 ) . similarly , when both the e - faced and the faced scores were applied to the series of 651 patients from the latin american cohorts , the auc - rocs were 0.84 ( 0.800.88 ) and 0.81 ( 0.770.86 ) , respectively , which are as excellent as those of the construction cohort , with no statistically significant differences between the two . furthermore , in the case of the prognostic capacity for respiratory mortality , the e - faced presented an auc - roc of 0.87 ( 0.830.91 ) and the faced one of 0.84 ( 0.800.88 ) ( figure 4 ) . it was also demonstrated ( figure 5 ) that the three tertiles of both the faced and the e - faced scores accurately differentiated patients into three groups of increasing severity , with no significant differences between the classifications created by the faced and e - faced scores . similarly , the comparative division of this validation cohort into three tertiles in both the e - faced and the faced scores also presented an excellent discriminatory capacity in three groups of increasing severity , with no significant differences from the construction cohort ( figure 6 ) . according to the results of this study of two large series of patients with bronchiectasis , the addition to the faced score of a simple dichotomized variable relating to the presence of previous severe exacerbations ( hospitalizations ; e - faced score ) significantly increases the prognostic capacity for future exacerbations and hospitalizations over the course of 1 year , making it a valid score for detecting patients with more exacerbated bronchiectasis . furthermore , the addition of this variable does not make the score any more complex but does maintain its excellent prognostic capacity for both all - cause and respiratory mortality , while also retaining the capacity of faced score to discriminate different degrees of severity . by adding a variable that is potentially modifiable or preventable with treatment , the e - faced would broaden its clinical applicability and its potential use in research studies on new treatments for bronchiectasis . the faced score is a very simple ( and even memorizable ) multilevel system for evaluating ( without any need for a computer ) the initial severity and prognosis of patients with bronchiectasis.3 however , one of its limitations is the fact that it does not cover exacerbations . and although there is little literature on this topic , some important studies have shown this variable to have a decisive impact , especially in its more severe forms , on both the severity and prognosis of bronchiectasis.2,6,7 in this respect , chalmers et al,2 in a study of the construction and validation of the bsi ( bronchiectasis severity index ) score , observed that both previous exacerbations ( at least two exacerbations ; hr ( hazard ratio ) 2.03 [ 1.024.03 ] ) and , above all , severe exacerbations ( hospitalization ; hr 2.43 [ 1.024.03 ] ) were significantly associated with an increase in all - cause mortality , with a very substantial relative weight ( above all severe exacerbations ) in the final value of the score . although the bsi score is more complex than the faced , it has demonstrated an excellent prognostic capacity for exacerbations / hospitalizations in patients with bronchiectasis.2 moreover , martnez - garca et al have demonstrated that , over and above any chronic bronchial infection or presence of systemic inflammation , severe exacerbations were capable of producing a steep drop in lung function , of up to 120 ml of fev1 in a year more than double the mean observed overall in patients with bronchiectasis ( ~50 ml / year).7 this substantial impact of exacerbations on bronchiectasis patients can be explained by the increase in diagnoses,19 related mortality,20 and hospitalizations due to bronchiectasis21,22 in recent years , even in industrialized countries , despite the excellent therapeutic arsenal at disposal . ringshausen et al,21 for example , found a rise in hospitalization from 9.4 to 39.4/100,000 inhabitants , especially in women and the elderly . similar findings have been observed in the usa , with an annual increase of 2.5%3% in the period 19932006.22 furthermore , patients who present the greatest number or severity of exacerbations are also the ones who accumulate a higher percentage of health costs . de la rosa et al10 found that the annual cost of treating a bronchiectasis patient in spain in 2012 was 4,671 euros , and ~10,000 euros in cases of severe bronchiectasis ( faced 57 points ) , with exacerbations being one of the variables that explained a higher percentage of cost . thus , the group of patients with more than two exacerbations ( 21.5% ) was responsible for 34.5% of the total cost of the 456 patients included , while those patients who required two or more hospitalizations ( 5.7% ) were responsible for 20.4% of the total cost . therefore , even without any definition of an exacerbatory clinical phenotype in bronchiectasis , the impact of exacerbations on these patients calls for a simple and prompt identification of the type of patients who will present a higher number or greater severity of exacerbations in the future . in this study , the e - faced score ( a simple score that only adds to the faced score one easily memorized dichotomic variable pertaining to the presence of hospitalizations in the year prior to the patient s diagnosis ) presents advantages over the faced . while maintaining the substantial prognostic capacity of the latter , for all - cause and respiratory mortality and for discrimination of degrees of severity , the e - faced is capable , without accruing any greater complexity , of significantly improving the early identification of patients who will have at least two exacerbations or one hospitalization over the course of a year , or both , despite receiving treatment . this means that it is important to establish a prompt and thorough preventive strategy for these patients . moreover , another advantage of the e - faced over the faced is its greater susceptibility to change after the administration of different treatments . this may make it more suitable for research studies , both because it incorporates the main variable customarily used in randomized clinical trials ( the number and severity of exacerbations ) and because it presents a great prognostic capacity for these factors . although the present study has several significant strengths , particularly large numbers of patients being included in both the series ( construction and external validation ) , it also presents a number of noteworthy limitations . on the one hand , exacerbations have been defined by the need for antibiotic treatment prescribed for changes in the volume or purulence of sputum or in the symptoms . they are therefore open to a more objective classification , although it should be emphasized that smaller exacerbations that do not require such treatment have also been recorded . it is nevertheless believed that the impact of this limitation on the conclusion can not be very significant as most patients with exacerbations of bronchiectasis are treated with antibiotics,3 and furthermore , more severe exacerbations have the greatest impact on patients.2,6 on the other hand , the e - faced was constructed using different cut - off points as regards the number of hospitalizations in the year prior to the patient s inclusion in the study , but no longitudinal data on the hospitalizations in the construction cohort ( n=819 ) in the subsequent years were available . such longitudinal data on the number of yearly exacerbations and hospitalizations are available , however , for the validation cohort ( n=651 ) , and , thanks to the way the e - faced was constructed , it has demonstrated an excellent prognostic capacity . moreover , we can not exclude the influence of the chronic treatments used on clinical outcomes , especially in the number or severity of exacerbations . however , this is a limitation of all studies that include historical cohorts of patients with data recorded longitudinally from the time of diagnosis . furthermore , no treatment has yet been proved to have any influence on the natural history of the disease . finally , data were not obtained from 2.4% of patients from the original faced score cohort and 3.1% of patients from the latin american series , mainly because of lack of information on their vital status , but such a small percentage of missing data can not be expected to change the conclusion of the study . in conclusion , the e - faced offers a more versatile alternative to the faced that is more susceptible to variations after medication , while maintaining the excellent prognostic capacity of the latter for mortality and its capacity to discriminate degrees of severity , without increasing the complexity of the score . moreover , it presents a greater prognostic capacity for exacerbations and hospitalizations , especially in patients in which these are more frequent . although further studies are required to investigate the applicability of the e - faced in research studies , it is considered that the greater capacity of this score for change makes it more suitable than the faced for use in clinical trials to evaluate different treatments for bronchiectasis . list of ethical committees and approval numbers this approval was obtained from the original database of faced score , published in eur respir j. 2014;43:1357 .

backgroundalthough the faced score has demonstrated a great prognostic capacity in bronchiectasis , it does not include the number or severity of exacerbations as a separate variable , which is important in the natural history of these patients.objectiveconstruction and external validation of a new index , the e - faced , to evaluate the predictive capacity of exacerbations and mortality.methodsthe new score was constructed on the basis of the complete cohort for the construction of the original faced score , while the external validation was undertaken with six cohorts from three countries ( brazil , argentina , and chile ) . the main outcome was the number of annual exacerbations / hospitalizations , with all - cause and respiratory - related deaths as the secondary outcomes . a statistical evaluation comprised the relative weight and ideal cut - off point for the number or severity of the exacerbations and was incorporated into the faced score ( e - faced ) . the results obtained after the application of faced and e - faced were compared in both the cohorts.resultsa total of 1,470 patients with bronchiectasis ( 819 from the construction cohorts and 651 from the external validation cohorts ) were followed up for 5 years after diagnosis . the best cut - off point was at least two exacerbations in the previous year ( two additional points ) , meaning that the e - faced has nine points of growing severity . e - faced presented an excellent prognostic capacity for exacerbations ( areas under the receiver operating characteristic curve : 0.82 for at least two exacerbations in 1 year and 0.87 for at least one hospitalization in 1 year ) that was statistically better than that of the faced score ( 0.72 and 0.78 , p<0.05 , respectively ) . the predictive capacities for all - cause and respiratory mortality were 0.87 and 0.86 , respectively , with both being similar to those of the faced.conclusione-faced score significantly increases the faced capacity to predict future yearly exacerbations while maintaining the score s simplicity and prognostic capacity for death .

Introduction Methods Design Patients Variables Follow-up and main endpoints Statistical analysis Results Construction of the E-FACED score Prognostic capacity for exacerbations All-cause mortality: comparison of E-FACED and FACED construction and validation cohorts Capacity to discriminate different degrees of severity Discussion Conclusion Supplementary material

bronchiectasis is a multidimensional disease , and hence , its severity or prognosis could not be defined with any precision using a single variable.13 accordingly , some recently published multidimensional scores combine clinical , functional , microbiological , and radiological variables to evaluate the prognosis and severity of bronchiectasis more comprehensively.2,3 one of these scores is the faced score,3 an acronym derived from five dichotomized variables ( f : forced expiratory volume in 1 s [ fev1 ] ; a : age ; c : chronic colonization by pseudomonas aeruginosa [ pa ] , e : radiological extension [ number of pulmonary lobes affected ] , and d : dyspnea ) . the faced score is easy to remember and to apply in clinical practice ; it presents a range of values of growing severity spanning 07 points , and it has demonstrated an excellent prognostic capacity for mortality ( both all - cause and respiratory ) in both medium and long term , and an excellent capacity to discriminate different degrees of severity.4 it also had a good external validation in europe as well as in various latin american countries in bronchiectasis patients with wide - ranging etiology and severity and with characteristics different from those of the initial cohort.46 nevertheless , some authors have found that the faced score does not predict exacerbations accurately.6 one of the key clinical aspects in the prognosis and severity of airway diseases is exacerbations , particularly serious ones that require hospitalization . in bronchiectasis , exacerbations have been associated with higher mortality2 and a greater impairment of lung function,7 as well as more severe forms of the disease.8,9 furthermore , another important aspect of exacerbations in these patients is the substantial concomitant health costs.10 as exacerbations are potentially preventable , exacerbations and hospitalizations are main outcomes in most of the clinical studies that seek to evaluate the efficacy and , above all , cost - efficiency of different treatments for bronchiectasis.1116 hence , tools that can predict future exacerbations and particularly those patients ar risk of presenting multiple exacerbations ( especially severe ones requiring hospitalizations ) are required . since the faced score was not constructed with the variable exacerbations , the present study aimed to analyze , in a wide - ranging series of patients different from the original faced cohort , the prognostic capacity for exacerbations ( and more particularly for patients with multiple exacerbations ) of a new score called e - faced and compare it with that of faced . it also sought to evaluate whether e - faced also maintains the faced score s excellent predictive capacity for all - cause and respiratory mortality , as well as its capacity to discriminate different degrees of severity of bronchiectasis . it comprised a total of 819 patients with bronchiectasis whose characteristics have previously been published.3 this series will be used as the construction cohort for the e - faced . meanwhile , 651 patients with bronchiectasis from six latin american centers ( four in brazil , one in argentina , and one in chile ) who were the subjects for the external validation of the faced score will also be used for the external validation of the e - faced score . the variables recorded in both the construction cohort ( n=819 ) and the validation cohort ( n=651 ) were the same as those used in the original studies for the construction , internal validation , and external validation of the faced score . the exacerbations , hospitalizations , and microbiological data of the construction cohort pertained to the year prior to the patients inclusion in the study , while these data were also available longitudinally for the validation cohort during the 5 years of the follow - up . the secondary outcomes were established as the predictive capacity for death from any cause or from respiratory causes , and the capacity to discriminate different degrees of severity . various logistic regressions were made to determine the optimal cut - off point for the number of exacerbations and hospitalizations that would add the best independent prognostic value to the faced score . the independent values in the construction series ( n=819 ) , apart from the value of the faced itself , the cut - off points of at least one , at least two , or more than two exacerbations or hospitalizations in the year prior to inclusion in the study , including a composite of at least two exacerbations or one hospitalization in the previous year . the prognostic capacity of the best score for the number or severity of the exacerbations was quantified by means of the odds ratio ( or ; 95% confidence interval [ 95% ci ] ) , while the value of the or , rounded up to its highest whole value , was used as a relative weight for the variable in the new score that was formed ( e - faced ) . in order to simplify the score , only different cut - off points were used for the number or severity of exacerbations , to make the variable dichotomic , as in the original faced score . once the range of the e - faced score had been calculated and obtained , its prognostic capacity for future exacerbations and hospitalizations over the course of a year ( according to different cut - off points for number and severity ) was determined by using the external validation database ( n=651 ) and calculating the corresponding areas under the receiver operating characteristic curve ( auc - roc ) and 95% ci . a prognostic capacity was considered excellent if the auc - roc is > 80% of the total area.18 the prognostic capacity of the e - faced for all - cause and respiratory mortality was then calculated , again by using the auc - roc . finally , the kaplan meier curves and the log - rank test for comparing the curves two by two were used to determine the capacity of the new index ( e - faced ) to discriminate the degree of severity , in order to divide bronchiectasis into mild ( first tertile ) , moderate ( second tertile ) , or severe ( third tertile ) . it comprised a total of 819 patients with bronchiectasis whose characteristics have previously been published.3 this series will be used as the construction cohort for the e - faced . meanwhile , 651 patients with bronchiectasis from six latin american centers ( four in brazil , one in argentina , and one in chile ) who were the subjects for the external validation of the faced score will also be used for the external validation of the e - faced score . the variables recorded in both the construction cohort ( n=819 ) and the validation cohort ( n=651 ) were the same as those used in the original studies for the construction , internal validation , and external validation of the faced score . the exacerbations , hospitalizations , and microbiological data of the construction cohort pertained to the year prior to the patients inclusion in the study , while these data were also available longitudinally for the validation cohort during the 5 years of the follow - up . the main variable was the prognostic capacity for exacerbations in a year , according to different cut - off points as regards number and severity . the secondary outcomes were established as the predictive capacity for death from any cause or from respiratory causes , and the capacity to discriminate different degrees of severity . various logistic regressions were made to determine the optimal cut - off point for the number of exacerbations and hospitalizations that would add the best independent prognostic value to the faced score . the independent values in the construction series ( n=819 ) , apart from the value of the faced itself , the cut - off points of at least one , at least two , or more than two exacerbations or hospitalizations in the year prior to inclusion in the study , including a composite of at least two exacerbations or one hospitalization in the previous year . the prognostic capacity of the best score for the number or severity of the exacerbations was quantified by means of the odds ratio ( or ; 95% confidence interval [ 95% ci ] ) , while the value of the or , rounded up to its highest whole value , was used as a relative weight for the variable in the new score that was formed ( e - faced ) . in order to simplify the score , only different cut - off points were used for the number or severity of exacerbations , to make the variable dichotomic , as in the original faced score . once the range of the e - faced score had been calculated and obtained , its prognostic capacity for future exacerbations and hospitalizations over the course of a year ( according to different cut - off points for number and severity ) was determined by using the external validation database ( n=651 ) and calculating the corresponding areas under the receiver operating characteristic curve ( auc - roc ) and 95% ci . a prognostic capacity was considered excellent if the auc - roc is > 80% of the total area.18 the prognostic capacity of the e - faced for all - cause and respiratory mortality was then calculated , again by using the auc - roc . finally , the kaplan meier curves and the log - rank test for comparing the curves two by two were used to determine the capacity of the new index ( e - faced ) to discriminate the degree of severity , in order to divide bronchiectasis into mild ( first tertile ) , moderate ( second tertile ) , or severe ( third tertile ) . in all cases , the construction cohort comprised 819 patients with bronchiectasis from the initial spanish cohort for the construction and internal validation of the faced , whereas the external validation cohort ( n=651 ) comprised the latin american cohort for the external validation of the faced . table 2 shows the results of the logistical regressions , which included different cut - off points for the number of exacerbations and hospitalizations in the previous year ( at least one , at least two , or more than two , as well as a composite of two exacerbations or at least one hospitalization ) to evaluate the faced score s independent prognostic capacity for all - cause mortality . as can be seen , the cut - off point that added the most statistically significant prognostic value to the faced score was at least one hospitalization in the previous year , with an or of 1.8 , and so this was the variable chosen to be added to the faced . this process therefore added two extra points to the faced score in cases of at least one hospitalization in the previous year , or none in cases of no hospitalizations . table 4 shows how the prognostic capacity of the e - faced score was significantly greater than that of the faced , in both the number and severity ( hospitalizations ) of the future exacerbations over the course of a year . the greatest benefit from e - faced was found in the identification of patients with future multiple exacerbations ( at least two exacerbations per year [ figure 2 ] , or at least two exacerbations or one hospitalization per year ) . figure 3 shows the auc - roc of both the e - faced and faced scores for all - cause mortality in the initial construction cohort ( n=819 ) . these are 0.87 ( 0.830.91 ) and 0.87 ( 0.820.91 ) , respectively , with no significant differences between them ; they are always > 0.80 , which means that they both present an excellent prognostic capacity . furthermore , in the case of prognostic capacity for respiratory deaths , the results were again similarly excellent : the e - faced score presented an auc - roc of 0.86 ( 0.810.9 ) , whereas the faced score presented one of 0.82 ( 0.780.87 ) . similarly , when both the e - faced and the faced scores were applied to the series of 651 patients from the latin american cohorts , the auc - rocs were 0.84 ( 0.800.88 ) and 0.81 ( 0.770.86 ) , respectively , which are as excellent as those of the construction cohort , with no statistically significant differences between the two . furthermore , in the case of the prognostic capacity for respiratory mortality , the e - faced presented an auc - roc of 0.87 ( 0.830.91 ) and the faced one of 0.84 ( 0.800.88 ) ( figure 4 ) . it was also demonstrated ( figure 5 ) that the three tertiles of both the faced and the e - faced scores accurately differentiated patients into three groups of increasing severity , with no significant differences between the classifications created by the faced and e - faced scores . similarly , the comparative division of this validation cohort into three tertiles in both the e - faced and the faced scores also presented an excellent discriminatory capacity in three groups of increasing severity , with no significant differences from the construction cohort ( figure 6 ) . table 2 shows the results of the logistical regressions , which included different cut - off points for the number of exacerbations and hospitalizations in the previous year ( at least one , at least two , or more than two , as well as a composite of two exacerbations or at least one hospitalization ) to evaluate the faced score s independent prognostic capacity for all - cause mortality . as can be seen , the cut - off point that added the most statistically significant prognostic value to the faced score was at least one hospitalization in the previous year , with an or of 1.8 , and so this was the variable chosen to be added to the faced . this process therefore added two extra points to the faced score in cases of at least one hospitalization in the previous year , or none in cases of no hospitalizations . table 4 shows how the prognostic capacity of the e - faced score was significantly greater than that of the faced , in both the number and severity ( hospitalizations ) of the future exacerbations over the course of a year . the greatest benefit from e - faced was found in the identification of patients with future multiple exacerbations ( at least two exacerbations per year [ figure 2 ] , or at least two exacerbations or one hospitalization per year ) . figure 3 shows the auc - roc of both the e - faced and faced scores for all - cause mortality in the initial construction cohort ( n=819 ) . these are 0.87 ( 0.830.91 ) and 0.87 ( 0.820.91 ) , respectively , with no significant differences between them ; they are always > 0.80 , which means that they both present an excellent prognostic capacity . furthermore , in the case of prognostic capacity for respiratory deaths , the results were again similarly excellent : the e - faced score presented an auc - roc of 0.86 ( 0.810.9 ) , whereas the faced score presented one of 0.82 ( 0.780.87 ) . similarly , when both the e - faced and the faced scores were applied to the series of 651 patients from the latin american cohorts , the auc - rocs were 0.84 ( 0.800.88 ) and 0.81 ( 0.770.86 ) , respectively , which are as excellent as those of the construction cohort , with no statistically significant differences between the two . furthermore , in the case of the prognostic capacity for respiratory mortality , the e - faced presented an auc - roc of 0.87 ( 0.830.91 ) and the faced one of 0.84 ( 0.800.88 ) ( figure 4 ) . it was also demonstrated ( figure 5 ) that the three tertiles of both the faced and the e - faced scores accurately differentiated patients into three groups of increasing severity , with no significant differences between the classifications created by the faced and e - faced scores . similarly , the comparative division of this validation cohort into three tertiles in both the e - faced and the faced scores also presented an excellent discriminatory capacity in three groups of increasing severity , with no significant differences from the construction cohort ( figure 6 ) . according to the results of this study of two large series of patients with bronchiectasis , the addition to the faced score of a simple dichotomized variable relating to the presence of previous severe exacerbations ( hospitalizations ; e - faced score ) significantly increases the prognostic capacity for future exacerbations and hospitalizations over the course of 1 year , making it a valid score for detecting patients with more exacerbated bronchiectasis . furthermore , the addition of this variable does not make the score any more complex but does maintain its excellent prognostic capacity for both all - cause and respiratory mortality , while also retaining the capacity of faced score to discriminate different degrees of severity . and although there is little literature on this topic , some important studies have shown this variable to have a decisive impact , especially in its more severe forms , on both the severity and prognosis of bronchiectasis.2,6,7 in this respect , chalmers et al,2 in a study of the construction and validation of the bsi ( bronchiectasis severity index ) score , observed that both previous exacerbations ( at least two exacerbations ; hr ( hazard ratio ) 2.03 [ 1.024.03 ] ) and , above all , severe exacerbations ( hospitalization ; hr 2.43 [ 1.024.03 ] ) were significantly associated with an increase in all - cause mortality , with a very substantial relative weight ( above all severe exacerbations ) in the final value of the score . although the bsi score is more complex than the faced , it has demonstrated an excellent prognostic capacity for exacerbations / hospitalizations in patients with bronchiectasis.2 moreover , martnez - garca et al have demonstrated that , over and above any chronic bronchial infection or presence of systemic inflammation , severe exacerbations were capable of producing a steep drop in lung function , of up to 120 ml of fev1 in a year more than double the mean observed overall in patients with bronchiectasis ( ~50 ml / year).7 this substantial impact of exacerbations on bronchiectasis patients can be explained by the increase in diagnoses,19 related mortality,20 and hospitalizations due to bronchiectasis21,22 in recent years , even in industrialized countries , despite the excellent therapeutic arsenal at disposal . therefore , even without any definition of an exacerbatory clinical phenotype in bronchiectasis , the impact of exacerbations on these patients calls for a simple and prompt identification of the type of patients who will present a higher number or greater severity of exacerbations in the future . while maintaining the substantial prognostic capacity of the latter , for all - cause and respiratory mortality and for discrimination of degrees of severity , the e - faced is capable , without accruing any greater complexity , of significantly improving the early identification of patients who will have at least two exacerbations or one hospitalization over the course of a year , or both , despite receiving treatment . this may make it more suitable for research studies , both because it incorporates the main variable customarily used in randomized clinical trials ( the number and severity of exacerbations ) and because it presents a great prognostic capacity for these factors . it is nevertheless believed that the impact of this limitation on the conclusion can not be very significant as most patients with exacerbations of bronchiectasis are treated with antibiotics,3 and furthermore , more severe exacerbations have the greatest impact on patients.2,6 on the other hand , the e - faced was constructed using different cut - off points as regards the number of hospitalizations in the year prior to the patient s inclusion in the study , but no longitudinal data on the hospitalizations in the construction cohort ( n=819 ) in the subsequent years were available . such longitudinal data on the number of yearly exacerbations and hospitalizations are available , however , for the validation cohort ( n=651 ) , and , thanks to the way the e - faced was constructed , it has demonstrated an excellent prognostic capacity . in conclusion , the e - faced offers a more versatile alternative to the faced that is more susceptible to variations after medication , while maintaining the excellent prognostic capacity of the latter for mortality and its capacity to discriminate degrees of severity , without increasing the complexity of the score . although further studies are required to investigate the applicability of the e - faced in research studies , it is considered that the greater capacity of this score for change makes it more suitable than the faced for use in clinical trials to evaluate different treatments for bronchiectasis .

kaposi s sarcoma ( ks ) was first described in the 1800s as a rare , fairly indolent tumor of specific populations . this form of ks , now referred to as classic ks , usually presents on the skin of the lower extremities of elderly men of specific european regions and religious origins . in the middle of the twentieth century ks became endemic in parts of africa and is currently one of the most common tumors in parts of central africa ( wabinga et al . in the late twentieth century ks became one of the first aids defining illnesses and is the most common tumor of aids patients world - wide . aids associated ks is generally far more aggressive than classic ks , arising on the skin in many parts of the body as well as in the oral cavity and can occur on internal organs where it is often fatal . while they differ in aggressiveness , all forms of ks are relatively indistinguishable at the histological level . grossly , the tumors have a characteristic red to purple hue , indicative of the high vascularization of the tumor . histologically , the tumor exhibits large vascular slits lined by flattened endothelium ; the slits are often , but not always , filled with blood cells . there are discernable levels of extravasated red blood cells and infiltrating lymphocytes in the tumor . while a number of cell types are present , the tumor is predominantly made up of elongated spindle cells . the spindle cells express endothelial cell markers on their surface including cd31 and cd34 , but express low levels of factor viiira ( russell jones et al . , recent expression data and array studies have found that spindle cells most closely resemble lymphatic endothelium , expressing vegf receptor 3 ( vegfr3 ) , a specific marker of lymphatic endothelial cells ( jussila et al . , 1998 ; skobe et al . , 1999 other lymphatic endothelial cell specific markers , including lyve-1 , podoplanin , and prox-1 , are also expressed by the spindle cells ( carroll et al . based on the epidemiology and the multicentric nature of the tumor , ks was predicted to have an infectious cause ( beral et al . , , kshv was discovered associated with all ks tumors and is now considered to be the etiologic agent ( chang et al . , 1994 ) . kshv was the eighth human herpesvirus discovered and is sub - classified as a gamma herpesvirus . all of the greater than 90 viral genes are expressed , the virus replicates rapidly , produces infectious virions and ultimately causes cell death likely due to a combination of host cell shut off and virus production . during viral latency in endothelial cells , this locus includes lana , vcyc , vflip , a family of proteins from a repeat region called the kaposins , and 12 pre - micrornas encoding 17 or more mature mirnas . these latent genes are responsible for the maintenance of the latent viral episome as well as the survival of latently infected cells . in later - stage ks tumors , , 1995 ; staskus et al . , 1997 ; dupin et al . , 1999 as expected the virus is predominantly found in the latent state in spindle cells where the limited number of latent genes and mirnas are expressed ( staskus et al . , 1997 ; . however , approximately 15% of the spindle cells express lytic viral genes and produce infectious virus . in addition to spindle cells , kshv is also found in other cell types in the ks lesion including monocytes ( blasig et al . , kshv can only sporadically be detected in the endothelium lining the vascular slits in the ks tumor ( dupin et al . , 1999 ) . kshv is also associated with two lymphoproliferative diseases , primary effusion lymphoma ( pel ) a pleural cavity solid b - cell lymphoma , and plasmablastic multicentric castleman s disease , a lymph node b - cell growth ( cesarman et al . because ks is an angioproliferative disease and the ks tumors are highly vascularized even at early stages , it has been proposed that kshv may directly induce angiogenesis . endothelial cells of the vascular system are normally maintained in a quiescent , non - proliferating state . however , during solid tumor formation , the secretion of pro - angiogenic cytokines by tumor cells can activate nearby endothelial cells to induce new blood vessel formation . many of the vascular slits identified in histological sections of early stage ks lesions are lined by uninfected endothelium , suggesting they are formed by endothelial cells activated in a paracrine fashion ( dupin et al . , 1999 ) . these uninfected cells may later become infected , as kshv has , in some cases , been detected in the cells surrounding the vascular spaces of later - stage nodular ks ( boshoff et al . despite the evidence for paracrine activation of uninfected endothelial cells , kshv also likely activates infected endothelial cells in an autocrine or cell autonomous fashion . because ks spindle cells are endothelial in origin , induction of the ks tumor cell is similar to the processes of angiogenesis . many of the characteristics of activated endothelial cells and angiogenesis are also associated with oncogenesis , including proliferation , migration , and metalloprotease expression . this review discusses the recent evidence that suggests that ( 1 ) kshv promotes continual neovascularization through paracrine factors and ( 2 ) kshv may drive tumor cell growth through autocrine and cell autonomous activation of angiogenic phenotypes . the vascular endothelial growth factor ( vegf ) family of cytokines plays a prominent role in regulation of angiogenesis ( breen , 2007 ) . vegf - a and its receptors are required for embryonic vascular development and are important for vascular permeability , proliferation , and survival of newly formed vasculature . vegf - a expression is detected in spindle cells of ks lesions , and its secretion is known to be increased by inflammatory cytokines that are present in the ks lesions ( samaniego et al . , 1998 ) . vegf - a is also expressed by kshv - infected pel cell lines and conditioned media from these cells is sufficient to induce capillary morphogenesis by endothelial cells ( liu et al . , 2001 ; akula et al . , 2005 ; subramanian et al . , 2010 ) . infection of endothelial cells with kshv directly leads to increased expression of vegf - a ( masood et al . , 2002 ; sivakumar et al . , 2008 ; wang and damania , 2008 ) . further , kshv conditioned media has been shown to regulate angiogenic phenotypes in endothelial cells ( sharma - walia et al . , 2010 ) . therefore , kshv induction of vegf - a is likely to be critical for both the induction of angiogenesis as well as the activation of infected spindle cells . although the mechanisms by which kshv induces vegf - a expression and secretion are still unclear , several potential pathways have been uncovered . hypoxia induced factor ( hif)-1 is a transcription factor that has been shown to be important for upregulation of vegf - a ( sodhi et al . , 2000 ; shin et al . , 2008 ) . however , during hypoxia , hif-1 is stabilized and can induce expression of genes through hypoxia responsive elements , including vegf - a . interestingly , kshv infection of endothelial cells induces the expression of hif-1 during normoxia which leads to increased hif transcriptional activity ( carroll et al . , 2006 ) . other studies have shown that kshv encodes proteins that can lead to increased stability of hif-1 . the kshv latency associated nuclear protein ( lana-1 ) can stabilize hif-1 , through both degradation of its suppressors , von hippel lindau protein and p53 ( cai et al . , 2006 ) , and through direct interaction between hif-1 and lana-1 ( cai et al . , 2007 ) . additionally , the virally encoded interferon response factor ( virf3 ) can , like lana-1 , interact with and stabilize hif-1 , leading to increased vegf - a expression ( shin et al . , 2008 ) . the kshv viral g - protein coupled receptor ( vgpcr ) is able to increase the activity of hif-1 as a transcription factor through activation of the mapk and p38 signaling pathways and subsequent phosphorylation of hif-1 ( sodhi et al . while induction of hif mrna expression by kshv infection has been shown , stabilization of hif directly by kshv infection of endothelial cells has yet to be clearly shown . pro - angiogenic factors that are induced by kshv are indicated in red ovals , pro - lymphangiogenic factors induced by kshv are indicated in blue ovals . white ovals indicate the activated pathway necessary for kshv induction of blood to lymphatic endothelial cell differentiation . the upper left corner indicates phenotypes of angiogenesis that are activated in normal endothelium by kshv - infected cells and the gradient of cytokines secreted by kshv - infected cells is indicated by the red gradient . the blue gradient in the upper right hand corner indicates the gradient of lymphangiogenic cytokines induced by kshv - infected cells that could induce lymphangiogenesis . other host proteins have been shown to be involved in the induction of vegf - a during kshv infection of endothelial cells as well . for example , emmprin is a membrane - associated glycoprotein that promotes matrix metalloproteinase expression . its expression in kshv - infected cells promotes cell invasiveness through activation of the pi3k / akt and mapk pathways ( qin et al . further work is ongoing in multiple labs to determine the cellular pathways essential for kshv induction of vegf - a . several kshv genes expressed during lytic replication have been implicated in regulation of vegf - a expression ( table 1 ) . in bcbl-1 cells ( a pleural effusion lymphoma cell line ) , glycoprotein b ( gb ) and k8.1 are required for enhanced vegf expression ( subramanian et al . , 2010 ) . treatment of these cells with sirna or neutralizing antibodies to gb or k8.1 significantly reduced vegf - a production . vgpcr is a constitutively active signaling receptor that has been linked to a variety of cell survival and pro - angiogenic signaling pathways ( arvanitakis et al . when injected into mice , nih3t3 cells expressing vgpcr form highly vascularized tumors with some similarities to ks and this may be due , at least in part , to increased vegf - a secretion ( bais et al . , 1998 ; guo et al . vgpcr upregulates vegf - a through activation of mapk and p38 , which , as described above , promotes hif-1 activity ( sodhi et al . , 2000 ) . transgenic mice expressing vgpcr also form highly vascularized tumors that are reminiscent of ks tumors . however , cell lines derived from these tumors expressed the lymphatic growth factor vegf - c , rather than vegf - a ( guo et al . , 2003 ) . increased vegf - a expression in cells expressing vgpcr is associated with constitutive activation of its receptor , vegfr2/kdr and downstream activation of pi3k / akt , contributing to endothelial cell survival ( montaner et al . , 2001 ; bais et al . , 2003 ) . however , gb , k8.1 , and vgpcr have only been detected in cells supporting lytic kshv infection whereas the bulk of the tumor cells are latently infected . the kshv glycoprotein k1 also induces increased vegf - a expression in endothelial cells and is capable of immortalizing primary endothelial cells ( wang et al . while there is evidence that k1 is expressed at very low levels during latency , the majority of its expression occurs during lytic infection ( chandriani and ganem , 2010 ) . in summary , the lytic phase of kshv infection might play a role in the paracrine induction of angiogenesis through increased secretion of vegf - a into the tumor milieu . in addition to vegf - a , kshv - infected endothelial cells also express other angiogenic cytokines . angiopoietin-1 and -2 are ligands for the receptor tyrosine kinase tie2 . although less is known about the functions of angiopoietins and tie2 , their signaling is required for proper vascular development during embryogenesis ( dumont et al . , 1994 ) . angiopoietin-1 is an agonist for the tie2 receptor , promoting endothelial cell survival and stability . in contrast , ang2 acts as an antagonist for tie2 , although its role is context - dependent . ang2 is upregulated during pathologic angiogenesis and this expression is thought to destabilize endothelial cells , allowing them to be activated by other pro - angiogenic stimuli , such as vegf , see figure 1 , circle 1 ( gale et al . , 2002 ) . interestingly , ang2 is expressed in ks lesions , and is upregulated in endothelial cells infected with kshv ( brown et al . , 2000 ; this induction can be activated by the kshv genes vgpcr and vil-6 , and can occur through a paracrine mechanism ( vart et al . , 2007 ) . another study suggests that the mapk pathway activation of transcription factors ap-1 and ets-1 is involved ( ye et al . , 2007 ) . in addition to ang2 , cells transfected with the vgpcr gene expressed increased levels of angiopoietin - like 4 , a member of the angiopoietin - like proteins that may play a role in vascular permeability and angiogenesis ( ma et al . , 2010 ) . kshv induces a number of other cytokines known to be involved in angiogenesis in other systems . these include interleukin 6 ( il-6 ) , monocyte chemoattractant protein-1 ( mcp-1 ) , pax-1 , and prostaglandin e2 ( schwarz and murphy , 2001 ; polson et al . , 2002 ; xie et al . , 2005 ; caselli et al . , 2007 ; cyclooxygenase enzymes catalyze the rate limiting step in the conversion of arachidonic acid into prostaglandins . prostaglandins signal through g - protein coupled receptors to regulate a variety of functions , including metabolic , neuronal , and immune functions . cyclooxygenase-2 ( cox-2 ) expression is induced early during kshv infection of endothelial cells and plays a role in the establishment of latency ( naranatt et al . , 2004 ; sharma - walia et al . , this expression is associated with increased secretion of prostaglandin e2 , which promotes inflammatory cytokine expression , cell survival , and angiogenesis ( sharma - walia et al . , 2010 ) . an additional cellular factor associated with angiogenesis , angiogenin , is induced in endothelial cells by the latent protein , lana-1 . angiogenin was recently shown to aid in induction of angiogenesis by both vegf and basic fibroblast growth factor ( sadagopan et al . , 2009 ) . kshv - induced angiogenin was able to promote endothelial cell migration and capillary morphogenesis ( sadagopan et al . , 2009 ) . since angiogenin is internalized by both infected and uninfected cells , the authors suggested angiogenin may work in both paracrine and autocrine fashions . in fact , all kshv - induced cytokines that act on endothelial cells have the potential to promote angiogenesis - like phenotypes on the endothelial - derived spindle cells . kshv not only upregulates pro - angiogenic cytokines , it may also promote angiogenesis through repression of angiogenic inhibitors . the kshv latent locus encodes for 17 mirnas which may play a role in downregulation of angiogenic gene expression ( cai et al . , 2005 ; expression of 10 of these mirnas in 293 cells altered the expression of 81 genes ( samols et al . , 2007 ) . thrombospondin-1 plays multiple roles in the repression of angiogenesis , however one of its main functions is activation of the anti - angiogenic growth factor transforming growth factor- ( tgf- ) . this study found that thrombospondin-1 contains 34 putative mirna binding sites , and can be downregulated by multiple kshv mirnas ( samols et al . , 2007 ) . therefore , downregulation of anti - angiogenic factors may be an important way by which kshv promotes continual neovascularization . the kshv genome itself encodes for cytokine and chemokine - like factors that activate endothelial cells and stimulate angiogenesis ( table 1 ) . among these factors are three genes with homology to the cellular chemokine macrophage inflammatory protein , the vmips i iii ( boshoff et al . in addition to having chemoattractant properties , these proteins promoted neovascularization in the chick chorio - allantoic membrane angiogenesis assay ( boshoff et al . , 1997 ; stine et al . , 2000 ) . kshv also encodes a viral homolog of interleukin 6 ( il-6 ) , a pro - inflammatory and pro - angiogenic cytokine . this cytokine , when expressed in nih3t3 cells , promoted secretion of vegf - a ( aoki et al . furthermore , when these cells were injected into mice , they gave rise to tumors more quickly than control cells and the tumors were more highly vascularized ( aoki et al . the viral il-6 ( vil-6 ) is mostly detected in endothelial cells and spindle cells undergoing lytic replication but like k1 it has been shown to be expressed at very low levels in latently infected pel cells and to be induced during latency only under specific conditions ( chatterjee et al . , 2002 ; chandriani et al . , 2010 ) . in summary , conditioned media from kshv - infected cells can induce angiogenic phenotypes in uninfected endothelial cells as indicated by the red gradient in figure 1 . kshv infection of endothelial cells induces expression of a number of cytokines that are capable of inducing angiogenesis in a paracrine fashion . paramount among these is vegf - a , an angiogenic cytokine that is induced by kshv infection of endothelial cells . while the predominant viral mechanism of vegf - a induction is unknown , a number of lytic kshv genes are sufficient to induce vegf - a when overexpressed . kshv - infected cells also produce a number of other angiogenic cytokines of cellular and viral origin that likely play a role in the induction of angiogenesis . taken together , all of the cytokines and induced pathways likely create a milieu that is beneficial to the induction of new blood vessels and play a significant role in the high vascularization of ks tumors . therefore ks tumor formation is likely to include increased angiogenic capacity of the spindle cells . there is growing evidence demonstrating the manipulation of host cell phenotypes by kshv and the role of these changes in the promotion of angiogenesis related phenotypes . these infected cell phenotypes include increased stability of tubules formed by macrovascular endothelial cells , induction of capillary morphogenesis in low growth factor conditions , and enhanced migration and invasion ( qian et al . , 2007 ; sadagopan et al . , 2007 , 2009 ; wang and damania , 2008 ; couty et al . , 2009 ; dimaio et al . , additionally , kshv induces the expression of vegf receptors on the surface of infected endothelial cells as discussed below . breakdown of these junctions is necessary for initiation of angiogenesis , immune cell extravasation , and tumor cell metastasis . interestingly , several studies have evaluated the adherens junctions of kshv - infected endothelial cells and found them to be perturbed ( mansouri et al . , 2008 ; qian et al . , 2008 ; guilluy et al . , , 2008 ; qian et al . , 2008 ) as well as disruption of ve - cadherin therefore , kshv infection can directly initiate a key angiogenic step , the breakdown of cell cell adherence . while the direct mechanism of kshv - induced disruption of adherens junctions during latency is not known , there are a number of candidate kshv genes that could be involved ( table 1 ) . the kshv - encoded ubiquitin ligase protein , k5 , targets ve - cadherin for degradation ( mansouri et al . , 2008 ) . overexpression of the kshv vgpcr induces endothelial cell permeability and downregulation of cell surface ve - cadherin as well ( dwyer et al . , 2011 ) . k5 also targets other cellular proteins , including platelet / endothelial cell adhesion molecule-1 ( pecam-1 , cd31 ) , a transmembrane protein important for endothelial cell cell communication , which could contribute to barrier dysfunction and increased permeability ( tomescu et al . , 2003 ; mansouri et al . , k1 , a primarily lytic protein that may also be expressed at low levels during latency was also shown to initiate signaling similar to that required for disruption of cadherin signaling ( guilluy et al . , 2011 ) . while the exact viral mechanism of disruption of adherens junctions by kshv infection is not known , the virus encodes multiple genes capable of altering cadherin . during angiogenesis , endothelial cells migrate from the pre - existing vasculature toward the site of angiogenic stimulus . endothelial cells exhibit enhanced migration and invasion following latent kshv infection ( dimaio et al . kshv - infected cells also express increased levels of the matrix metalloproteinases mmp-1 , -2 , and -9 ( qian et al . , 2007 ) . mmp proteins break down the extracellular matrix supporting stable vasculature to allow for invasion and migration of endothelial cells during angiogenesis ( figure 1 , circle 2 ) . expression of mmp proteins induced by kshv allows for increased invasion of both infected and uninfected endothelial cells ( wang et al . while these processes constitute one component of angiogenesis , they are also known to play roles in oncogenesis ( gialeli et al . , 2011 ) indicating that kshv activation of angiogenic phenotypes in endothelial cells may lead to enhanced oncogenesis as well . endothelial cells grown in three - dimensional culture will migrate and organize into capillary - like structures . this activity is dependent , at least in part , on growth factors and cytokines present in the matrix or growth media . kshv - infected cells are able to undergo capillary morphogenesis in low growth factor conditions to a greater extent than uninfected cells ( wang and damania , 2008 ) . this could be partially due to increased cytokine secretion from kshv - infected cells . in fact , when endothelial cells are cultured in the presence of conditioned media from kshv - infected bcbl-1 cells , their ability to organize into capillary - like structures is increased ( wang and damania , 2008 ) . however , the effect of bcbl-1 conditioned media was greater on kshv - infected endothelial cells than on mock - infected cells , suggesting that infected cells are more receptive to angiogenic growth factors . in addition , this same study found that capillary - like structures formed by kshv - infected endothelial cells are more persistent than mock - infected cells , indicative of the promotion of cell survival and continual angiogenesis by kshv ( wang and damania , 2008 and our unpublished results ) . kshv latent infection of endothelial cells also induces vegf receptor expression , which may allow infected cells to respond more robustly to vegfs . vegf receptors 1 and 2 play roles in angiogenesis while 2 and 3 play a role in lymphangiogenesis ( described below ) . while kshv infection has not been reported to alter the expression levels of vegfr2 ( kdr ) , vegfr1 expression is significantly increased following kshv endothelial cell infection ( carroll et al . , 2004 ) . drugs that inhibited hif-1 activation and signaling also inhibited vegfr1 upregulation ( carroll et al . , 2006 ) . vegfr1 has been described as both a positive and negative regulator of angiogenesis depending on the context . vegfr1 mouse knockouts have higher levels of angiogenesis ( fong et al . , 1995 ) . however , in cell culture models , vegfr1 has been shown to potentiate angiogenesis ( cao , 2009 ) . more studies will be needed to determine the importance of increased vegfr1 expression in kshv infection and ks tumor formation . interestingly , expression of vegfr3 , the main receptor for vegf - c and d is also significantly increased by kshv infection ( carroll et al . , 2004 ; hong et al . , 2004 ) vegfr3 , a receptor specific to lymphatic endothelium and critical for lymphangiogenesis will be discussed below . importantly , endothelial tip cells at the leading edge of new vascular protrusions are the only main adult cell type known to express both the blood endothelial cell receptor , vegfr1 , and the lymphatic endothelial cell receptor , vegfr3 ( tammela et al . , 2008 ) : kshv infection of endothelial cells directly induces expression of both of these receptors . the mechanisms by which kshv induces angiogenic phenotypes in latently infected cells are largely unknown . a number of angiogenic phenotypes are likely to be a direct result of the cytokine milieu of the infected cells . as described above , kshv - infected cells secrete both viral and host cytokines that are sufficient to induce angiogenic phenotypes . however , it is also apparent that some of the angiogenic effects seen in latently infected cells are cell autonomous , independent of either paracrine or autocrine factors . as described above , conditioned media from pel cells had stronger effects on tubule formation of kshv - infected endothelial cells ( akula et al . , 2005 ) . we have also recently found that kshv infection induces the pro - angiogenic integrin , integrin 3 , during latent infection ( dimaio et al . , 2011 ) . induction of integrin 3 leads to increased cell surface expression of the v3 integrin heterodimer . we have further shown that latently infected endothelial cells become more adherent to the integrin ligands fibronectin and vitronectin , and are more migratory than mock - infected cells . although both uninfected and infected cells organize in three - dimensional cultures in complete media , infected cells are more sensitive to inhibitors of integrin 3 and its downstream signaling molecules , such as src kinase ( dimaio et al . , 2011 ) . this suggests that during latent kshv infection there is a shift in endothelial cell signaling that results in a more angiogenic phenotype dependent on v3 expression on the surface of the cell ( figure 1 , center ) . therefore , kshv alteration of endothelial cell signaling pathways can dramatically affect how the cell responds to intra- and extra - cellular signals . these changes that lead to alterations in angiogenic properties are likely to play a role in the growth and cell cell interactions of infected cells , thereby playing a role in ks tumor formation . during development of the vascular system , a subset of endothelial cells in the cardinal vein begin to express markers of lymphatic differentiation , including the master regulatory gene , prox-1 . these cells then bud from the cardinal vein , differentiate into lymphatic endothelial cells , and form the lymphatic vascular system ( wigle and oliver , 1999 ) . immunohistochemistry of ks tumors showed that spindle cells express markers of lymphatic endothelium , suggesting these cells may arise from primary infection of lymphatic endothelial cells , rather than blood endothelial cells ( jussila et al . , 1998 ; skobe et al . , 1999 ; weninger et al . , 1999 ; pyakurel et al . , 2006 ) . an alternative hypothesis is that kshv infection of blood endothelial cells drives differentiation toward a more lymphatic phenotype . this idea is supported by evidence that kshv infection of blood endothelial cells promotes expression of lymphatic - specific genes , including prox-1 , vegfr3 , podoplanin , and lyve-1 , effectively driving the reprogramming of blood endothelial cells to become lymphatic endothelium ( carroll et al . microarray analysis comparing kshv - infected blood endothelial cells to blood and lymphatic endothelial cells indicate that kshv - infected blood endothelial cells have gene expression profiles that align more closely to lymphatic endothelial cells than that of blood endothelial cells ( carroll et al . , 2004 ; the kshv latent gene kaposin b can directly promote the stability of prox-1 mrna ( yoo et al . , 2010 ) leading to increased expression of prox-1 . however , this effect was not sufficient to induce prox-1 expression in blood endothelial cells . we recently found that induction of blood to lymphatic endothelial cell reprogramming requires signaling through the cellular receptor gp130 . endothelial cells that are latently infected with kshv have increased expression and signaling of gp130 ( morris et al . , 2008 ) . this leads to activation of the jak / stat3 pathway and the pi3k / akt pathway leading to expression of lymphatic - specific genes starting with prox-1 . inhibition of this pathway by sirnas that target gp130 or akt or pharmacological inhibitors that block pi3 kinase or jak2/stat3 signaling is sufficient to block lymphatic differentiation ( see figure 1 , center ) . kshv vil-6 is sufficient to induce gp130 activation and we recently found that vil-6 is sufficient to induce lymphatic differentiation ( morris et al . , 2012 ) . however , kshv lacking vil-6 is still able to cause blood to lymphatic endothelial cell differentiation , indicating that kshv has evolved multiple strategies to activate gp130 and induce blood to lymphatic endothelial cell differentiation ( morris et al . , 2008 ) . induction of lymphatic differentiation by kshv is only part of the story , however . despite the expression of lymphatic - specific genes , blood endothelial cells infected with kshv retain expression of some blood specific markers ( wang et al . , 2004a ) . additionally , infection of lymphatic endothelial cells with kshv induces expression of blood specific markers ( wang et al . , 2004a ) . kshv mirnas were found to target the transcription factor maf ( hansen et al . , 2010 ) . downregulation of maf in lymphatic endothelial cells by sirna restored expression of blood endothelial markers , such as vegfr1 and cxcr4 . thus , infection of blood or lymphatic endothelial cells by kshv alters host gene expression to an intermediate state between the two cell types . as described above , this intermediate phenotype with both vegfr1 and r3 expression is only present in the leading tip of endothelial cells involved in active neo - angiogenesis . in the ks lesions only lana+ cells expressed prox-1 , indicating that this effect requires kshv gene expression ( hong et al . , 2004 ) . this suggests that differentiation toward lymphatic endothelial cells may specifically allow the spindle cells to respond to lymphangiogenic growth factors . in fact , kshv infection of endothelial cells induces both vegf - a and vegf - c ( sivakumar et al . , 2008 ) . therefore , induction of both vegfr1 and r3 allow kshv - infected cells to respond to key angiogenic and lymphangiogenic factors in the tumor environment . the direct role of kshv reprogramming of blood endothelial cells to lymphatic in induction of angiogenic and lymphangiogenic phenotypes is still under investigation . the highly vascular nature of ks tumors and the large amounts of neo - angiogenesis in the tumor led to the proposal that the etiologic agent of the tumor might directly induce angiogenesis . in accordance with this hypothesis kshv infection of endothelial cells , in particular , kshv induces the expression of vegf - a and -c and other cytokines as well as encoding angiogenic cytokines from its own genome ( boshoff et al . , 1997 ; aoki et al . , 1999 ; brown et al . , 2000 ; stine et al . , 2000 ; schwarz and murphy , 2001 ; masood et al . , 2002 ; polson et al . , 2002 ; wang et al . , 2004b ; xie et al . , 2005 ; caselli et al . , 2007 ; vart et al . , 2007 ; ye et al . , 2007 sivakumar et al . , 2008 ; wang and damania , 2008 ; sadagopan et al . additionally , because the tumor cell is endothelial in nature , induction of angiogenic cytokines may also activate the tumor cells and aid in the growth of ks tumors . kshv also induces angiogenic phenotypes directly in latently infected cells in a cell autonomous fashion , indicating that angiogenic activation of the infected endothelial cell may directly play a role in tumor formation . while kshv activates many growth - signaling properties and in general the induction of angiogenic phenotypes supports endothelial cell proliferation , in most cultures kshv does not induce increases in endothelial cell proliferation . it is possible that the cell culture milieu simply does not match the tumor milieu . ks spindle cells are not fully transformed ex vivo and , except in very rare cases , have a limited life span indicating that factors in the tumor environment that come from other cells types could be necessary to maintain ks spindle cell growth . the increase in growth could also be masked by the fact that endothelial cells in culture are rapidly dividing and therefore do not need additional growth signals . along those lines , that being said , the endothelial cell transforming potential of kshv in culture can be unmasked given specific conditions . dermal microvascular endothelial cells that were immortalized with the e6 and e7 genes from papillomavirus are readily transformed by kshv , including increased proliferation ( moses et al . , 1999 ) . therefore , kshv activation of endothelial cells can induce a proliferative advantage in the correct genetic environment . however , it is unknown if viral induction of angiogenic phenotypes is necessary for the growth in the e6/e7 immortalized endothelial cells . in general , viruses do not evolve to cause cancer , as it is likely a dead end for transmission . kshv likely evolved to activate the cell where it is maintained to ensure survival and spread of the virus . a major side effect of this activation may be providing an ideal environment for angiogenesis leading to increased vascularization of small tumor growths and expansion of ks tumors . while the study of viral induction of angiogenesis can lead to a better understanding of how kshv causes endothelial cell tumors , information gleaned from the study of viral mechanisms of induction of angiogenesis and lymphangiogenesis will also lead to a better understanding of endothelial cell activation and tumor angiogenesis in general . thus , the study of kshv infection of endothelial cells provides a controlled system for analyzing the regulation and induction of angiogenic phenotypes that will likely shed light on the field of tumor angiogenesis . 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 .

kaposi s sarcoma ( ks ) is a highly vascularized tumor supporting large amounts of neo - angiogenesis . the major cell type in ks tumors is the spindle cell , a cell that expresses markers of lymphatic endothelium . kshv , the etiologic agent of ks , is found in the spindle cells of all ks tumors . considering the extreme extent of angiogenesis in ks tumors at all stages it has been proposed that kshv directly induces angiogenesis in a paracrine fashion . in accordance with this theory , kshv infection of endothelial cells in culture induces a number of host pathways involved in activation of angiogenesis and a number of kshv genes themselves can induce pathways involved in angiogenesis . spindle cells are phenotypically endothelial in nature , and therefore , activation through the induction of angiogenic and/or lymphangiogenic phenotypes by the virus may also be directly involved in spindle cell growth and tumor induction . accordingly , kshv infection of endothelial cells induces cell autonomous angiogenic phenotypes to activate host cells . kshv infection can also reprogram blood endothelial cells to lymphatic endothelium . however , kshv induces some blood endothelial specific genes upon infection of lymphatic endothelial cells creating a phenotypic intermediate between blood and lymphatic endothelium . induction of pathways involved in angiogenesis and lymphangiogenesis are likely to be critical for tumor cell growth and spread . thus , induction of both cell autonomous and non - autonomous changes in angiogenic and lymphangiogenic pathways by kshv likely plays a key role in the formation of ks tumors .

Kaposis Sarcoma and KSHV Paracrine Induction of Angiogenesis by KSHV KSHV Induction of Angiogenic Phenotypes within the Infected Cell Angiogenesis vs. Lymphangiogenesis Summary Conflict of Interest Statement

kaposi s sarcoma ( ks ) was first described in the 1800s as a rare , fairly indolent tumor of specific populations . while a number of cell types are present , the tumor is predominantly made up of elongated spindle cells . the spindle cells express endothelial cell markers on their surface including cd31 and cd34 , but express low levels of factor viiira ( russell jones et al . , recent expression data and array studies have found that spindle cells most closely resemble lymphatic endothelium , expressing vegf receptor 3 ( vegfr3 ) , a specific marker of lymphatic endothelial cells ( jussila et al . , 1999 other lymphatic endothelial cell specific markers , including lyve-1 , podoplanin , and prox-1 , are also expressed by the spindle cells ( carroll et al . , , kshv was discovered associated with all ks tumors and is now considered to be the etiologic agent ( chang et al . all of the greater than 90 viral genes are expressed , the virus replicates rapidly , produces infectious virions and ultimately causes cell death likely due to a combination of host cell shut off and virus production . during viral latency in endothelial cells , this locus includes lana , vcyc , vflip , a family of proteins from a repeat region called the kaposins , and 12 pre - micrornas encoding 17 or more mature mirnas . , 1999 as expected the virus is predominantly found in the latent state in spindle cells where the limited number of latent genes and mirnas are expressed ( staskus et al . however , approximately 15% of the spindle cells express lytic viral genes and produce infectious virus . in addition to spindle cells , kshv is also found in other cell types in the ks lesion including monocytes ( blasig et al . kshv is also associated with two lymphoproliferative diseases , primary effusion lymphoma ( pel ) a pleural cavity solid b - cell lymphoma , and plasmablastic multicentric castleman s disease , a lymph node b - cell growth ( cesarman et al . because ks is an angioproliferative disease and the ks tumors are highly vascularized even at early stages , it has been proposed that kshv may directly induce angiogenesis . endothelial cells of the vascular system are normally maintained in a quiescent , non - proliferating state . however , during solid tumor formation , the secretion of pro - angiogenic cytokines by tumor cells can activate nearby endothelial cells to induce new blood vessel formation . many of the vascular slits identified in histological sections of early stage ks lesions are lined by uninfected endothelium , suggesting they are formed by endothelial cells activated in a paracrine fashion ( dupin et al . despite the evidence for paracrine activation of uninfected endothelial cells , kshv also likely activates infected endothelial cells in an autocrine or cell autonomous fashion . because ks spindle cells are endothelial in origin , induction of the ks tumor cell is similar to the processes of angiogenesis . this review discusses the recent evidence that suggests that ( 1 ) kshv promotes continual neovascularization through paracrine factors and ( 2 ) kshv may drive tumor cell growth through autocrine and cell autonomous activation of angiogenic phenotypes . the vascular endothelial growth factor ( vegf ) family of cytokines plays a prominent role in regulation of angiogenesis ( breen , 2007 ) . vegf - a expression is detected in spindle cells of ks lesions , and its secretion is known to be increased by inflammatory cytokines that are present in the ks lesions ( samaniego et al . infection of endothelial cells with kshv directly leads to increased expression of vegf - a ( masood et al . further , kshv conditioned media has been shown to regulate angiogenic phenotypes in endothelial cells ( sharma - walia et al . therefore , kshv induction of vegf - a is likely to be critical for both the induction of angiogenesis as well as the activation of infected spindle cells . hypoxia induced factor ( hif)-1 is a transcription factor that has been shown to be important for upregulation of vegf - a ( sodhi et al . however , during hypoxia , hif-1 is stabilized and can induce expression of genes through hypoxia responsive elements , including vegf - a . interestingly , kshv infection of endothelial cells induces the expression of hif-1 during normoxia which leads to increased hif transcriptional activity ( carroll et al . the kshv viral g - protein coupled receptor ( vgpcr ) is able to increase the activity of hif-1 as a transcription factor through activation of the mapk and p38 signaling pathways and subsequent phosphorylation of hif-1 ( sodhi et al . while induction of hif mrna expression by kshv infection has been shown , stabilization of hif directly by kshv infection of endothelial cells has yet to be clearly shown . white ovals indicate the activated pathway necessary for kshv induction of blood to lymphatic endothelial cell differentiation . the upper left corner indicates phenotypes of angiogenesis that are activated in normal endothelium by kshv - infected cells and the gradient of cytokines secreted by kshv - infected cells is indicated by the red gradient . the blue gradient in the upper right hand corner indicates the gradient of lymphangiogenic cytokines induced by kshv - infected cells that could induce lymphangiogenesis . other host proteins have been shown to be involved in the induction of vegf - a during kshv infection of endothelial cells as well . transgenic mice expressing vgpcr also form highly vascularized tumors that are reminiscent of ks tumors . however , gb , k8.1 , and vgpcr have only been detected in cells supporting lytic kshv infection whereas the bulk of the tumor cells are latently infected . in summary , the lytic phase of kshv infection might play a role in the paracrine induction of angiogenesis through increased secretion of vegf - a into the tumor milieu . ang2 is upregulated during pathologic angiogenesis and this expression is thought to destabilize endothelial cells , allowing them to be activated by other pro - angiogenic stimuli , such as vegf , see figure 1 , circle 1 ( gale et al . interestingly , ang2 is expressed in ks lesions , and is upregulated in endothelial cells infected with kshv ( brown et al . , 2000 ; this induction can be activated by the kshv genes vgpcr and vil-6 , and can occur through a paracrine mechanism ( vart et al . kshv induces a number of other cytokines known to be involved in angiogenesis in other systems . cyclooxygenase-2 ( cox-2 ) expression is induced early during kshv infection of endothelial cells and plays a role in the establishment of latency ( naranatt et al . an additional cellular factor associated with angiogenesis , angiogenin , is induced in endothelial cells by the latent protein , lana-1 . thrombospondin-1 plays multiple roles in the repression of angiogenesis , however one of its main functions is activation of the anti - angiogenic growth factor transforming growth factor- ( tgf- ) . the viral il-6 ( vil-6 ) is mostly detected in endothelial cells and spindle cells undergoing lytic replication but like k1 it has been shown to be expressed at very low levels in latently infected pel cells and to be induced during latency only under specific conditions ( chatterjee et al . in summary , conditioned media from kshv - infected cells can induce angiogenic phenotypes in uninfected endothelial cells as indicated by the red gradient in figure 1 . kshv infection of endothelial cells induces expression of a number of cytokines that are capable of inducing angiogenesis in a paracrine fashion . paramount among these is vegf - a , an angiogenic cytokine that is induced by kshv infection of endothelial cells . while the predominant viral mechanism of vegf - a induction is unknown , a number of lytic kshv genes are sufficient to induce vegf - a when overexpressed . kshv - infected cells also produce a number of other angiogenic cytokines of cellular and viral origin that likely play a role in the induction of angiogenesis . taken together , all of the cytokines and induced pathways likely create a milieu that is beneficial to the induction of new blood vessels and play a significant role in the high vascularization of ks tumors . therefore ks tumor formation is likely to include increased angiogenic capacity of the spindle cells . there is growing evidence demonstrating the manipulation of host cell phenotypes by kshv and the role of these changes in the promotion of angiogenesis related phenotypes . these infected cell phenotypes include increased stability of tubules formed by macrovascular endothelial cells , induction of capillary morphogenesis in low growth factor conditions , and enhanced migration and invasion ( qian et al . , additionally , kshv induces the expression of vegf receptors on the surface of infected endothelial cells as discussed below . breakdown of these junctions is necessary for initiation of angiogenesis , immune cell extravasation , and tumor cell metastasis . interestingly , several studies have evaluated the adherens junctions of kshv - infected endothelial cells and found them to be perturbed ( mansouri et al . , 2008 ) as well as disruption of ve - cadherin therefore , kshv infection can directly initiate a key angiogenic step , the breakdown of cell cell adherence . while the direct mechanism of kshv - induced disruption of adherens junctions during latency is not known , there are a number of candidate kshv genes that could be involved ( table 1 ) . , k1 , a primarily lytic protein that may also be expressed at low levels during latency was also shown to initiate signaling similar to that required for disruption of cadherin signaling ( guilluy et al . while the exact viral mechanism of disruption of adherens junctions by kshv infection is not known , the virus encodes multiple genes capable of altering cadherin . expression of mmp proteins induced by kshv allows for increased invasion of both infected and uninfected endothelial cells ( wang et al . , 2011 ) indicating that kshv activation of angiogenic phenotypes in endothelial cells may lead to enhanced oncogenesis as well . in fact , when endothelial cells are cultured in the presence of conditioned media from kshv - infected bcbl-1 cells , their ability to organize into capillary - like structures is increased ( wang and damania , 2008 ) . however , the effect of bcbl-1 conditioned media was greater on kshv - infected endothelial cells than on mock - infected cells , suggesting that infected cells are more receptive to angiogenic growth factors . in addition , this same study found that capillary - like structures formed by kshv - infected endothelial cells are more persistent than mock - infected cells , indicative of the promotion of cell survival and continual angiogenesis by kshv ( wang and damania , 2008 and our unpublished results ) . kshv latent infection of endothelial cells also induces vegf receptor expression , which may allow infected cells to respond more robustly to vegfs . vegfr1 has been described as both a positive and negative regulator of angiogenesis depending on the context . interestingly , expression of vegfr3 , the main receptor for vegf - c and d is also significantly increased by kshv infection ( carroll et al . , 2004 ) vegfr3 , a receptor specific to lymphatic endothelium and critical for lymphangiogenesis will be discussed below . importantly , endothelial tip cells at the leading edge of new vascular protrusions are the only main adult cell type known to express both the blood endothelial cell receptor , vegfr1 , and the lymphatic endothelial cell receptor , vegfr3 ( tammela et al . , 2008 ) : kshv infection of endothelial cells directly induces expression of both of these receptors . the mechanisms by which kshv induces angiogenic phenotypes in latently infected cells are largely unknown . a number of angiogenic phenotypes are likely to be a direct result of the cytokine milieu of the infected cells . however , it is also apparent that some of the angiogenic effects seen in latently infected cells are cell autonomous , independent of either paracrine or autocrine factors . as described above , conditioned media from pel cells had stronger effects on tubule formation of kshv - infected endothelial cells ( akula et al . this suggests that during latent kshv infection there is a shift in endothelial cell signaling that results in a more angiogenic phenotype dependent on v3 expression on the surface of the cell ( figure 1 , center ) . therefore , kshv alteration of endothelial cell signaling pathways can dramatically affect how the cell responds to intra- and extra - cellular signals . these changes that lead to alterations in angiogenic properties are likely to play a role in the growth and cell cell interactions of infected cells , thereby playing a role in ks tumor formation . during development of the vascular system , a subset of endothelial cells in the cardinal vein begin to express markers of lymphatic differentiation , including the master regulatory gene , prox-1 . these cells then bud from the cardinal vein , differentiate into lymphatic endothelial cells , and form the lymphatic vascular system ( wigle and oliver , 1999 ) . immunohistochemistry of ks tumors showed that spindle cells express markers of lymphatic endothelium , suggesting these cells may arise from primary infection of lymphatic endothelial cells , rather than blood endothelial cells ( jussila et al . an alternative hypothesis is that kshv infection of blood endothelial cells drives differentiation toward a more lymphatic phenotype . this idea is supported by evidence that kshv infection of blood endothelial cells promotes expression of lymphatic - specific genes , including prox-1 , vegfr3 , podoplanin , and lyve-1 , effectively driving the reprogramming of blood endothelial cells to become lymphatic endothelium ( carroll et al . microarray analysis comparing kshv - infected blood endothelial cells to blood and lymphatic endothelial cells indicate that kshv - infected blood endothelial cells have gene expression profiles that align more closely to lymphatic endothelial cells than that of blood endothelial cells ( carroll et al . however , this effect was not sufficient to induce prox-1 expression in blood endothelial cells . we recently found that induction of blood to lymphatic endothelial cell reprogramming requires signaling through the cellular receptor gp130 . this leads to activation of the jak / stat3 pathway and the pi3k / akt pathway leading to expression of lymphatic - specific genes starting with prox-1 . however , kshv lacking vil-6 is still able to cause blood to lymphatic endothelial cell differentiation , indicating that kshv has evolved multiple strategies to activate gp130 and induce blood to lymphatic endothelial cell differentiation ( morris et al . induction of lymphatic differentiation by kshv is only part of the story , however . despite the expression of lymphatic - specific genes , blood endothelial cells infected with kshv retain expression of some blood specific markers ( wang et al . additionally , infection of lymphatic endothelial cells with kshv induces expression of blood specific markers ( wang et al . downregulation of maf in lymphatic endothelial cells by sirna restored expression of blood endothelial markers , such as vegfr1 and cxcr4 . thus , infection of blood or lymphatic endothelial cells by kshv alters host gene expression to an intermediate state between the two cell types . as described above , this intermediate phenotype with both vegfr1 and r3 expression is only present in the leading tip of endothelial cells involved in active neo - angiogenesis . this suggests that differentiation toward lymphatic endothelial cells may specifically allow the spindle cells to respond to lymphangiogenic growth factors . in fact , kshv infection of endothelial cells induces both vegf - a and vegf - c ( sivakumar et al . therefore , induction of both vegfr1 and r3 allow kshv - infected cells to respond to key angiogenic and lymphangiogenic factors in the tumor environment . the direct role of kshv reprogramming of blood endothelial cells to lymphatic in induction of angiogenic and lymphangiogenic phenotypes is still under investigation . the highly vascular nature of ks tumors and the large amounts of neo - angiogenesis in the tumor led to the proposal that the etiologic agent of the tumor might directly induce angiogenesis . in accordance with this hypothesis kshv infection of endothelial cells , in particular , kshv induces the expression of vegf - a and -c and other cytokines as well as encoding angiogenic cytokines from its own genome ( boshoff et al . additionally , because the tumor cell is endothelial in nature , induction of angiogenic cytokines may also activate the tumor cells and aid in the growth of ks tumors . kshv also induces angiogenic phenotypes directly in latently infected cells in a cell autonomous fashion , indicating that angiogenic activation of the infected endothelial cell may directly play a role in tumor formation . while kshv activates many growth - signaling properties and in general the induction of angiogenic phenotypes supports endothelial cell proliferation , in most cultures kshv does not induce increases in endothelial cell proliferation . ks spindle cells are not fully transformed ex vivo and , except in very rare cases , have a limited life span indicating that factors in the tumor environment that come from other cells types could be necessary to maintain ks spindle cell growth . the increase in growth could also be masked by the fact that endothelial cells in culture are rapidly dividing and therefore do not need additional growth signals . along those lines , that being said , the endothelial cell transforming potential of kshv in culture can be unmasked given specific conditions . dermal microvascular endothelial cells that were immortalized with the e6 and e7 genes from papillomavirus are readily transformed by kshv , including increased proliferation ( moses et al . therefore , kshv activation of endothelial cells can induce a proliferative advantage in the correct genetic environment . however , it is unknown if viral induction of angiogenic phenotypes is necessary for the growth in the e6/e7 immortalized endothelial cells . kshv likely evolved to activate the cell where it is maintained to ensure survival and spread of the virus . while the study of viral induction of angiogenesis can lead to a better understanding of how kshv causes endothelial cell tumors , information gleaned from the study of viral mechanisms of induction of angiogenesis and lymphangiogenesis will also lead to a better understanding of endothelial cell activation and tumor angiogenesis in general . thus , the study of kshv infection of endothelial cells provides a controlled system for analyzing the regulation and induction of angiogenic phenotypes that will likely shed light on the field of tumor angiogenesis .

for more than a decade , mass distribution of mectizan(ivermectin , msd ) has been recommended for prevention of blindness caused by infection with onchocerca volvulus . the remarkable efficacy of mectizanagainst the microfilariae of o. volvulus , as well as the safety and effectiveness of the drug against a broad range of intestinal helminths and ectoparasites , has resulted in its acceptance at the community level . however , in recent years , serious adverse events ( saes ) associated with mass distribution of mectizanin cameroon have caused widespread concern . many of these saes were characterized by progressive neurologic decline and encephalopathy within a few days of taking mectizan [ , mectizandonation program ( mdp ) , unpublished data ] . affected persons lived in loiasis - endemic areas and , based on their post - treatment l. loa microfilarial loads , are presumed to have had high densities of l. loa microfilaremia prior to treatment with mectizan(mdp , unpublished data ) . the mectizanexpert committee ( mec ) limits mass distribution of mectizanto areas considered meso- or hyper - endemic for onchocerciasis , as determined by the technique known as rapid epidemiologic mapping for onchocerciasis ( remo ) . remo is based on rapid epidemiologic assessment ( rea ) for the presence of onchocercal nodules in samples of 30 adult men in selected indicator villages . villages with a nodule prevalence of 40% , 2039% , or < 20% are defined as hyper- , meso- , or hypo - endemic , respectively . these estimates of onchocerciasis endemicity are projected onto larger geographical areas with similar ecologic characteristics . although residents of areas classified as hypo - endemic do not receive mass treatment with mectizan , they are eligible for individual clinic - based treatment if they are diagnosed with onchocerciasis . remo projections sometimes result in the inclusion of hypo - endemic villages into areas designated for mass distribution of mectizan . until recently , such misclassifications of hypo - endemic villages had few , if any , known negative consequences . individuals living in these villages benefited from mectizan 's action against intestinal helminths and ectoparasites , even if few of them suffered from onchocercal skin or eye disease . however , when cases of l. loa encephalopathy occurred following mass distribution of mectizan , the specificity of remo ( i.e. its ability to exclude hypo - endemic villages from mass drug distribution ) was no longer considered adequate . during the mid-1990s , reviews of the clinical features of mectizan - associated l. loa encephalopathy revealed that basic supportive care ( i.e. , hydration , feeding , and nursing care ) was critical for patient recovery ; indeed , prompt and sustained supportive care is the most important therapeutic measure . thus , mass distribution of mectizanin l. loa - endemic areas should be preceded by community education to increase awareness of possible saes following treatment . during and after mass drug distribution , intensified surveillance for saes is required , as is a referral system to appropriate health facilities for any saes that may occur . in affected areas , these requirements have led to a temporary shift away from the strategy of community - directed treatment with ivermectin ( cdti ) currently favored by the african programme for onchocerciasis control ( apoc ) . cdti emphasizes community decision - making on the timing and manner of drug distribution by community volunteers ( rather than by nurses or other medical personnel ) . in 2000 , the mec and the technical consultative committee ( tcc ) of apoc recommended a new approach to mass treatment for onchocerciasis in areas co - endemic for loiasis . this approach begins with an environmental risk model based on remote sensing data for central and west africa . the model , developed by madeleine thomson and colleagues at the liverpool school of tropical medicine , predicts the prevalence of l. loa infection based on vegetation and other land characteristics associated with a suitable environment for the presence of chrysops spp . the model generates a map that , although still in the process of being validated , serves as an important operational starting point when considering whether a given area is likely to be endemic for loiasis . for high - risk areas on the map , the mec / tcc recommendations for areas co - endemic for onchocerciasis and loiasis currently require village - by - village rea before initiating mass treatment with mectizan . mass distribution of mectizanis recommended for all villages that remain classified as meso- or hyper - endemic for onchocerciasis by rea but only after enhanced health education and training of community members , and only if sae surveillance by community distributors and medical preparedness for sae management can be ensured . in villages confirmed as hypo - endemic on the basis of rea , mass treatment is not recommended , although mectizanis made available to treat individuals with clinical onchocerciasis after health workers assess the individual 's burden of l. loa microfilariae and related risk of l. loa encephalopathy . increased costs result from village - by - village rea before treatment , as well as from safeguards required to reduce risk of death and disability from l. loa encephalopathy in hyper- and meso - endemic areas . further , communities classified as hypo - endemic may be surrounded by meso- and hyper - endemic communities , and this can complicate the public health messages and interventions in both areas . the current mec / tcc approach assumes maximum risk of l. loa encephalopathy in all areas predicted as high risk on the environmental risk model map , and it uses village - by - village rea to increase programmatic certainty that " enough " onchocerciasis exists to warrant mass treatment with mectizan . this strategy focuses the mass treatment decision on the health benefits of preventing blindness rather than on the risk of l. loa encephalopathy following treatment . it was adopted because the only way to reliably predict risk of encephalopathy was to microscopically examine blood samples from all residents of a treatment area for l. loa microfilaremia before mass treatment . a new technique , known as raploa , has been developed that does not require collection of blood to detect areas with high - density l. loa microfilaremia . a recent multi - center study showed that 1 ) the prevalence of l. loa infection is associated with microfilarial density ; and 2 ) the prevalence of individuals with high - level microfilaremia in a community is associated with the percentage of community members who respond positively to questions about previous experience with a migrating eyeworm . this study found that the most sensitive and specific rapid assessment tool was based on a " restricted " definition of eyeworm passage , in which the subject must recognize a photograph of eyeworm as the same condition that he or she had experienced , and the eyeworm episode must have lasted no more than 7 days . in three different ecologic settings in nigeria and cameroon , investigators found that the prevalence of persons with high microfilarial densities ( 30,000 microfilariae per milliliter [ mf / ml ] of blood ) remained low when the prevalence of a history of eyeworm was less than 40% , but that it increased rapidly above this level ( figure 1 , excerpted from the multi - country report on raploa ) . raploa is now being suggested as an alternate approach to rea in areas that are predicted by the environmental risk model map to be co - endemic for l. loa . conceptually , such an approach , which bases the mass treatment decision on a defined risk of l. loa encephalopathy following mectizantreatment , seems more appealing than one based solely on an assessment of how much onchocerciasis is " enough " at the village level to justify an unknown risk of treatment - related l. loa encephalopathy . a threshold of 40% of persons with a history of eyeworm has been suggested for distinguishing between high - risk areas for l. loa encephalopathy ( which would require enhanced community education , sae surveillance , and supportive medical care ) from those at low risk . presumably , the traditional cdti approach to mass drug distribution could be used in areas categorized as low risk . a close examination of figure 1 reveals that in some villages where < 40% of persons have a history of eyeworm , as many as 2% of persons also have l. loa microfilarial densities of 30,000 per ml . these persons would be at risk of l. loa encephalopathy if mass distribution of mectizanwere implemented . use of 20% prevalence of a history of eyeworm as the threshold ( raploa-20 ) would substantially reduce ( to near zero , based on figure 1 ) the prevalence of persons with microfilarial densities of 30,000 per ml in areas classified as low risk . another alternative , suggested by some , would be to apply both rea and raploa-40 simultaneously in a given area to gather the maximum available information about both onchocerciasis and loiasis . with this strategy , once communities are confirmed by rea as hyper- or meso - endemic for onchocerciasis , raploa-40 would be used to determine whether they are at high risk for l. loa encephalopathy following treatment with mectizan . decision models can be useful to guide programmatic decisions , identify new research needs , identify critical decision points , and determine factors that most strongly influence both cost and safety . the primary purpose of the current model is to provide a structured framework to understand and estimate the potential health risks and benefits of alternative approaches to mass treatment strategies in areas that are co - endemic for onchocerciasis and loiasis . the broad conceptual framework underlying the model , described above , is shown graphically in figure 2 . the underlying assumptions of the model are based on the scientific literature and , when scientific data are lacking , on expert opinion and information available to the mdp ( see additional file 1 : assumptions used in the decision model ) . decision models can be useful to guide programmatic decisions , identify new research needs , identify critical decision points , and determine factors that most strongly influence both cost and safety . the primary purpose of the current model is to provide a structured framework to understand and estimate the potential health risks and benefits of alternative approaches to mass treatment strategies in areas that are co - endemic for onchocerciasis and loiasis . the broad conceptual framework underlying the model , described above , is shown graphically in figure 2 . the underlying assumptions of the model are based on the scientific literature and , when scientific data are lacking , on expert opinion and information available to the mdp ( see additional file 1 : assumptions used in the decision model ) . the model was constructed in the standard fashion using treeage software ( williamstown , ma ) . four strategies ( rea , raploa-40 , raploa-20 , and combined rea / raploa-40 ) were examined ( figure 3 ) under four different ecologic conditions with varying intensities of o. volvulus and l. loa transmission ( appendix - see additional file 1 ) . we examined three principal outcomes : 1 ) onchocercal blindness ; 2 ) chronic neurologic complications or death associated with l. loa encephalopathy following mectizantreatment ; and 3 ) disability - adjusted life years ( dalys ) , which were influenced by onchocercal blindness and skin disease as well as by treatment - related l. loa encephalopathy , death , and chronic neurologic sequelae ( appendix - see additional file 1 ) . daly losses for onchocercal blindness and skin disease were extrapolated from the global burden of disease estimates ( appendix - see additional file 1 ) . , authors ' estimates were used to reflect a short - term serious event ( l. loa encephalopathy ) , a chronic severe event ( permanent neurologic sequelae ) , and death at unknown age . economic outcomes , particularly the costs associated with the various approaches , were not included because data were not available . the risk of l. loa encephalopathy temporally related to mectizantreatment was assumed to be a function of a threshold prevalence of high intensity microfilaremia , defined as 30,000 mf / ml of blood . this threshold was suggested previously by ducorps and colleagues and boussinesq and co - workers . other investigators have suggested thresholds as high as 50,000 mf / ml and as low as 10,000 mf / ml . we assumed that the risk of l. loa encephalopathy following mectizantreatment was reduced by 50% in the second year of treatment . the estimated risk of l. loa encephalopathy following mectizantreatment is based on two factors : 1 ) the percentage of communities with different portions of their population having microfilarial loads of 30,000 mf / ml ( which we categorized as 0% , 0.10.9% , 1.01.9% , 2.03.9% , 4.07.9% , and 8% ) ; and 2 ) risk of l. loa encephalopathy following mectizantreatment in individuals with microfilarial loads above the 30,000 mf / ml threshold , which we assumed to be 1% ( appendix - see additional file 1 ) . the model estimates the prevalence of individuals with microfilarial loads of 30,000 mf / ml within communities that receive 1 ) mass treatment without additional education or enhanced sae surveillance ; 2 ) mass treatment with additional education and enhanced surveillance ; or 3 ) clinic - based treatment for individuals diagnosed with onchocerciasis . these estimates are based on the estimated sensitivity and specificity of raploa ( figure 1 ) , the performance of rea , and the underlying distribution of high - intensity l. loa microfilaremia within communities ( appendix - see additional file 1 ) . four scenarios of varying l. loa prevalence were examined , two in which 60% of communities had 1 resident with high - density l. loa microfilaremia and two in which this figure was 10% ( appendix - see additional file 1 ) . based on the estimated prevalence of individuals with high intensity infection , the community risk of l. loa encephalopathy following mectizantreatment was estimated . three ultimate outcomes were considered : death , chronic or permanent neurological damage , and no adverse outcome . we assumed that the outcome of l. loa encephalopathy following mectizantreatment was influenced by the presence or absence of adequate supportive care for these patients ( appendix - see additional file 1 ) . the model assumes that the lifetime risk of onchocercal blindness and skin disease is determined by the estimated community prevalence of onchocerciasis as determined by rea . in areas where the sowda form of onchocerciasis predominates , this assumption would underestimate risk of skin disease . in the high - prevalence scenarios for o. volvulus , the percentage of communities that were classified as hyper- , meso- and hypo - endemic for onchocerciasis was 60% , 30% , and 10% , respectively . for low - prevalence areas , these figures were 30% , 30% and 40% , respectively ( appendix - see additional file 1 ) . since treatment halts progression of disease in those who already have clinical manifestations and prevents disease manifestations in those already infected or those not yet infected , we assumed that treatment is completely effective in eliminating the risk of morbidity due to onchocerciasis among treated individuals . we assumed that there was no impact of mass distribution on transmission or on risk of blindness or skin disease among persons who did not take mectizan . thus , non - treated individuals were assumed to receive no benefit from mass treatment in their village . the risk of l. loa encephalopathy temporally related to mectizantreatment was assumed to be a function of a threshold prevalence of high intensity microfilaremia , defined as 30,000 mf / ml of blood . this threshold was suggested previously by ducorps and colleagues and boussinesq and co - workers . other investigators have suggested thresholds as high as 50,000 mf / ml and as low as 10,000 mf / ml . we assumed that the risk of l. loa encephalopathy following mectizantreatment was reduced by 50% in the second year of treatment . the estimated risk of l. loa encephalopathy following mectizantreatment is based on two factors : 1 ) the percentage of communities with different portions of their population having microfilarial loads of 30,000 mf / ml ( which we categorized as 0% , 0.10.9% , 1.01.9% , 2.03.9% , 4.07.9% , and 8% ) ; and 2 ) risk of l. loa encephalopathy following mectizantreatment in individuals with microfilarial loads above the 30,000 mf / ml threshold , which we assumed to be 1% ( appendix - see additional file 1 ) . the model estimates the prevalence of individuals with microfilarial loads of 30,000 mf / ml within communities that receive 1 ) mass treatment without additional education or enhanced sae surveillance ; 2 ) mass treatment with additional education and enhanced surveillance ; or 3 ) clinic - based treatment for individuals diagnosed with onchocerciasis . these estimates are based on the estimated sensitivity and specificity of raploa ( figure 1 ) , the performance of rea , and the underlying distribution of high - intensity l. loa microfilaremia within communities ( appendix - see additional file 1 ) . four scenarios of varying l. loa prevalence were examined , two in which 60% of communities had 1 resident with high - density l. loa microfilaremia and two in which this figure was 10% ( appendix - see additional file 1 ) . based on the estimated prevalence of individuals with high intensity infection , the community risk of l. loa encephalopathy following mectizantreatment was estimated . three ultimate outcomes were considered : death , chronic or permanent neurological damage , and no adverse outcome . we assumed that the outcome of l. loa encephalopathy following mectizantreatment was influenced by the presence or absence of adequate supportive care for these patients ( appendix - see additional file 1 ) . the model assumes that the lifetime risk of onchocercal blindness and skin disease is determined by the estimated community prevalence of onchocerciasis as determined by rea . in areas where the sowda form of onchocerciasis predominates , this assumption would underestimate risk of skin disease . in the high - prevalence scenarios for o. volvulus , the percentage of communities that were classified as hyper- , meso- and hypo - endemic for onchocerciasis was 60% , 30% , and 10% , respectively . for low - prevalence areas , these figures were 30% , 30% and 40% , respectively ( appendix - see additional file 1 ) . since treatment halts progression of disease in those who already have clinical manifestations and prevents disease manifestations in those already infected or those not yet infected , we assumed that treatment is completely effective in eliminating the risk of morbidity due to onchocerciasis among treated individuals . we assumed that there was no impact of mass distribution on transmission or on risk of blindness or skin disease among persons who did not take mectizan . thus , non - treated individuals were assumed to receive no benefit from mass treatment in their village . in our model , onchocerciasis reduced average disability adjusted life years ( dalys ) by 1.52 and 0.88 years per person in the high- and low - prevalence areas , respectively ( table 1 ) . although our model was conservative in assuming systematic annual drug coverage of only 65% , mass treatment with mectizanresulted in a net gain of 0.5 to 1.0 dalys per person ( reducing lost dalys from 1.52 to 0.53 and from 0.88 to 0.31 in high - and low - prevalence areas , respectively ) , even in the absence of measures to reduce the risk of l. loa - associated death and permanent disability ( table 1 ) . similarly , in our model , mass treatment with mectizanreduced the lifetime risk of blindness from 166 to 58 cases per 1000 population in areas with a high prevalence of onchocerciasis and from 94 to 33 cases per 1000 population in low prevalence areas . because rea - based strategies to reduce mectizan - related l. loa encephalopathy result in fewer people receiving mectizan , these reductions in blindness were somewhat diminished in the presence of such strategies ( table 2 ) . in the absence of any approach to reduce complications of l. loa encephalopathy following mass treatment with mectizan , the estimated risk of l. loa associated death or permanent neurologic sequelae was 85.1 and 26.0 cases per 1 million population in high- and low - prevalence areas for loiasis , respectively . all four risk - reduction strategies dramatically reduced the risk of these complications ( table 3 ) . regardless of the prevalence of onchocerciasis or loiasis , the current risk reduction strategy , rea , led to the greatest reductions ( 94% 99% ) in death and permanent disability associated with l. loa encephalopathy . similar reductions ( 9398% ) were observed for raploa-20 . raploa-40 and the combined rea / raploa-40 approach were less effective in reducing risk ( table 3 , figure 4 ) . risk of l. loa associated death and disability was 2.0 to 6.7 times greater with raploa-40 or rea / raploa-40 than with rea or raploa-20 ( table 3 ) . figure 4 illustrates the relationship between the estimated number of cases of blindness and the number of deaths and chronic disabilities resulting from l. loa encephalopathy in the context of 1 ) no mass treatment ; 2 ) mass treatment with no risk reduction strategies ; and 3 ) mass treatment with each of the four risk reduction strategies in the model . in our model , onchocerciasis reduced average disability adjusted life years ( dalys ) by 1.52 and 0.88 years per person in the high- and low - prevalence areas , respectively ( table 1 ) . although our model was conservative in assuming systematic annual drug coverage of only 65% , mass treatment with mectizanresulted in a net gain of 0.5 to 1.0 dalys per person ( reducing lost dalys from 1.52 to 0.53 and from 0.88 to 0.31 in high - and low - prevalence areas , respectively ) , even in the absence of measures to reduce the risk of l. loa - associated death and permanent disability ( table 1 ) . similarly , in our model , mass treatment with mectizanreduced the lifetime risk of blindness from 166 to 58 cases per 1000 population in areas with a high prevalence of onchocerciasis and from 94 to 33 cases per 1000 population in low prevalence areas . because rea - based strategies to reduce mectizan - related l. loa encephalopathy result in fewer people receiving mectizan , these reductions in blindness were somewhat diminished in the presence of such strategies ( table 2 ) . in the absence of any approach to reduce complications of l. loa encephalopathy following mass treatment with mectizan , the estimated risk of l. loa associated death or permanent neurologic sequelae was 85.1 and 26.0 cases per 1 million population in high- and low - prevalence areas for loiasis , respectively . all four risk - reduction strategies dramatically reduced the risk of these complications ( table 3 ) . regardless of the prevalence of onchocerciasis or loiasis , the current risk reduction strategy , rea , led to the greatest reductions ( 94% 99% ) in death and permanent disability associated with l. loa encephalopathy . similar reductions ( 9398% ) were observed for raploa-20 . raploa-40 and the combined rea / raploa-40 approach were less effective in reducing risk ( table 3 , figure 4 ) . risk of l. loa associated death and disability was 2.0 to 6.7 times greater with raploa-40 or rea / raploa-40 than with rea or raploa-20 ( table 3 ) . figure 4 illustrates the relationship between the estimated number of cases of blindness and the number of deaths and chronic disabilities resulting from l. loa encephalopathy in the context of 1 ) no mass treatment ; 2 ) mass treatment with no risk reduction strategies ; and 3 ) mass treatment with each of the four risk reduction strategies in the model . the occurrence of l. loa encephalopathy following mass treatment with mectizanhas negatively affected onchocerciasis control efforts in central africa , which rely on a strategy of mass treatment with mectizanusing cdti . the lack of a rapid epidemiologic assessment tool to estimate risk of l. loa encephalopathy following mectizantreatment has severely limited attempts to expand onchocerciasis control into areas thought to be co - endemic for l. loa . validation of raploa is currently ongoing and discussions are now underway about how best to apply raploa programmatically to reduce risk of treatment - related l. loa encephalopathy and its complications . our model showed the substantial public health benefits of mectizandistribution , expressed either as dalys or as blindness prevention , even in areas with co - endemic loiasis in which no measures to reduce l. loa associated risk were implemented . the model also indicated that all four risk - reduction strategies would substantially decrease the risk of death and permanent disability from l. loa encephalopathy temporally related to mectizantreatment ( figure 4 ) . two approaches the currently recommended rea and raploa-20 resulted in the greatest reductions in death and permanent disability from l. loa encephalopathy . in comparison , risk of death and permanent disability was about twice as high for the strategy based on raploa-40 . we found no advantage to a strategy that would employ a combination of rea and raploa-40 . the currently recommended approach for reducing risk of death and permanent neurologic complications from l. loa encephalopathy incurs significant programmatic costs . these costs result from village - by - village assessments of onchocerciasis endemicity by rea , as well as the need to 1 ) educate at - risk communities about l. loa encephalopathy , 2 ) establish surveillance for treatment - related saes , and 3 ) provide appropriate supportive medical and nursing care for those who develop l. loa encephalopathy following mass treatment . community education , surveillance , and supportive medical care are key elements in all four risk - reduction strategies evaluated in our model . village - by - village raploa would likely take about as long to execute as rea and would consume comparable financial and human resources . thus , it is not yet certain that the results from a raploa assessment in a small sample of villages would accurately predict the risk of saes throughout an entire area . in the absence of such a sampling scheme , village - by - village assessment using raploa would be necessary and would likely provide no significant cost savings over village - by - village rea . if raploa is validated and found to be operationally reproducible and feasible , however , it would offer several advantages . data collected through raploa could significantly enhance our understanding of the epidemiology of high - density l. loa microfilaremia and provide an opportunity to refine the predictions of the environmental risk model map . the current approach of rea assumes that the map is 100% accurate in identifying areas that are highly endemic for loiasis . in these areas , onchocerciasis control programs are required invariably to carry out rea in every village and , in all villages where mass treatment with mectizanis offered , to bear the additional costs of enhanced training , sae surveillance , and reinforcing health systems for the clinical management of l. loa encephalopathy . however , the environmental risk model map predicts risk of loiasis on the basis of environmental features that indicate favorable habitat for the chrysops vector . it is possible ( and likely ) that many areas conducive to the vector do not have significant transmission of l. loa . use of raploa may reveal the absence of high - density l. loa microfilaremia ( as predicted by history of eyeworm ) throughout much of central africa where the environmental risk model map currently predicts l. loa to be highly endemic . in these areas , mass treatment with mectizancould be instituted with no need for enhanced sae training , surveillance , and medical systems . this would result in large cost savings at the regional level and vastly simplified program operations . because the actual prevalence of high - density l. loa microfilaremia throughout much of central africa is unknown and cost data were not available , we could not account for these potential advantages of raploa in the decision model . finally , the raploa approach may prove useful in the global programme to eliminate lymphatic filariasis for assessing risk of treatment - related l. loa encephalopathy in areas that are co - endemic for l. loa and wuchereria bancrofti . in most of sub - saharan africa , this programme relies on mass treatment with both mectizanand albendazole . a major challenge to all four risk - reduction strategies is the potential for misclassification , which may be substantial . unfortunately , the cost of being able to rapidly detect and treat all cases of l. loa encephalopathy temporally related to mectizantreatment appears to be the establishment of surveillance and medical systems that , in many areas , may be unnecessary . the principal disadvantage of raploa-20 is its relatively low specificity , which could result in large areas of central africa being misclassified as high risk for l. loa encephalopathy and unnecessary expenditures of health resources . because raploa-40 is more specific however , our model suggests that raploa-40 could result in twice as many l. loa associated deaths and cases of chronic disability , because this technique allows communities classified as low risk to have as many as 2% of persons with 30,000 l. loa mf / ml blood . these communities could therefore receive mectizanin the absence of adequate surveillance and systems to rapidly detect and treat l. loa encephalopathy . the magnitude of this problem would depend on the degree to which communities with persons at risk for l. loa encephalopathy are geographically clustered within areas classified as high risk by raploa-40 . if they are not tightly clustered , but rather scattered in relatively small numbers throughout central africa , mectizan - related l. loa encephalopathy will be difficult to predict and even harder to prevent . the apparent clustering of reported cases of l. loa encephalopathy in certain regions of cameroon is a reassuring sign in this regard . further research on the performance of raploa and on the geographic clustering of communities with persons with high microfilarial loads is urgently needed . as with any decision model , the accuracy of our conclusions is limited by the uncertainty surrounding our assumptions . for many of our assumptions , published data are lacking and we relied on expert opinion and unpublished data available to the mdp . even when data were available , especially on the epidemiology and health risks of l. loa , they were limited to a few studies in specific areas ; the degree to which these data are representative for other areas in central africa is not known . when we performed sensitivity analyses for the key variables in our model , our basic conclusions did not substantially change ( data not shown ) . we considered as treatment - related benefits only prevention of onchocercal blindness and skin disease , and omitted benefits resulting from mectizan 's action against intestinal helminths , strongyloides stercoralis , wuchereria bancrofti , and a variety of ectoparasites . our assumption that drug coverage was 65% is consistent with population - based coverage figures reported from onchocerciasis control programs in africa . however , by assuming that this coverage was systematic , 35% of persons in affected communities would never have received mectizan . this assumption limited both the health benefits of mectizanand the number of persons with l. loa - associated encephalopathy . in a sensitivity analysis that assumed a random coverage pattern , with up to 100% of those targeted for mass treatment receiving one or more doses of mectizan , the conclusions of our model did not change . the relative effectiveness of each of the four strategies in reducing onchocercal blindness and the risk of l. loa associated death and disability remained constant ( data not shown ) . in central africa where onchocerciasis and loiasis are co - endemic , l. loa encephalopathy has occurred following mass treatment with mectizan . in some cases , a new rapid assessment technique called raploa may provide programme managers with an important tool with which to assess l. loa endemicity and the risk of treatment - related l. loa encephalopathy . our decision model suggests that use of raploa-20 or the current approach of rea would lead to the greatest reductions in death and permanent disability associated with l. loa encephalopathy . however , use of rea would result in fewer communities receiving mectizan . compared to rea , the usefulness of raploa , both in preventing blindness and avoiding l. loa encephalopathy following mectizantreatment in co - endemic areas , is highly dependent on the raploa cutoff point . full programmatic implementation of raploa will require testing of the method in other areas of central africa , an improved understanding of geographic clustering of high - density l. loa microfilaremia , sampling strategies that would avoid the need for raploa assessments in all communities , cost analyses , and confirmation that the questionnaire can be administered easily and reliably by field staff . apoc african programme for onchocerciasis control cdti community - directed treatment with ivermectin daly disability - adjusted life year mec mectizanexpert committee mdp mectizandonation program msd merck , sharpe , and dohme , inc . raploa rapid assessment procedure for loa loa . the raploa technique to which we refer in this paper uses a " restricted definition " of eyeworm : the subject must recognize a photograph of eyeworm as the same condition that he or she is reporting , and rea rapid epidemiologic assessment ( for onchocerciasis ) remo rapid epidemiologic mapping of onchocerciasis sae serious adverse event tcc technical consultative committee of the african programme for onchocerciasis control all authors participated fully in extensive critical discussions as the model developed and in writing the paper . da conceived of the study , helped develop the decision model , and was the primary author of the paper . nayt - d conceived of the study , provided data , helped develop the decision model , and helped write the paper . for conceived of the study , provided critical data and perspective , helped refine the model , and helped write the paper . bjorn thylefors , director , mectizandonation program , for helpful discussions on study conceptualization and for his expert opinion on parameters for the model . prevalence of persons with l. loa microfilaremia 30,000 per ml as a function of the prevalence of a restricted definition of eyeworm , from figure 10 of the multi - center report on raploa . see raploa in the list of abbreviations for details on the restricted definition of eyeworm . conceptual framework for current and alternative approaches to minimizing risks and maximizing benefits of mass treatment with mectizanin areas suspected of being co - endemic for onchocerciasis and loiasis . implementation of training , surveillance , and supportive care , indicated in shaded boxes , are assumed to reduce the risk of death associated with saes . decision model of four strategies to reduce risk of l. loa encephalopathy following treatment with mectizanin areas that are 1 ) thought to be endemic for loiasis on the basis of remote sensing ( the environmental risk model map ) ; and 2 ) determined to be hyper- or meso - endemic for onchocerciasis by remo . [ + ] indicates truncation of a branch to simplify the figure . for the remainder of this branch , see the structure of similarly labelled branches elsewhere in the figure . estimated rates of lifetime blindness ( per 1000 population ) and of death and chronic disability from l. loa encephalopathy , under conditions of 1 ) no mass treatment with mectizan ; 2 ) mass treatment with no risk reduction strategy ; and 3 ) mass treatment using rea , raploa-40 , raploa-20 , or combined rea / raploa-40 . data are shown for two levels of loiasis co - endemicity , both with highly endemic onchocerciasis . similar patterns are observed in areas of low onchocerciasis endemicity ( data not shown ) . estimated mean disability - adjusted life years ( dalys ) lost on a per - capita basis as a result of onchocercal blindness , skin disease , and l. loa encephalopathy , under conditions of : 1 ) no mass treatment with mectizan ; 2 ) mass treatment with no risk reduction strategy ; and 3 ) mass treatment using rea , raploa-40 , raploa-20 , or combined rea / raploa-40 to reduce risk of l. loa encephalopathy . four scenarios are considered based on high and low prevalence of loiasis and onchocerciasis , respectively ( as defined in the appendix - see additional file 1 ) . estimated risk of blindness per 1000 population in four scenarios based on high and low prevalence of loiasis and onchocerciasis , under conditions of : 1 ) no mass treatment with mectizan ; 2 ) mass treatment with no risk reduction strategy ; and 3 ) mass treatment using rea , raploa-40 , raploa-20 , or combined rea / raploa-40 to reduce risk of l. loa encephalopathy . estimated risk of death or chronic disability per 1 million population resulting from l. loa encephalopathy following mass treatment with mectizan , under conditions of : 1 ) no mass treatment ; 2 ) mass treatment with no risk reduction strategy ; and 3 ) mass treatment using rea , raploa-40 , raploa-20 , or combined rea / raploa-40 to reduce risk of l. loa encephalopathy . four scenarios are considered based on high and low prevalence of loiasis and onchocerciasis , respectively ( defined in the appendix - see additional file 1 ) .

backgroundthe occurrence of loa loa encephalopathy following mass treatment of onchocerciasis with mectizan has adversely affected onchocerciasis control efforts in central africa . persons with very high densities of l. loa microfilaremia are at increased risk of encephalopathy , but little is known about the geographic distribution of these persons within central africa . raploa , a new technique that correlates the proportion of community members reporting a history of eyeworm with the prevalence of high - intensity l. loa microfilaremia in that community , may be useful for rapid assessment of areas at potential risk of treatment - related l. loa encephalopathy . validation of raploa is ongoing . the operational and risk - reduction advantages of raploa over the current technique of village - by - village rapid epidemiologic assessment for onchocerciasis ( rea ) are unknown.methodswe developed a decision model to compare four strategies for minimizing sequelae of l. loa encephalopathy following mass treatment with mectizan in areas co - endemic for onchocerciasis and loiasis : rea ; raploa with threshold eyeworm prevalences of 40% and 20% ( raploa-40 and raploa-20 , respectively ) ; and combined rea / raploa-40.resultsin the model , all four strategies significantly reduced risk of death and neurologic complications from l. loa encephalopathy , but raploa-20 and rea resulted in half as many such cases as did raploa-40 or combined rea / raploa-40.conclusionraploa is likely to be useful programmatically in reducing risk of l. loa encephalopathy following mass treatment with mectizan. it also may be cost - saving . before full - scale implementation , additional data are needed on geographic clustering of high - density l. loa microfilaremia and on raploa 's reliability and cost .

Background Rationale for a decision analysis model Methods Risk of Risk of Onchocerciasis Results Health benefits of Mectizan Reduction in Discussion Conclusion List of abbreviations Competing interests Authors' contributions Supplementary Material Acknowledgements Figures and Tables

affected persons lived in loiasis - endemic areas and , based on their post - treatment l. loa microfilarial loads , are presumed to have had high densities of l. loa microfilaremia prior to treatment with mectizan(mdp , unpublished data ) . in 2000 , the mec and the technical consultative committee ( tcc ) of apoc recommended a new approach to mass treatment for onchocerciasis in areas co - endemic for loiasis . for high - risk areas on the map , the mec / tcc recommendations for areas co - endemic for onchocerciasis and loiasis currently require village - by - village rea before initiating mass treatment with mectizan . mass distribution of mectizanis recommended for all villages that remain classified as meso- or hyper - endemic for onchocerciasis by rea but only after enhanced health education and training of community members , and only if sae surveillance by community distributors and medical preparedness for sae management can be ensured . in villages confirmed as hypo - endemic on the basis of rea , mass treatment is not recommended , although mectizanis made available to treat individuals with clinical onchocerciasis after health workers assess the individual 's burden of l. loa microfilariae and related risk of l. loa encephalopathy . increased costs result from village - by - village rea before treatment , as well as from safeguards required to reduce risk of death and disability from l. loa encephalopathy in hyper- and meso - endemic areas . the current mec / tcc approach assumes maximum risk of l. loa encephalopathy in all areas predicted as high risk on the environmental risk model map , and it uses village - by - village rea to increase programmatic certainty that " enough " onchocerciasis exists to warrant mass treatment with mectizan . a new technique , known as raploa , has been developed that does not require collection of blood to detect areas with high - density l. loa microfilaremia . a recent multi - center study showed that 1 ) the prevalence of l. loa infection is associated with microfilarial density ; and 2 ) the prevalence of individuals with high - level microfilaremia in a community is associated with the percentage of community members who respond positively to questions about previous experience with a migrating eyeworm . in three different ecologic settings in nigeria and cameroon , investigators found that the prevalence of persons with high microfilarial densities ( 30,000 microfilariae per milliliter [ mf / ml ] of blood ) remained low when the prevalence of a history of eyeworm was less than 40% , but that it increased rapidly above this level ( figure 1 , excerpted from the multi - country report on raploa ) . raploa is now being suggested as an alternate approach to rea in areas that are predicted by the environmental risk model map to be co - endemic for l. loa . conceptually , such an approach , which bases the mass treatment decision on a defined risk of l. loa encephalopathy following mectizantreatment , seems more appealing than one based solely on an assessment of how much onchocerciasis is " enough " at the village level to justify an unknown risk of treatment - related l. loa encephalopathy . a threshold of 40% of persons with a history of eyeworm has been suggested for distinguishing between high - risk areas for l. loa encephalopathy ( which would require enhanced community education , sae surveillance , and supportive medical care ) from those at low risk . these persons would be at risk of l. loa encephalopathy if mass distribution of mectizanwere implemented . use of 20% prevalence of a history of eyeworm as the threshold ( raploa-20 ) would substantially reduce ( to near zero , based on figure 1 ) the prevalence of persons with microfilarial densities of 30,000 per ml in areas classified as low risk . with this strategy , once communities are confirmed by rea as hyper- or meso - endemic for onchocerciasis , raploa-40 would be used to determine whether they are at high risk for l. loa encephalopathy following treatment with mectizan . the primary purpose of the current model is to provide a structured framework to understand and estimate the potential health risks and benefits of alternative approaches to mass treatment strategies in areas that are co - endemic for onchocerciasis and loiasis . the primary purpose of the current model is to provide a structured framework to understand and estimate the potential health risks and benefits of alternative approaches to mass treatment strategies in areas that are co - endemic for onchocerciasis and loiasis . four strategies ( rea , raploa-40 , raploa-20 , and combined rea / raploa-40 ) were examined ( figure 3 ) under four different ecologic conditions with varying intensities of o. volvulus and l. loa transmission ( appendix - see additional file 1 ) . we examined three principal outcomes : 1 ) onchocercal blindness ; 2 ) chronic neurologic complications or death associated with l. loa encephalopathy following mectizantreatment ; and 3 ) disability - adjusted life years ( dalys ) , which were influenced by onchocercal blindness and skin disease as well as by treatment - related l. loa encephalopathy , death , and chronic neurologic sequelae ( appendix - see additional file 1 ) . the risk of l. loa encephalopathy temporally related to mectizantreatment was assumed to be a function of a threshold prevalence of high intensity microfilaremia , defined as 30,000 mf / ml of blood . the estimated risk of l. loa encephalopathy following mectizantreatment is based on two factors : 1 ) the percentage of communities with different portions of their population having microfilarial loads of 30,000 mf / ml ( which we categorized as 0% , 0.10.9% , 1.01.9% , 2.03.9% , 4.07.9% , and 8% ) ; and 2 ) risk of l. loa encephalopathy following mectizantreatment in individuals with microfilarial loads above the 30,000 mf / ml threshold , which we assumed to be 1% ( appendix - see additional file 1 ) . these estimates are based on the estimated sensitivity and specificity of raploa ( figure 1 ) , the performance of rea , and the underlying distribution of high - intensity l. loa microfilaremia within communities ( appendix - see additional file 1 ) . based on the estimated prevalence of individuals with high intensity infection , the community risk of l. loa encephalopathy following mectizantreatment was estimated . the risk of l. loa encephalopathy temporally related to mectizantreatment was assumed to be a function of a threshold prevalence of high intensity microfilaremia , defined as 30,000 mf / ml of blood . the estimated risk of l. loa encephalopathy following mectizantreatment is based on two factors : 1 ) the percentage of communities with different portions of their population having microfilarial loads of 30,000 mf / ml ( which we categorized as 0% , 0.10.9% , 1.01.9% , 2.03.9% , 4.07.9% , and 8% ) ; and 2 ) risk of l. loa encephalopathy following mectizantreatment in individuals with microfilarial loads above the 30,000 mf / ml threshold , which we assumed to be 1% ( appendix - see additional file 1 ) . these estimates are based on the estimated sensitivity and specificity of raploa ( figure 1 ) , the performance of rea , and the underlying distribution of high - intensity l. loa microfilaremia within communities ( appendix - see additional file 1 ) . although our model was conservative in assuming systematic annual drug coverage of only 65% , mass treatment with mectizanresulted in a net gain of 0.5 to 1.0 dalys per person ( reducing lost dalys from 1.52 to 0.53 and from 0.88 to 0.31 in high - and low - prevalence areas , respectively ) , even in the absence of measures to reduce the risk of l. loa - associated death and permanent disability ( table 1 ) . similarly , in our model , mass treatment with mectizanreduced the lifetime risk of blindness from 166 to 58 cases per 1000 population in areas with a high prevalence of onchocerciasis and from 94 to 33 cases per 1000 population in low prevalence areas . in the absence of any approach to reduce complications of l. loa encephalopathy following mass treatment with mectizan , the estimated risk of l. loa associated death or permanent neurologic sequelae was 85.1 and 26.0 cases per 1 million population in high- and low - prevalence areas for loiasis , respectively . regardless of the prevalence of onchocerciasis or loiasis , the current risk reduction strategy , rea , led to the greatest reductions ( 94% 99% ) in death and permanent disability associated with l. loa encephalopathy . risk of l. loa associated death and disability was 2.0 to 6.7 times greater with raploa-40 or rea / raploa-40 than with rea or raploa-20 ( table 3 ) . figure 4 illustrates the relationship between the estimated number of cases of blindness and the number of deaths and chronic disabilities resulting from l. loa encephalopathy in the context of 1 ) no mass treatment ; 2 ) mass treatment with no risk reduction strategies ; and 3 ) mass treatment with each of the four risk reduction strategies in the model . although our model was conservative in assuming systematic annual drug coverage of only 65% , mass treatment with mectizanresulted in a net gain of 0.5 to 1.0 dalys per person ( reducing lost dalys from 1.52 to 0.53 and from 0.88 to 0.31 in high - and low - prevalence areas , respectively ) , even in the absence of measures to reduce the risk of l. loa - associated death and permanent disability ( table 1 ) . similarly , in our model , mass treatment with mectizanreduced the lifetime risk of blindness from 166 to 58 cases per 1000 population in areas with a high prevalence of onchocerciasis and from 94 to 33 cases per 1000 population in low prevalence areas . in the absence of any approach to reduce complications of l. loa encephalopathy following mass treatment with mectizan , the estimated risk of l. loa associated death or permanent neurologic sequelae was 85.1 and 26.0 cases per 1 million population in high- and low - prevalence areas for loiasis , respectively . regardless of the prevalence of onchocerciasis or loiasis , the current risk reduction strategy , rea , led to the greatest reductions ( 94% 99% ) in death and permanent disability associated with l. loa encephalopathy . raploa-40 and the combined rea / raploa-40 approach were less effective in reducing risk ( table 3 , figure 4 ) . risk of l. loa associated death and disability was 2.0 to 6.7 times greater with raploa-40 or rea / raploa-40 than with rea or raploa-20 ( table 3 ) . figure 4 illustrates the relationship between the estimated number of cases of blindness and the number of deaths and chronic disabilities resulting from l. loa encephalopathy in the context of 1 ) no mass treatment ; 2 ) mass treatment with no risk reduction strategies ; and 3 ) mass treatment with each of the four risk reduction strategies in the model . the occurrence of l. loa encephalopathy following mass treatment with mectizanhas negatively affected onchocerciasis control efforts in central africa , which rely on a strategy of mass treatment with mectizanusing cdti . the lack of a rapid epidemiologic assessment tool to estimate risk of l. loa encephalopathy following mectizantreatment has severely limited attempts to expand onchocerciasis control into areas thought to be co - endemic for l. loa . validation of raploa is currently ongoing and discussions are now underway about how best to apply raploa programmatically to reduce risk of treatment - related l. loa encephalopathy and its complications . the model also indicated that all four risk - reduction strategies would substantially decrease the risk of death and permanent disability from l. loa encephalopathy temporally related to mectizantreatment ( figure 4 ) . the currently recommended approach for reducing risk of death and permanent neurologic complications from l. loa encephalopathy incurs significant programmatic costs . these costs result from village - by - village assessments of onchocerciasis endemicity by rea , as well as the need to 1 ) educate at - risk communities about l. loa encephalopathy , 2 ) establish surveillance for treatment - related saes , and 3 ) provide appropriate supportive medical and nursing care for those who develop l. loa encephalopathy following mass treatment . data collected through raploa could significantly enhance our understanding of the epidemiology of high - density l. loa microfilaremia and provide an opportunity to refine the predictions of the environmental risk model map . in these areas , onchocerciasis control programs are required invariably to carry out rea in every village and , in all villages where mass treatment with mectizanis offered , to bear the additional costs of enhanced training , sae surveillance , and reinforcing health systems for the clinical management of l. loa encephalopathy . use of raploa may reveal the absence of high - density l. loa microfilaremia ( as predicted by history of eyeworm ) throughout much of central africa where the environmental risk model map currently predicts l. loa to be highly endemic . because the actual prevalence of high - density l. loa microfilaremia throughout much of central africa is unknown and cost data were not available , we could not account for these potential advantages of raploa in the decision model . finally , the raploa approach may prove useful in the global programme to eliminate lymphatic filariasis for assessing risk of treatment - related l. loa encephalopathy in areas that are co - endemic for l. loa and wuchereria bancrofti . unfortunately , the cost of being able to rapidly detect and treat all cases of l. loa encephalopathy temporally related to mectizantreatment appears to be the establishment of surveillance and medical systems that , in many areas , may be unnecessary . if they are not tightly clustered , but rather scattered in relatively small numbers throughout central africa , mectizan - related l. loa encephalopathy will be difficult to predict and even harder to prevent . the relative effectiveness of each of the four strategies in reducing onchocercal blindness and the risk of l. loa associated death and disability remained constant ( data not shown ) . in central africa where onchocerciasis and loiasis are co - endemic , l. loa encephalopathy has occurred following mass treatment with mectizan . in some cases , a new rapid assessment technique called raploa may provide programme managers with an important tool with which to assess l. loa endemicity and the risk of treatment - related l. loa encephalopathy . compared to rea , the usefulness of raploa , both in preventing blindness and avoiding l. loa encephalopathy following mectizantreatment in co - endemic areas , is highly dependent on the raploa cutoff point . full programmatic implementation of raploa will require testing of the method in other areas of central africa , an improved understanding of geographic clustering of high - density l. loa microfilaremia , sampling strategies that would avoid the need for raploa assessments in all communities , cost analyses , and confirmation that the questionnaire can be administered easily and reliably by field staff . the raploa technique to which we refer in this paper uses a " restricted definition " of eyeworm : the subject must recognize a photograph of eyeworm as the same condition that he or she is reporting , and rea rapid epidemiologic assessment ( for onchocerciasis ) remo rapid epidemiologic mapping of onchocerciasis sae serious adverse event tcc technical consultative committee of the african programme for onchocerciasis control all authors participated fully in extensive critical discussions as the model developed and in writing the paper . prevalence of persons with l. loa microfilaremia 30,000 per ml as a function of the prevalence of a restricted definition of eyeworm , from figure 10 of the multi - center report on raploa . conceptual framework for current and alternative approaches to minimizing risks and maximizing benefits of mass treatment with mectizanin areas suspected of being co - endemic for onchocerciasis and loiasis . decision model of four strategies to reduce risk of l. loa encephalopathy following treatment with mectizanin areas that are 1 ) thought to be endemic for loiasis on the basis of remote sensing ( the environmental risk model map ) ; and 2 ) determined to be hyper- or meso - endemic for onchocerciasis by remo . estimated rates of lifetime blindness ( per 1000 population ) and of death and chronic disability from l. loa encephalopathy , under conditions of 1 ) no mass treatment with mectizan ; 2 ) mass treatment with no risk reduction strategy ; and 3 ) mass treatment using rea , raploa-40 , raploa-20 , or combined rea / raploa-40 . estimated mean disability - adjusted life years ( dalys ) lost on a per - capita basis as a result of onchocercal blindness , skin disease , and l. loa encephalopathy , under conditions of : 1 ) no mass treatment with mectizan ; 2 ) mass treatment with no risk reduction strategy ; and 3 ) mass treatment using rea , raploa-40 , raploa-20 , or combined rea / raploa-40 to reduce risk of l. loa encephalopathy . estimated risk of blindness per 1000 population in four scenarios based on high and low prevalence of loiasis and onchocerciasis , under conditions of : 1 ) no mass treatment with mectizan ; 2 ) mass treatment with no risk reduction strategy ; and 3 ) mass treatment using rea , raploa-40 , raploa-20 , or combined rea / raploa-40 to reduce risk of l. loa encephalopathy . estimated risk of death or chronic disability per 1 million population resulting from l. loa encephalopathy following mass treatment with mectizan , under conditions of : 1 ) no mass treatment ; 2 ) mass treatment with no risk reduction strategy ; and 3 ) mass treatment using rea , raploa-40 , raploa-20 , or combined rea / raploa-40 to reduce risk of l. loa encephalopathy .

pregnancy is a progressively hyperglycemic period of life , accompanied by increasing insulin resistance as from mid - gestation , with the hyperglycemia serving a highly important role in the nutrition and development of the fetus by providing it with adequate levels of glucose . gestational diabetes mellitus is a common pathologic state that increases the incidence of complications in both the mother and the fetus . furthermore , gdm and gestational dysregulation of blood glucose levels expose the women affected to higher risk for subsequent development of type 2 diabetes mellitus and cardiovascular disease later in their lives [ 35 ] , the risk being proportional to the degree of the dysregulation . glucose tolerance and metabolism as well as insulin resistance are altered in type ii diabetes mellitus ( t2 dm ) , gestational diabetes mellitus ( gdm ) , and the postpartum period of a pregnancy complicated by pgdm . t2 dm and pgdm have the same predisposing factors , namely , high body mass index before pregnancy , elevated levels of fasting glucose , and a degree of hyperglycemia in pregnancy , these leading to dysglycemia 1 to 4 months after delivery and recurrent gestational diabetes mellitus [ 626 ] . although the pathophysiologic mechanisms responsible for these changes are not as yet completely understood , growing insight into the processes involved has been gained over the last few years . there are two main pathways leading to gdm , t2 dm , and possibly pgdm : insulin resistance and chronic subclinical inflammation . insulin resistance is caused by the inability of tissues to respond to insulin and the deficient secretion of insulin by pancreatic beta cells [ 2729 ] . the deficient secretion can not compensate for the pregnancy - induced insulin resistance , this resulting in gdm , a condition which sometimes persists after delivery [ 812 , 30 ] . with regard to the contribution of inflammatory processes to the pathogenesis of dysglycemia conditions , it has been reported that long - term activation of the acute phase inflammatory response is a risk factor for t2 dm and cardiovascular disease . furthermore , obesity has a role in the development of both t2 dm and gdm through chronic subclinical inflammation , low - grade activation of the acute phase response , and dysregulation of adipokines [ 3133 ] . increased levels of inflammatory agents during and after pregnancy have been reported in patients with gdm , while increased body fat has been strongly associated with inflammation and adipocyte necrosis , hypoxia , and release of chemokines which cause macrophages to infiltrate adipose tissue . macrophages secrete cytokines which activate the subsequent secretion of inflammation mediating agents , specifically interleukin-6 ( il-6 ) and c - reactive protein ( crp ) . moreover , other molecules such as plasminogen activator inhibitor 1 ( pai-1 ) and sialic acid lead to dysregulations of metabolism , hyperglycemia , insulin resistance , and , finally , overt t2 dm [ 31 , 3446 ] . crucially , hormones produced by the placenta and increased maternal fat mass have been reported to play a major role in gdm . in this context , the gaps in the mechanisms underlying glucose metabolism in pregnancy and nonpregnancy states have initiated research efforts to uncover other potential mediators of insulin resistance , namely , the adipokines . these are a group of substances , knowledge about which is continuously expanding , that are produced mainly in the adipose tissue . the group includes leptin , adiponectin , tumor necrosis factor alpha ( tnf- ) , retinol - binding protein-4 ( rbp-4 ) , resistin , visfatin , and apelin . these molecules are involved in a wide range of physiological processes including lipid metabolism , atherosclerosis , blood pressure regulation , insulin sensitivity , and angiogenesis , while they also influence immunity and inflammation . their levels in pathologic states appear increased , with the exception of adiponectin which shows decreased levels . in this paper we summarize the data available concerning the role of these mediators in women with pgdm . adipokines , whose significant role in the pathogenesis of numerous pathologic conditions has recently been recognized , are adipose tissue - derived substances mediating communication and endocrine function between this metabolically active tissue and other sites throughout the body . a summary of the levels of adipokines and other inflammatory mediators in cases of pgdm is shown in table 1 . it has insulin - sensitizing action , stimulating glucose uptake in skeletal muscle , and reduces hepatic glucose production through amp - activated protein kinase , while it also possesses antiatherogenic and anti - inflammatory properties [ 51 , 52 ] . the levels of adiponectin decrease as visceral fat increases [ 5356 ] in such conditions as central obesity , insulin resistance , and diabetes mellitus ; lower adiponectin levels have notably been associated with subclinical inflammation . it has been shown that adiponectin levels begin to decrease early in the pathogenesis of diabetes , as adipose tissue increases in tandem with reduction in insulin sensitivity . hypoadiponectinemia has also been associated with beta cell dysfunction [ 58 , 59 ] , while it has additionally been linked to future development of insulin resistance and type 2 diabetes mellitus [ 6164 ] , in the development of which adiponectin appears to have a causative role . adiponectin has also been studied in animal experiments in which it was demonstrated that it can reduce insulin resistance and enhance the action of insulin in liver , resulting in lowering of glucose blood levels [ 6567 ] . in normal human pregnancy , adiponectin levels have been found to be unchanged or decreased [ 6872 ] and negatively correlated with maternal bmi and insulin sensitivity . in gdm pregnancies adiponectin appears to be decreased independently of maternal body mass index ( bmi ) or insulin sensitivity [ 68 , 7485 ] . additionally , the fact that low circulating levels of adiponectin are found early in pregnancy has been associated with subsequent development of gdm . meanwhile , low levels of adiponectin in pregnancy , which have incidentally been associated with certain ethnic groups such as women of south asian origin , have a significant impact on the development of gdm [ 8688 ] . while hypoadiponectinemia is strongly associated with beta - cell dysfunction in pregnancy , the levels of adiponectin after delivery have been investigated in only a few studies . in one of them the investigators measured adiponectin levels in 89 women with pgdm at both 3 and 12 months postpartum and compared the values obtained with normal controls ( women with normal pregnancies ) . they found that adiponectin levels were lower in women with pgdm at 3 months postpartum . this registered decrease remains valid even after correction for body fat mass ( bfm ) . the lower values of adiponectin are also associated with decreased insulin sensitivity and low hdl . it is of note that plasma adiponectin further decreased by 10% after 1 year in insulin - resistant women with pgdm . other investigators studying 25 women with pgdm and comparing their adiponectin levels with those of 23 women with normal pregnancies at least 1 year after the index pregnancy ( mean 4 years ) found that the levels were significantly lower in women with pgdm compared to normal controls . this study also showed that adiponectin was negatively correlated to other inflammatory markers , namely , crp , pai-1 , and il-6 , the which correlations remained unchanged even after adjustment for bmi . in another study , 140 women with previous gdm and various states of glucose tolerance after delivery ( 8 with diabetes mellitus , 60 with impaired glucose tolerance and 72 with normal glucose tolerance ) the authors reported lower adiponectin levels in the women with pgdm 1.57 years postdelivery , while it is also of interest that the levels of adiponectin were progressively lower , the postpartum glucose tolerance values ranging from normality to impaired glucose tolerance and finally diabetes mellitus after gdm ( p for linearity = 0.006 ) . plasma adiponectin was moreover negatively correlated with fasting glucose , fasting insulin , and rbp-4 levels . another recent study examined 60 women with gdm at 30 weeks of gestation and 6 weeks and 6 months postpartum and compared the results with normal pregnancies . they did not find significantly different levels of adiponectin during and after pregnancy in the group of gdm women , but they did observe a significant difference in the group of non - gdm women ( both at 6 weeks and 6 months postpartum , p < 0.01 ) . leptin is an adipokine that is produced by the ob ( obese ) gene in adipose tissue cells , especially in white adipose tissue , its action at the hypothalamus resulting in decreasing food intake and increasing energy consumption [ 93 , 94 ] . its mechanism of action is to increase insulin sensitivity by influencing insulin secretion , glucose utilization , glycogen synthesis , and fatty acid metabolism [ 9395 ] . furthermore , it adjusts gonadotropin releasing - hormone secretion from the hypothalamus and activates the sympathetic nervous system . in normal pregnancy , leptin concentration increases from early pregnancy onwards and decreases to normal pre - pregnancy levels before delivery [ 9699 ] . this initial increase may be due to placental synthesis , since it occurs before the rise in maternal bmi and rapidly falls after delivery . the function of increased maternal leptin is to enhance the mobilization of maternal fat stores thus enabling access of lipid substrates to the fetus . in gdm , leptin has a more debatable role , since it appears to be elevated in women with gdm [ 101103 ] but , after adjustment for bmi and insulin resistance [ 104 , 105 ] , it is shown to be decreased or even unaltered , while it has also been associated with insulin resistance in pregnancy [ 101 , 103 , 105 ] . there is to date an insufficient number of studies examining the role of leptin in the postpartum period following a gdm pregnancy . in one study , 89 women with pgdm were followed and found to have significantly increased plasma leptin at 3 months after delivery ( p < 0.003 ) compared to controls . leptin levels were negatively associated with adiponectin but this association was not significant after the adjustment for bfm . meanwhile , other authors have failed to find any difference in leptin levels between pgdm and normal pregnancies 18 months after delivery . leptin levels were studied during pregnancy and 6 weeks and 6 months after delivery both in normal pregnancies and in pregnancies complicated by gdm . there were significantly higher levels of leptin in previous gdm pregnancies compared with normal pregnancies both at 6 weeks and 6 months postpartum . rbp-4 , which is an adipokine synthesized in hepatocytes and adipocytes , serves as a carrier for retinol in blood and is postulated to play a role in regulating glucose metabolism and insulin sensitivity [ 122 , 123 ] . in pathologic glucose tolerance states ( such as obesity , insulin resistance , polycystic ovary syndrome , and cardiovascular disease ) , rbp-4 concentration has been shown to be elevated [ 106 , 124128 ] . other studies have reported low levels of the protein in humans with t2 dm and have determined that rbp-4 concentration does not relate to insulin sensitivity in calorie restricted obese individuals [ 129131 ] . it has moreover been demonstrated that overexpression of rbp-4 in normal mice increases insulin resistance , whereas genetic disruption of this adipokine increases insulin sensitivity . in normal pregnancy , rbp-4 increases significantly between early and late pregnancy with a parallel decrease in insulin sensitivity , although other authors have reported a decrease in the levels of this hormone after early gestation . gdm has been associated with increased , decreased , or even stable levels of rbp-4 [ 134138 ] . in pgdm it has been shown that rbp-4 was significantly higher more than 18 months postpartum in women with normal or impaired glucose tolerance or with diabetes mellitus in the postpartum period compared with women without gdm . a trend was also documented of increasing rbp-4 values from normality to dm in the pgdm group ( p for linearity = 0.006 ) . furthermore , rbp-4 was positively correlated with fasting insulin , whereas the correlation to adiponectin was negative . finally , rbp-4 concentration was significantly higher in women with metabolic syndrome than in those without . another study that measured rpb-4 levels in 60 women with gdm during pregnancy as well as 6 weeks and 6 months after delivery found a positive correlation of rbp-4 with fasting insulin levels . additionally , there was a significant reduction in rbp-4 in the control group between delivery and 6 weeks and 6 months postpartum , although there was no respective decrease in the gdm group . between the two groups , a significant difference in the levels of rbp-4 ( p < 0.05 ) was not observed until 6 weeks postpartum . resistin , a hormone expressed by adipocytes as well as monocytes and macrophages , appears to have levels that parallel the mass of adipose tissue [ 107109 ] . in obesity and insulin resistance , the role of resistin is as yet highly controversial [ 110 , 111 ] , although its function has been associated with impaired glucose tolerance [ 106 , 111 ] . the results in animal experiments are indicative of insulin resistance induction in animals , but the same is not true in humans [ 107 , 111113 ] . in normal pregnancy it is expressed in human placenta , with plasma resistin levels in pregnant women being significantly higher as compared to normal controls . it increases in the third trimester [ 68 , 76 , 114116 ] and may regulate energy metabolism during pregnancy . in gdm its levels have been found either elevated or decreased [ 76 , 117120 ] , with some studies having reported elevated maternal resistin in gdm [ 68 , 118 , 121 , 147 ] , while others found lower [ 73 , 118 ] or unaltered values . with regard to pgdm , one study showed that resistin was significantly higher 18 months postpartum in women with normal or impaired glucose tolerance who had pgdm compared to a group of women who did not develop dm during pregnancy . this could be explained by a biphasic effect of insulin on the release of resistin , whereby low concentration of insulin increases the release of resistin , while this is reduced at higher insulin levels . the authors also reported that plasma resistin levels correlated with bmi , fat quantity , and plasma insulin and that the presence of metabolic syndrome was not significantly associated with plasma resistin levels in the postpartum period . visfatin is an adipocytokine produced mainly in visceral fat , as compared to subcutaneous fat , which exerts insulin mimetic action and , additionally , plays a pro - inflammatory role . it has higher concentrations in cases of obesity or insulin resistance , including t2 dm and metabolic syndrome [ 139 , 141 , 142 ] . conversely , other studies showed no relation of visfatin to insulin sensitivity or increased bmi and visceral fat mass [ 143 , 144 ] . in pregnancy , although this hormone has been reported by some authors to maintain the same levels in the third trimester as in the non - pregnant state , other studies have demonstrated an increase [ 72 , 146 ] . visfatin levels peak between 19 and 26 weeks of gestation , while at between 27 and 34 weeks visfatin has the lowest serum concentrations . reported that visfatin may have a paracrine or autocrine action since it is locally increased in omental fat without increased plasma levels in pregnancy . some studies have shown elevated levels in women with gdm [ 149151 ] with a further increase of these levels detected in the presence of high maternal blood glucose levels . in contrast , a number of other studies found that visfatin levels are lower in gdm [ 146 , 152154 ] . tnf- and il-6 are inflammatory mediators produced by monocytes and macrophages in the adipose tissue . these cytokines are increased in obesity and have multiple effects on insulin sensitivity in muscles , liver , or beta cells of the pancreas , ultimately leading to insulin resistance [ 32 , 168 ] . in pregnancy , tnf- and il-6 production occurs in placenta [ 163 , 164 ] , while it is considered that a chronic inflammatory process in the adipose tissue may contribute to pregnancy - induced insulin resistance [ 164167 , 169 ] . placental production of tnf has been shown to be maximized late in pregnancy and to decline rapidly after pregnancy , this being in accordance with placental production of tnf- . in early pregnancy tnf- mrna later in pregnancy and specifically near term , tnf- mrna also appears in villous stromal cells and tnf- transcripts are present in maternal cells in the decidua adjacent to the extracellular membranes . most of the tnf- produced by the placenta is delivered to maternal circulation and by comparison only a small amount to the fetal compartment . though in a gdm pregnancy il-6 and tnf- rarely increase [ 165167 ] , when this does occur , the increase is caused by the oxidative stress and the inflammation associated with the hyperglycemia . conversely , tnf- possibly inhibits insulin secretion and insulin regulated glucose uptake in gdm [ 84 , 104 , 165 , 166 , 170 ] . an in vitro experiment showed that placentas from women with gdm released more tnf- in response to a glucose stimulus than placentas from women with normal glucose tolerance . il-6 levels are significantly higher in women with pgdm at least 1 year postdelivery ( mean 4 years postpartum ) compared with normal controls , this difference becoming nonsignificant when it was adjusted for bmi . it is interesting to note that when nonobese women with pgdm were examined , the difference remained significant for il-6 ( bmi played no role ) and that il-6 was positively related to crp , this persisting even after adjustment for bmi . tnf- was not significantly different in pgdm 3 months after delivery compared to women with normal glucose tolerance during pregnancy . by contrast , il-6 was significantly higher before adjusting for bfm but after adjustment for bfm adiponectin did not correlate to il-6 . hauguel - de mouzon et al . reported in a recent study that tnf- rose significantly 6 weeks postpartum both in women with normal pregnancies and in gdm pregnancies when compared to antepartum values . the increase for tnf- was not significant for both groups 6 months postpartum . in another study , 18 women with pgdm at least 12 months after delivery were compared with normal controls and women with polycystic ovary syndrome ( pcos ) . they found no significant difference in tnf- between the pgdm cases and controls , although the difference between pcos and controls was significant . crp , an inflammatory agent common in numerous pathologic conditions , has been associated with metabolic abnormal states such as insulin resistance , hyperglycemia , and t2 dm [ 31 , 3436 ] , while it also appears to be associated with central obesity [ 44 , 46 ] . in the first trimester of pregnancy the levels of crp are increased and have been related to higher risk for gdm development [ 156 , 157 ] , this association also being valid with measurement of crp later in the course of pregnancy [ 160 , 170 ] . furthermore , crp is increased in maternal obesity , insulin resistance , and maternal dysglycemia [ 158 , 161 , 162 ] . the pathophysiologic role of inflammatory proteins and adiponectin seem to be the gradual impairment of beta cell function and increasing insulin resistance , which results in ineffective plasma glucose regulation and subsequent dysglycemia in the months and years that follow pregnancy . as concerns pgdm , there is some evidence that women with a history of prior gdm have postpartum increased crp that manifests chronic subclinical inflammation [ 4046 ] . increased crp levels in women with pgdm , which have also been related to metabolic syndrome , have been reported in several studies [ 4346 ] . the postpartum period which is complicated by gestational diabetes is a period of chronic subclinical inflammation . some investigators have shown significantly increased levels of crp 3 months after delivery in women with pgdm compared with controls . other authors also found a negative correlation between adiponectin levels and crp , but this correlation became nonsignificant after adjustment for bfm , the latter being explained by the finding that crp is also related to central obesity [ 44 , 46 ] . the nhanes iii study showed that adjustment of crp values for waist circumference attenuated differences in women with pgdm and normal women . another study recruiting 46 women with pgdm 3 years postpartum reported that women with pgdm had significantly elevated high - sensitivity crp ( hs - crp ) compared with controls . studied 25 women with pgdm and found that in a mean period of 4 years postpartum they had significantly higher crp levels compared to controls . crp was negatively related to adiponectin and positively related to il-6 and these associations remained unchanged after adjustment for bmi . in another study , 18 women with pgdm at least 12 months after index delivery were found to have no significant difference in hs - crp compared with normal controls . on the other hand , other authors studied 70 women with pgdm 6 years after their pregnancy and found significantly higher crp levels in women with pgdm in the presence of abdominal obesity ; they also found abnormal glucose tolerance compared with the women without a history of gdm . this was further confirmed in another study where significantly elevated crp levels and fibrinogen were detected in 26 women with pgdm as compared with controls . plasminogen activator inhibitor 1 ( pai-1 ) is a protein that in humans is encoded by the serpine1 gene and is mainly produced by the endothelial cells , though it is also secreted by other tissue types , such as adipose tissue . its main function is to inhibit tissue plasminogen activator ( tpa ) and urokinase ( upa ) , the activators of plasminogen , and hence fibrinolysis . pai-1 is increased in various disease states , such as obesity , ms , insulin resistance , and t2 dm [ 31 , 3436 ] . pai-1 is increased in women with pgdm compared with normal women 3 months after delivery . in this study pai-1 remained higher after adjustment for bfm , while the authors also found a negative correlation between adiponectin levels and pai-1 . another study reported that women with pgdm had significantly elevated pai-1 compared with controls 3 years after delivery . other authors studying 74 women with pgdm 3 months after delivery found them to exhibit increased pai-1 levels when they had impaired insulin sensitivity postpartum , while tpa was also observed to be elevated . in another study , 25 women with pgdm demonstrated significantly higher pai-1 levels compared to controls in a mean period of 4 years postpartum , although the difference lost significance after adjustment for bmi . women with pgdm are characterized by chronic subclinical inflammation which is associated with insulin resistance and abnormality in glucose metabolism . approximately 30% of these women have metabolic syndrome and many of them will develop t2 dm within 5 years of diagnosis [ 26 , 31 , 173 ] . the conversion rates from gdm to t2 dm range from 2.6% to 70% over a period of 6 weeks to 28 years postpartum . the problem of gestational diabetes is common and its incidence is exhibiting an increasing prevalence . early recognition and management of women predisposed to develop t2 dm later in their lives is thus crucial in the development of primary health care strategies , modification of lifestyle , and dietary habits significantly enabling prevention or delay of appearance of glucose intolerance states in predisposed women .

previous gestational diabetes mellitus ( pgdm ) is a common condition and has been associated with future development of type 2 diabetes mellitus ( t2 dm ) and metabolic syndrome ( ms ) in women affected . the pathogenesis and risk factors implicated in the development of these conditions later in the lives of women with pgdm are not as yet fully understood . research has recently focused on a group of substances produced mainly by adipose tissue called adipokines , this group including , among others , adiponectin , leptin , retinol - binding protein-4 ( rbp-4 ) , and resistin . these substances as well as other inflammatory mediators ( crp , il-6 , pai-1 , tnf- ) seem to play an important role in glucose tolerance and insulin sensitivity dysregulation in women with pgdm . we summarize the data available on the role of these molecules .

1. Introduction 2. Adipokines in pGDM 3. Other Mediators of Inflammation in pGDM 4. Conclusions

pregnancy is a progressively hyperglycemic period of life , accompanied by increasing insulin resistance as from mid - gestation , with the hyperglycemia serving a highly important role in the nutrition and development of the fetus by providing it with adequate levels of glucose . gestational diabetes mellitus is a common pathologic state that increases the incidence of complications in both the mother and the fetus . furthermore , gdm and gestational dysregulation of blood glucose levels expose the women affected to higher risk for subsequent development of type 2 diabetes mellitus and cardiovascular disease later in their lives [ 35 ] , the risk being proportional to the degree of the dysregulation . glucose tolerance and metabolism as well as insulin resistance are altered in type ii diabetes mellitus ( t2 dm ) , gestational diabetes mellitus ( gdm ) , and the postpartum period of a pregnancy complicated by pgdm . t2 dm and pgdm have the same predisposing factors , namely , high body mass index before pregnancy , elevated levels of fasting glucose , and a degree of hyperglycemia in pregnancy , these leading to dysglycemia 1 to 4 months after delivery and recurrent gestational diabetes mellitus [ 626 ] . although the pathophysiologic mechanisms responsible for these changes are not as yet completely understood , growing insight into the processes involved has been gained over the last few years . there are two main pathways leading to gdm , t2 dm , and possibly pgdm : insulin resistance and chronic subclinical inflammation . with regard to the contribution of inflammatory processes to the pathogenesis of dysglycemia conditions , it has been reported that long - term activation of the acute phase inflammatory response is a risk factor for t2 dm and cardiovascular disease . furthermore , obesity has a role in the development of both t2 dm and gdm through chronic subclinical inflammation , low - grade activation of the acute phase response , and dysregulation of adipokines [ 3133 ] . increased levels of inflammatory agents during and after pregnancy have been reported in patients with gdm , while increased body fat has been strongly associated with inflammation and adipocyte necrosis , hypoxia , and release of chemokines which cause macrophages to infiltrate adipose tissue . macrophages secrete cytokines which activate the subsequent secretion of inflammation mediating agents , specifically interleukin-6 ( il-6 ) and c - reactive protein ( crp ) . moreover , other molecules such as plasminogen activator inhibitor 1 ( pai-1 ) and sialic acid lead to dysregulations of metabolism , hyperglycemia , insulin resistance , and , finally , overt t2 dm [ 31 , 3446 ] . crucially , hormones produced by the placenta and increased maternal fat mass have been reported to play a major role in gdm . in this context , the gaps in the mechanisms underlying glucose metabolism in pregnancy and nonpregnancy states have initiated research efforts to uncover other potential mediators of insulin resistance , namely , the adipokines . these are a group of substances , knowledge about which is continuously expanding , that are produced mainly in the adipose tissue . the group includes leptin , adiponectin , tumor necrosis factor alpha ( tnf- ) , retinol - binding protein-4 ( rbp-4 ) , resistin , visfatin , and apelin . these molecules are involved in a wide range of physiological processes including lipid metabolism , atherosclerosis , blood pressure regulation , insulin sensitivity , and angiogenesis , while they also influence immunity and inflammation . in this paper we summarize the data available concerning the role of these mediators in women with pgdm . adipokines , whose significant role in the pathogenesis of numerous pathologic conditions has recently been recognized , are adipose tissue - derived substances mediating communication and endocrine function between this metabolically active tissue and other sites throughout the body . a summary of the levels of adipokines and other inflammatory mediators in cases of pgdm is shown in table 1 . it has insulin - sensitizing action , stimulating glucose uptake in skeletal muscle , and reduces hepatic glucose production through amp - activated protein kinase , while it also possesses antiatherogenic and anti - inflammatory properties [ 51 , 52 ] . the levels of adiponectin decrease as visceral fat increases [ 5356 ] in such conditions as central obesity , insulin resistance , and diabetes mellitus ; lower adiponectin levels have notably been associated with subclinical inflammation . it has been shown that adiponectin levels begin to decrease early in the pathogenesis of diabetes , as adipose tissue increases in tandem with reduction in insulin sensitivity . hypoadiponectinemia has also been associated with beta cell dysfunction [ 58 , 59 ] , while it has additionally been linked to future development of insulin resistance and type 2 diabetes mellitus [ 6164 ] , in the development of which adiponectin appears to have a causative role . in normal human pregnancy , adiponectin levels have been found to be unchanged or decreased [ 6872 ] and negatively correlated with maternal bmi and insulin sensitivity . in gdm pregnancies adiponectin appears to be decreased independently of maternal body mass index ( bmi ) or insulin sensitivity [ 68 , 7485 ] . additionally , the fact that low circulating levels of adiponectin are found early in pregnancy has been associated with subsequent development of gdm . meanwhile , low levels of adiponectin in pregnancy , which have incidentally been associated with certain ethnic groups such as women of south asian origin , have a significant impact on the development of gdm [ 8688 ] . while hypoadiponectinemia is strongly associated with beta - cell dysfunction in pregnancy , the levels of adiponectin after delivery have been investigated in only a few studies . in one of them the investigators measured adiponectin levels in 89 women with pgdm at both 3 and 12 months postpartum and compared the values obtained with normal controls ( women with normal pregnancies ) . they found that adiponectin levels were lower in women with pgdm at 3 months postpartum . the lower values of adiponectin are also associated with decreased insulin sensitivity and low hdl . it is of note that plasma adiponectin further decreased by 10% after 1 year in insulin - resistant women with pgdm . other investigators studying 25 women with pgdm and comparing their adiponectin levels with those of 23 women with normal pregnancies at least 1 year after the index pregnancy ( mean 4 years ) found that the levels were significantly lower in women with pgdm compared to normal controls . this study also showed that adiponectin was negatively correlated to other inflammatory markers , namely , crp , pai-1 , and il-6 , the which correlations remained unchanged even after adjustment for bmi . in another study , 140 women with previous gdm and various states of glucose tolerance after delivery ( 8 with diabetes mellitus , 60 with impaired glucose tolerance and 72 with normal glucose tolerance ) the authors reported lower adiponectin levels in the women with pgdm 1.57 years postdelivery , while it is also of interest that the levels of adiponectin were progressively lower , the postpartum glucose tolerance values ranging from normality to impaired glucose tolerance and finally diabetes mellitus after gdm ( p for linearity = 0.006 ) . plasma adiponectin was moreover negatively correlated with fasting glucose , fasting insulin , and rbp-4 levels . they did not find significantly different levels of adiponectin during and after pregnancy in the group of gdm women , but they did observe a significant difference in the group of non - gdm women ( both at 6 weeks and 6 months postpartum , p < 0.01 ) . leptin is an adipokine that is produced by the ob ( obese ) gene in adipose tissue cells , especially in white adipose tissue , its action at the hypothalamus resulting in decreasing food intake and increasing energy consumption [ 93 , 94 ] . its mechanism of action is to increase insulin sensitivity by influencing insulin secretion , glucose utilization , glycogen synthesis , and fatty acid metabolism [ 9395 ] . in normal pregnancy , leptin concentration increases from early pregnancy onwards and decreases to normal pre - pregnancy levels before delivery [ 9699 ] . in gdm , leptin has a more debatable role , since it appears to be elevated in women with gdm [ 101103 ] but , after adjustment for bmi and insulin resistance [ 104 , 105 ] , it is shown to be decreased or even unaltered , while it has also been associated with insulin resistance in pregnancy [ 101 , 103 , 105 ] . there is to date an insufficient number of studies examining the role of leptin in the postpartum period following a gdm pregnancy . in one study , 89 women with pgdm were followed and found to have significantly increased plasma leptin at 3 months after delivery ( p < 0.003 ) compared to controls . rbp-4 , which is an adipokine synthesized in hepatocytes and adipocytes , serves as a carrier for retinol in blood and is postulated to play a role in regulating glucose metabolism and insulin sensitivity [ 122 , 123 ] . in pathologic glucose tolerance states ( such as obesity , insulin resistance , polycystic ovary syndrome , and cardiovascular disease ) , rbp-4 concentration has been shown to be elevated [ 106 , 124128 ] . other studies have reported low levels of the protein in humans with t2 dm and have determined that rbp-4 concentration does not relate to insulin sensitivity in calorie restricted obese individuals [ 129131 ] . in normal pregnancy , rbp-4 increases significantly between early and late pregnancy with a parallel decrease in insulin sensitivity , although other authors have reported a decrease in the levels of this hormone after early gestation . gdm has been associated with increased , decreased , or even stable levels of rbp-4 [ 134138 ] . in pgdm it has been shown that rbp-4 was significantly higher more than 18 months postpartum in women with normal or impaired glucose tolerance or with diabetes mellitus in the postpartum period compared with women without gdm . a trend was also documented of increasing rbp-4 values from normality to dm in the pgdm group ( p for linearity = 0.006 ) . finally , rbp-4 concentration was significantly higher in women with metabolic syndrome than in those without . another study that measured rpb-4 levels in 60 women with gdm during pregnancy as well as 6 weeks and 6 months after delivery found a positive correlation of rbp-4 with fasting insulin levels . resistin , a hormone expressed by adipocytes as well as monocytes and macrophages , appears to have levels that parallel the mass of adipose tissue [ 107109 ] . in obesity and insulin resistance , the role of resistin is as yet highly controversial [ 110 , 111 ] , although its function has been associated with impaired glucose tolerance [ 106 , 111 ] . it increases in the third trimester [ 68 , 76 , 114116 ] and may regulate energy metabolism during pregnancy . with regard to pgdm , one study showed that resistin was significantly higher 18 months postpartum in women with normal or impaired glucose tolerance who had pgdm compared to a group of women who did not develop dm during pregnancy . this could be explained by a biphasic effect of insulin on the release of resistin , whereby low concentration of insulin increases the release of resistin , while this is reduced at higher insulin levels . the authors also reported that plasma resistin levels correlated with bmi , fat quantity , and plasma insulin and that the presence of metabolic syndrome was not significantly associated with plasma resistin levels in the postpartum period . visfatin is an adipocytokine produced mainly in visceral fat , as compared to subcutaneous fat , which exerts insulin mimetic action and , additionally , plays a pro - inflammatory role . it has higher concentrations in cases of obesity or insulin resistance , including t2 dm and metabolic syndrome [ 139 , 141 , 142 ] . conversely , other studies showed no relation of visfatin to insulin sensitivity or increased bmi and visceral fat mass [ 143 , 144 ] . in pregnancy , although this hormone has been reported by some authors to maintain the same levels in the third trimester as in the non - pregnant state , other studies have demonstrated an increase [ 72 , 146 ] . some studies have shown elevated levels in women with gdm [ 149151 ] with a further increase of these levels detected in the presence of high maternal blood glucose levels . tnf- and il-6 are inflammatory mediators produced by monocytes and macrophages in the adipose tissue . these cytokines are increased in obesity and have multiple effects on insulin sensitivity in muscles , liver , or beta cells of the pancreas , ultimately leading to insulin resistance [ 32 , 168 ] . in pregnancy , tnf- and il-6 production occurs in placenta [ 163 , 164 ] , while it is considered that a chronic inflammatory process in the adipose tissue may contribute to pregnancy - induced insulin resistance [ 164167 , 169 ] . placental production of tnf has been shown to be maximized late in pregnancy and to decline rapidly after pregnancy , this being in accordance with placental production of tnf- . in early pregnancy tnf- mrna later in pregnancy and specifically near term , tnf- mrna also appears in villous stromal cells and tnf- transcripts are present in maternal cells in the decidua adjacent to the extracellular membranes . conversely , tnf- possibly inhibits insulin secretion and insulin regulated glucose uptake in gdm [ 84 , 104 , 165 , 166 , 170 ] . an in vitro experiment showed that placentas from women with gdm released more tnf- in response to a glucose stimulus than placentas from women with normal glucose tolerance . il-6 levels are significantly higher in women with pgdm at least 1 year postdelivery ( mean 4 years postpartum ) compared with normal controls , this difference becoming nonsignificant when it was adjusted for bmi . it is interesting to note that when nonobese women with pgdm were examined , the difference remained significant for il-6 ( bmi played no role ) and that il-6 was positively related to crp , this persisting even after adjustment for bmi . tnf- was not significantly different in pgdm 3 months after delivery compared to women with normal glucose tolerance during pregnancy . by contrast , il-6 was significantly higher before adjusting for bfm but after adjustment for bfm adiponectin did not correlate to il-6 . reported in a recent study that tnf- rose significantly 6 weeks postpartum both in women with normal pregnancies and in gdm pregnancies when compared to antepartum values . in another study , 18 women with pgdm at least 12 months after delivery were compared with normal controls and women with polycystic ovary syndrome ( pcos ) . crp , an inflammatory agent common in numerous pathologic conditions , has been associated with metabolic abnormal states such as insulin resistance , hyperglycemia , and t2 dm [ 31 , 3436 ] , while it also appears to be associated with central obesity [ 44 , 46 ] . in the first trimester of pregnancy the levels of crp are increased and have been related to higher risk for gdm development [ 156 , 157 ] , this association also being valid with measurement of crp later in the course of pregnancy [ 160 , 170 ] . furthermore , crp is increased in maternal obesity , insulin resistance , and maternal dysglycemia [ 158 , 161 , 162 ] . the pathophysiologic role of inflammatory proteins and adiponectin seem to be the gradual impairment of beta cell function and increasing insulin resistance , which results in ineffective plasma glucose regulation and subsequent dysglycemia in the months and years that follow pregnancy . increased crp levels in women with pgdm , which have also been related to metabolic syndrome , have been reported in several studies [ 4346 ] . the postpartum period which is complicated by gestational diabetes is a period of chronic subclinical inflammation . some investigators have shown significantly increased levels of crp 3 months after delivery in women with pgdm compared with controls . other authors also found a negative correlation between adiponectin levels and crp , but this correlation became nonsignificant after adjustment for bfm , the latter being explained by the finding that crp is also related to central obesity [ 44 , 46 ] . the nhanes iii study showed that adjustment of crp values for waist circumference attenuated differences in women with pgdm and normal women . another study recruiting 46 women with pgdm 3 years postpartum reported that women with pgdm had significantly elevated high - sensitivity crp ( hs - crp ) compared with controls . studied 25 women with pgdm and found that in a mean period of 4 years postpartum they had significantly higher crp levels compared to controls . in another study , 18 women with pgdm at least 12 months after index delivery were found to have no significant difference in hs - crp compared with normal controls . on the other hand , other authors studied 70 women with pgdm 6 years after their pregnancy and found significantly higher crp levels in women with pgdm in the presence of abdominal obesity ; they also found abnormal glucose tolerance compared with the women without a history of gdm . this was further confirmed in another study where significantly elevated crp levels and fibrinogen were detected in 26 women with pgdm as compared with controls . plasminogen activator inhibitor 1 ( pai-1 ) is a protein that in humans is encoded by the serpine1 gene and is mainly produced by the endothelial cells , though it is also secreted by other tissue types , such as adipose tissue . its main function is to inhibit tissue plasminogen activator ( tpa ) and urokinase ( upa ) , the activators of plasminogen , and hence fibrinolysis . pai-1 is increased in various disease states , such as obesity , ms , insulin resistance , and t2 dm [ 31 , 3436 ] . pai-1 is increased in women with pgdm compared with normal women 3 months after delivery . another study reported that women with pgdm had significantly elevated pai-1 compared with controls 3 years after delivery . other authors studying 74 women with pgdm 3 months after delivery found them to exhibit increased pai-1 levels when they had impaired insulin sensitivity postpartum , while tpa was also observed to be elevated . in another study , 25 women with pgdm demonstrated significantly higher pai-1 levels compared to controls in a mean period of 4 years postpartum , although the difference lost significance after adjustment for bmi . women with pgdm are characterized by chronic subclinical inflammation which is associated with insulin resistance and abnormality in glucose metabolism . approximately 30% of these women have metabolic syndrome and many of them will develop t2 dm within 5 years of diagnosis [ 26 , 31 , 173 ] . the conversion rates from gdm to t2 dm range from 2.6% to 70% over a period of 6 weeks to 28 years postpartum . the problem of gestational diabetes is common and its incidence is exhibiting an increasing prevalence . early recognition and management of women predisposed to develop t2 dm later in their lives is thus crucial in the development of primary health care strategies , modification of lifestyle , and dietary habits significantly enabling prevention or delay of appearance of glucose intolerance states in predisposed women .

new technology has been defined as anything that does nt quite work yet.1 while in some instances accurate , this definition would justifiably elicit public outcry if applied to the latest medical technology . and yet , as will be described below , many new medical devices ( i.e. , medical technology ) make it to market without the kind of rigorous evidence expected for drug approval . in this paper , we review the existing process of medical device approval , and we discuss how independent , systematic assessments of newly approved medical devices can evaluate the extent and quality of the evidence for medical devices , thereby supporting the safe and effective use of emerging medical technology . medical technology , regulated as devices , includes items as mundane as bedpans and latex gloves , and as complex as magnetic resonance imaging scanners and hybrid devices such as drug - eluting stents . the explosive growth of medical technology has paved the way for fields as disparate as organ transplantation and preimplantation genetic diagnosis.2 by the late 1990s , the us food and drug administration ( fda ) had approved about 500,000 medical device models produced by approximately 23,000 different manufacturers . it has been estimated that 4% of the population in the usa has at least 1 implanted medical device.3,4 in spite of the ubiquity and impact of emerging medical technology , few physicians , and even fewer patients , understand the process by which new medical devices are evaluated and approved by the fda . although there have been some serious and well - publicized problems ( e.g. , rofecoxib ) , the process for evaluating pharmaceuticals before their release into the market is much more thorough than the analogous process for new medical devices.59 whereas all new drugs must pass rigorous premarketing approval testing with randomized clinical trials , such testing is required for relatively few new devices . devices are defined according to the federal food , drug and cosmetic act , section 201 , as an instrument , apparatus , implement , machine , contrivance , implant , in vitro reagent , or other similar article that is intended for use in the diagnosis of disease or other conditions , or in the cure , mitigation , treatment , or prevention of disease.3,10 this drug / device split in testing extends throughout the health care system to health plans and hospitals , as formulary committees for drug coverage have few device coverage correlates . the somewhat artificial distinction between devices and drugs in contrast , in the uk , the national institute for health and clinical excellence ( nice ) evaluates both drugs and devices according to the same protocol . recently , there has been a call to address the issue in the usa with a center for comparative effectiveness reviews.11 within the fda , the center for devices and radiological health ( cdrh ) has primary responsibility for the premarket assessment of new medical technology . the cdrh not only considers the risks of new devices , but also monitors them via a nationwide postmarket surveillance system . new devices are classified as low ( i ) , moderate ( ii ) , or high risk ( iii ) . among the thousands of device applications submitted annually , fewer than 100those considered to be novel and high - risk undergo a premarket approval application ( pma ) analogous to the rigorous scrutiny required for new drugs.12 the bulk of new device approval applications are instead initiated via a premarket notification application [ 510(k ) ] , an fda process based on the assumption that most devices are essentially equivalent to those already approved . for example , via the 510(k ) pathway , the manufacturer of an approved laser may not be required to provide safety and efficacy data to employ a second - generation laser for treating conditions beyond those listed in the initial pma approval.13 computed tomographic ( ct ) colonography ( virtual colonoscopy ) , which received 510(k ) fda clearance in may of 2003 , exemplifies an application of ct for which neither validity nor clinical benefit had to be demonstrated before marketing and clinical application.14,15 this pathway to approval does not usually require clinical data derived from randomized trials regarding the effectiveness or safety of a device for a given use or population of patients.3,1618 for the fiscal year 2005 , the office of device evaluation reported receiving 3,130 510(k ) applications ( ca . 250 per month ) , whereas only 43 original pmas requiring rigorous study were submitted.12 the 510(k ) process leads to the imprimatur fda approved on many devices for which there may be only limited data in support of clinical effectiveness or safety for that specific clinical indication ( see fig . 1 for an overview of the fda approval process for medical devices).3figure 1overview of the medical device approval process.3 overview of the medical device approval process.3 there are concerns about inadequate scrutiny of devices at the fda , resulting in part from limited resources . in fact , the fda issued a report stating that medical device program resources have been reduced in recent years , and there have been indications that review performance has begun to decline.19 the institute of medicine ( iom ) has described all of the fda evaluation programs as severely underfunded , which has led to a potential increase in public risk and growing reliance on industry - paid user fees . these fees will account in 2008 for more than 40% of the drug regulation resources and almost a quarter of the resources needed for the review of medical devices.9,1921 some have expressed concern that user fees render the fda more accountable to the very drug and device manufacturers it is supposed to be regulating and that the manufacturers are replacing the public as the fda s primary clients.21 in addition , the fda has limited ability and authority to conduct postmarketing surveillance for both devices and pharmaceuticals ; it falls on the manufacturers and academic and clinical investigators to initiate the bulk of recalls and failure reports.3,16,2225 this situation leaves ample room for those with potential and real financial conflicts of interest to exert undue influence on the regulatory process . in a recent initiative to reduce conflicts of interest among members serving on device regulatory advisory committees , guidelines have been drafted that suggest an eligibility cutoff of us $ 50,000 as the maximum financial interest of any potential committee member under fda consideration.26 furthermore , the fda explicitly does not regulate physician behavior or approve particular clinical procedures.27 once a technology is approved , it can be used in clinical scenarios that fall outside the patient- and clinical - inclusion criteria of the pivotal trials . in the recent controversy over the long - term safety of drug - eluting stents , it was revealed that up to 55% of these devices are implanted for such off - label use.28,29 the stent uproar has been said to represent both a success and a failure of the fda medical - device regulatory system : while a panel was rapidly assembled to respond to concerns , much still remains unknown about the long - term safety of drug - eluting stents , and millions of these devices have already been implanted.25 recognizing that all health care sectors need to cooperate to critically assess the available evidence to ensure the safety and effectiveness of medical care , the institute of medicine ( iom ) addressed the quality chasm by initiating a roundtable on evidence - based medicine based on the findings of numerous studies showing that ... far too much of our health care spending is devoted to activities that do not improve health , and far too little investment is devoted to better understanding the relative advantages among various intervention choices . this gap in knowledge about what approaches deliver the best results will only be compounded as the pace of technology development quickens ... 30,31 . ... far too much of our health care spending is devoted to activities that do not improve health , and far too little investment is devoted to better understanding the relative advantages among various intervention choices . this gap in knowledge about what approaches deliver the best results will only be compounded as the pace of technology development quickens ... 30,31 . one means of addressing the problem of our lack of information about the relative efficacy and safety of various medical devices is to use an evidence - based medicine ( ebm ) approach that encourages the uniform application of consistent scientific methodology to inform and tailor care.3234 ebm - based assessments of new medical technology have the potential to enhance safety and efficacy by advocating that clinical practice be informed by analyses of well - designed , randomized , controlled trials optimally conducted at more than 1 center ; these features of ebm may increase transparency and reduce opportunities for bias and conflicts of interest . however , in some circumstances well - designed observational studies may suffice for evaluation of diagnostic tests.35 medical technology assessment may amplify conflicts of interest related to investments , reimbursement for clinical services , research support , political and lobbying influences , and other factors affecting all stakeholders : manufacturers , stockholders , physicians , patients , insurance carriers , and even the fda itself . fortunately , several existing medical technology assessment groups provide complementary critical analyses of the efficacy and safety of new medical devices ( tables 1 , 2 , and 3 ) . among the government - sponsored entities other than the fda that have contributed to the research and dissemination of technology assessments , the most prominent is the centers for medicare and medicaid services ( cms ) , which often initiates coverage of medical devices with an evidence - development process ultimately aimed at synthesizing large amounts of data from various clinical experiences.36 other groups include the iom , the national institutes of health , the us preventive services task force , and the agency for healthcare research and quality ( ahrq ) , which itself includes a network for developing evidence to inform decisions about effectiveness ( decide ) , centers for education and research on therapeutics ( certs ) , and several evidence - based practice centers ( epcs ) funded by the ahrq that not only review relative effectiveness and safety , but also identify areas requiring further study . a number of foreign federal and private technology assessment agencies ( e.g. , nice in the uk ) also make important contributions . table 1major evidence - based sources of information on medical technologycenterwebsiteservicescommentsfood and drug administration center for devices and radiological health databases ( fda cdrh)http://www.fda.gov / cdrh / databases.htmlfull listing of medical devices in commercial distribution by both domestic and foreign manufacturers , as well as medical devices at various stages of premarket approval , and those devices which have been recalled from the market . the manufacturer and user facility device experience ( maude ) database provides information on reported adverse events for individual devices.fdacdrh reports are lengthy and dense ; they often do not provide a synthesis of the data or the information . the cdrh also assures that non - medical radiation - emitting products such as cell phones , lasers , and tv sets meet radiation safety standardsaddresses topics such as fda approval , use and purchase of specific medical devices and diagnostic tests , manufacturer contact information , device instructions , post - approval alterations of products , and information about devices applied in the treatment and diagnosis of specific diseasescenters for medicare and medicaid ( cms)http://www.cms.hhs.gov / mcd / index_list.asp?list_type = techinformation on medicare coverage decisions including the rationale . often provides cost analysismedicare advisory committee technology assessments : decisions may be influenced by political pressures , process is not always transparent , limited number of topics reviewedthe cochrane library health technology assessment databasehttp://www.mrw.interscience.wiley.com / cochrane / cochrane_clhta_articles_fs.htmlextensive number of topics covered . access to assessments from other english - speaking countries , including canada , uk and australia . cost often included in the assessmentsreviews lengthy and do not all use the same criteria / standards ; many non - technology topics are covered . reviews are referenced in the literature , but not as primary documents for medical technology evaluations ; thus do not directly influence policyrequires a subscription to access the database , though reviews are often available for downloadtable 2selection of technology assessment entitiescenterwebsiteservicescommentsagency for healthcare research and quality ( ahrq)http://www.ahrq.govfocus on evidence , high standards , and technology s impact on health outcomes . tec is an evidence - based practice center ( epc ) designated by the ahrq.often lengthy assessments , but with a brief summary . there is a public route for submitting input to ahrq , although it remains unclear how this input is incorporated into the decision - making process . blue shield associationhttp://www.bcbs.com / tec / whatistec.htmlcalifornia technology assessment forum - blue shield of california foundation ( ctaf)http://www.ctaf.orgevidence - based reviews with expert and community input ; focus on identifying medical technologies that will improve healthno cost analyses . stated goal is to educate the public , but assessments are often highly technicalhayeshttp://www.hayesinc.comindependent , professional reviews ; consultation about specific health technology topicsmust subscribe in order to read assessmentsinstitute of clinical systems improvement ( icsi)http://www.icsi.orgindependent non - profit organization funded by all 6 of the health payers in minnesotafocus on health care services for people who live and work in the state of minnesota and in adjacent areas of surrounding statesecri institutehttp://www.ecri.org / pages / default.aspxnon - profit health research services agency that also operates an international medical device problem reporting system and a journal , health devices . medical devices safety reports ( mdsr ) are availablemdsr are selected and edited such that they provide general information . ecri does not serve as an alerting service , but weekly alerts of medical device hazards and recalls are available through membershiphttp://www.mdsr.ecri.org / information / about.aspxtable 3selection of professional societiescenterweb siteservicescommentsamerican society for bariatric surgery ( asbs)http://www.asbs.org / specialist panels of individuals with experience in a particular field are asked to weigh in on the approval of a new device in their field . members and specialist sites also provide information about post - approval application of new devicesfinancial conflicts of interest and/or other professional bias must be taken into consideration , as such conflicts could reduce objectivityamerican college of cardiology ( acc)http://www.acc.org / american rhinologic society ( ars)http://www.american - rhinologic.org / index.phtmlamerican society for therapeutic radiology and oncology ( astro)http://www.astro.org / american college of physicians ( acp)http://www.acponline.orgamerican academy of family physicians ( aafp)http://www.aafp.orgamerican academy of pediatrics ( aap)http://www.aap.orgamerican osteopathic association ( aoa)http://www.do - online.orgamerican thoracic society ( ats)http://www.thoracic.org major evidence - based sources of information on medical technology selection of technology assessment entities selection of professional societies private payers rely on a mix of both proprietary and publicly available technology assessments for their policy development ; some have their own proprietary technology assessment process or purchase proprietary technology assessments produced by independent , often for - profit , companies . an example of an independent , nonprofit organization that publishes technology assessments is the institute of clinical systems improvement ( icsi ) , with 56 members funded by all 6 of the health plans in minnesota . the blue cross / blue shield association ( bcbsa ) produces technology assessments through an epc , the technology evaluation center ( tec ) . tec assessments are available to the public at their website and are distributed to all of the blues plans as a common starting point for medical policy coverage development . technology assessment organizations , whether developed by specific payers or independent companies , variably engage specialty societies or local physicians for their input . payers typically convene a committee to consider either privately or publicly the findings of an assessment ; consumers , specialists , and practicing physicians may serve as contributors to the assessment itself , as members of an assessment committee , or as majority or minority voting members on a technology assessment . a pointed summary from the 2004 consensus conference of the american society for bariatric surgery ( asbs ) suggested that insurance providers are unwilling to invest in expensive treatments that yield no profits until after 4 years because 4 years is the median time for an individual to remain with an insurance company.37 whether such views are substantiated , it remains important to acknowledge the potential for vested interests . professional societies oversee the utilization of new technologies that fall under their domain , and they frequently provide checks and balances on other decision - making entities . their web sites can serve as a forum for hotly debated , complex topics that affect patient safety ( table 3).38,39 indeed , professional societies have often taken the lead in endorsing evidence - based reviews . for example , the american college of physicians ( acp ) announced its collaboration with the american academy of family physicians ( aafp ) , the american academy of pediatrics ( aap ) , and the american osteopathic association ( aoa ) to release joint principles for a patient - centered medical home , a program in which evidence - based medicine serves clinical decision making.40 the american thoracic society ( ats ) has been a strong advocate of applying and refining standard criteria for conducting systematic reviews with the aim of developing practice guidelines.41,42 professional societies not only provide a format for discussion , they also work toward improving the overall quality of debate . however , as with other evaluative entities , they are not immune to pressures and financial conflicts of interest that may influence their recommendations.43,44 two examples of professional society recommendation serve to highlight the potential for member self - interest to influence the evaluation of novel medical devices . in may of 2006 , an article in the lay press questioned patient testimonials on a manufacturer s web site extolling a new procedure , balloon dilation of the sinuses , a minimally invasive alternative to sinus surgery . this challenge was officially echoed in a position statement by the american rhinologic society ( ars ) , claiming that in spite of fda approval , ... the scientific literature has no data on the technology for long - term safety , indications , efficacy and outcomes.4547 what the ars did not say is that some of their members might experience a decline in income if the balloon dilation procedure were established as the standard of care over surgery . likewise , intensity - modulated radiation therapy ( imrt ) , an alternative to conventional 3d - conformal radiation therapy ( 3d - crt ) , has been broadly embraced by the radiation oncology professional organizations for treatment of prostate cancer despite the lack of any comparative studies in humans and no fda review process of specific applications of imrt for prostate cancer . the embrace of imrt may have been in part driven by its favorable reimbursement compared to that of standard therapies for prostate cancer.48 the safety and effectiveness of medical devices are not yet comprehensively evaluated by the fda , other federal entities , payers , or professional societies . therefore , a stance of critical self - analysis will remain crucial while advocating innovation . technology assessments by objective , independent , not - for - profit entities are necessary so that deficiencies in the clinical evidence and potential threats to patient safety are recognized in a manner that counterbalances market forces driving diffusion and demand . the california technology assessment forum ( ctaf ) is an independent , nonprofit entity whose mission is to provide transparent , objective reviews of new medical devices to better educate the public , clinicians , and policymakers ( see http://www.ctaf.org ) . the ctaf reviews are intended to complement the role played by professional societies and the fda . recent topics have included full - field digital mammography for breast cancer screening , gene expression profiling for the diagnosis of heart transplant rejection , wireless capsule endoscopy , and device - controlled breathing as a treatment for hypertension . ctaf contracts with general internists at academic medical centers to provide about 15 systematic reviews of new and emerging medical technologies per year . topics are selected through a consensus process based on impact of the technology and availability of relevant clinical data by an ad hoc committee consisting of representatives from the ctaf panel ( see below ) , the generalist physician consultants and the blue shield of ca health plan . potential topics may be brought to the attention of the committee by all potential stakeholders : the health plan , industry , professional societies , consumer groups , or ctaf staff . although the subject of these reviews is often highly specialized and technical , ctaf believes that generalist physicians are better suited to provide objective , evidence - based assessments than are subspecialists who , in spite of their superior technical knowledge , may be more likely to have preexisting biases or opinions . three times per year , ctaf invites manufacturers , patients , clinicians , and payers to openly debate the safety and effectiveness of 4 to 5 new devices ; these discussions typically include input ranging from anecdotal patient experience to expert testimony from the principal investigators of the pivotal randomized controlled trials . ctaf reviews are systematic analyses of the peer - reviewed literature that synthesize the results of all published clinical trials , case series , and randomized clinical trials , with an emphasis on comparative trials . the medline database , cochrane clinical trials database , cochrane reviews database , and the database of abstracts of reviews of effects ( dare ) are searched using relevant key words from 1966 up to the present . in addition , the bibliographies of systematic reviews and key articles are manually searched for additional references . each assessment is researched and written by the lead consultant with consultation and input from the other consultants as needed and are , generally , 25,000 to 30,000 words in length . the consultant must determine whether each new technology selected for review meets 5 criteria ( see table 4 ) according to a consistent , reproducible approach with a visible line of reasoning . the technology assessment should specify critical patient - centered outcomes , as well as identify which data are available that will ideally evaluate these outcomes , and where there is insufficient data . ctaf reviews are sent to subspecialty experts and to professional societies for critical appraisal and comment . members from both of these groups are invited to the ctaf meetings to contribute to the discussion in a public forum . other interested parties from the government ( e.g. , cms ) , health care organizations , academic centers , and the community are also invited to attend and participate in the discussion . efforts are also made to invite those who are advocates of a particular device ( such as manufacturers and inventors ) , as well as others who may be against it . table 4california technology assessment forum assessment criterianumberdescriptioncommentsta 1the technology must have final approval from the appropriate government regulatory bodiesapproval via 510(k ) process is streamlined for new devices that are similar to older devicesta 2the scientific evidence must permit conclusions concerning the effectiveness of the technology regarding health outcomesrange : levels 15 , e.g. , level 1 , randomized , controlled trials powered to demonstrate clinically significant outcomes ; level 5 , case series without controlsta 3the technology must improve net health outcomesdiagnostic tests must change management in ways that benefit patientsta 4the technology must be as beneficial as any established alternativesit remains unclear how to evaluate diagnostic / prognostic tests lacking a gold - standard referenceta 5the improvement must be attainable outside the investigational settingby definition , trials are conducted within an investigational setting , and therefore this criterion implies a speculation about potential , and not actually studied , applications california technology assessment forum assessment criteria the voting panel includes experts from a variety of disciplines : ethicists , consumer advocates , practicing clinicians , methodologists , and others . the consultants present a brief overview of the peer - reviewed literature to the panel and then recommend that the technology either meets or does not meet the 5 criteria for approval . if the recommendation can not be accepted as stated , the panel then entertains and votes on alternatives . approximately 25% of new technologies are approved ( i.e. , found to meet all 5 ctaf criteria for efficacy and safety ) . there is no formal appeal process , but technologies will be assessed again when more data become available and frequently are approved at that time . ctaf does not have the ability to formally track the costs or impact of new technology it has reviewed . private payers rely on a mix of both proprietary and publicly available technology assessments for their policy development ; some have their own proprietary technology assessment process or purchase proprietary technology assessments produced by independent , often for - profit , companies . an example of an independent , nonprofit organization that publishes technology assessments is the institute of clinical systems improvement ( icsi ) , with 56 members funded by all 6 of the health plans in minnesota . the blue cross / blue shield association ( bcbsa ) produces technology assessments through an epc , the technology evaluation center ( tec ) . tec assessments are available to the public at their website and are distributed to all of the blues plans as a common starting point for medical policy coverage development . technology assessment organizations , whether developed by specific payers or independent companies , variably engage specialty societies or local physicians for their input . payers typically convene a committee to consider either privately or publicly the findings of an assessment ; consumers , specialists , and practicing physicians may serve as contributors to the assessment itself , as members of an assessment committee , or as majority or minority voting members on a technology assessment . a pointed summary from the 2004 consensus conference of the american society for bariatric surgery ( asbs ) suggested that insurance providers are unwilling to invest in expensive treatments that yield no profits until after 4 years because 4 years is the median time for an individual to remain with an insurance company.37 whether such views are substantiated , it remains important to acknowledge the potential for vested interests . professional societies oversee the utilization of new technologies that fall under their domain , and they frequently provide checks and balances on other decision - making entities . their web sites can serve as a forum for hotly debated , complex topics that affect patient safety ( table 3).38,39 indeed , professional societies have often taken the lead in endorsing evidence - based reviews . for example , the american college of physicians ( acp ) announced its collaboration with the american academy of family physicians ( aafp ) , the american academy of pediatrics ( aap ) , and the american osteopathic association ( aoa ) to release joint principles for a patient - centered medical home , a program in which evidence - based medicine serves clinical decision making.40 the american thoracic society ( ats ) has been a strong advocate of applying and refining standard criteria for conducting systematic reviews with the aim of developing practice guidelines.41,42 professional societies not only provide a format for discussion , they also work toward improving the overall quality of debate . however , as with other evaluative entities , they are not immune to pressures and financial conflicts of interest that may influence their recommendations.43,44 two examples of professional society recommendation serve to highlight the potential for member self - interest to influence the evaluation of novel medical devices . in may of 2006 , an article in the lay press questioned patient testimonials on a manufacturer s web site extolling a new procedure , balloon dilation of the sinuses , a minimally invasive alternative to sinus surgery . this challenge was officially echoed in a position statement by the american rhinologic society ( ars ) , claiming that in spite of fda approval , ... the scientific literature has no data on the technology for long - term safety , indications , efficacy and outcomes.4547 what the ars did not say is that some of their members might experience a decline in income if the balloon dilation procedure were established as the standard of care over surgery . likewise , intensity - modulated radiation therapy ( imrt ) , an alternative to conventional 3d - conformal radiation therapy ( 3d - crt ) , has been broadly embraced by the radiation oncology professional organizations for treatment of prostate cancer despite the lack of any comparative studies in humans and no fda review process of specific applications of imrt for prostate cancer . the embrace of imrt may have been in part driven by its favorable reimbursement compared to that of standard therapies for prostate cancer.48 the safety and effectiveness of medical devices are not yet comprehensively evaluated by the fda , other federal entities , payers , or professional societies . therefore , a stance of critical self - analysis will remain crucial while advocating innovation . technology assessments by objective , independent , not - for - profit entities are necessary so that deficiencies in the clinical evidence and potential threats to patient safety are recognized in a manner that counterbalances market forces driving diffusion and demand . the california technology assessment forum ( ctaf ) is an independent , nonprofit entity whose mission is to provide transparent , objective reviews of new medical devices to better educate the public , clinicians , and policymakers ( see http://www.ctaf.org ) . the ctaf reviews are intended to complement the role played by professional societies and the fda . recent topics have included full - field digital mammography for breast cancer screening , gene expression profiling for the diagnosis of heart transplant rejection , wireless capsule endoscopy , and device - controlled breathing as a treatment for hypertension . ctaf contracts with general internists at academic medical centers to provide about 15 systematic reviews of new and emerging medical technologies per year . topics are selected through a consensus process based on impact of the technology and availability of relevant clinical data by an ad hoc committee consisting of representatives from the ctaf panel ( see below ) , the generalist physician consultants and the blue shield of ca health plan . potential topics may be brought to the attention of the committee by all potential stakeholders : the health plan , industry , professional societies , consumer groups , or ctaf staff . although the subject of these reviews is often highly specialized and technical , ctaf believes that generalist physicians are better suited to provide objective , evidence - based assessments than are subspecialists who , in spite of their superior technical knowledge , may be more likely to have preexisting biases or opinions . three times per year , ctaf invites manufacturers , patients , clinicians , and payers to openly debate the safety and effectiveness of 4 to 5 new devices ; these discussions typically include input ranging from anecdotal patient experience to expert testimony from the principal investigators of the pivotal randomized controlled trials . ctaf reviews are systematic analyses of the peer - reviewed literature that synthesize the results of all published clinical trials , case series , and randomized clinical trials , with an emphasis on comparative trials . the medline database , cochrane clinical trials database , cochrane reviews database , and the database of abstracts of reviews of effects ( dare ) are searched using relevant key words from 1966 up to the present . in addition , the bibliographies of systematic reviews and key articles are manually searched for additional references . each assessment is researched and written by the lead consultant with consultation and input from the other consultants as needed and are , generally , 25,000 to 30,000 words in length . the consultant must determine whether each new technology selected for review meets 5 criteria ( see table 4 ) according to a consistent , reproducible approach with a visible line of reasoning . the technology assessment should specify critical patient - centered outcomes , as well as identify which data are available that will ideally evaluate these outcomes , and where there is insufficient data . ctaf reviews are sent to subspecialty experts and to professional societies for critical appraisal and comment . members from both of these groups are invited to the ctaf meetings to contribute to the discussion in a public forum . other interested parties from the government ( e.g. , cms ) , health care organizations , academic centers , and the community are also invited to attend and participate in the discussion . efforts are also made to invite those who are advocates of a particular device ( such as manufacturers and inventors ) , as well as others who may be against it . table 4california technology assessment forum assessment criterianumberdescriptioncommentsta 1the technology must have final approval from the appropriate government regulatory bodiesapproval via 510(k ) process is streamlined for new devices that are similar to older devicesta 2the scientific evidence must permit conclusions concerning the effectiveness of the technology regarding health outcomesrange : levels 15 , e.g. , level 1 , randomized , controlled trials powered to demonstrate clinically significant outcomes ; level 5 , case series without controlsta 3the technology must improve net health outcomesdiagnostic tests must change management in ways that benefit patientsta 4the technology must be as beneficial as any established alternativesit remains unclear how to evaluate diagnostic / prognostic tests lacking a gold - standard referenceta 5the improvement must be attainable outside the investigational settingby definition , trials are conducted within an investigational setting , and therefore this criterion implies a speculation about potential , and not actually studied , applications california technology assessment forum assessment criteria the voting panel includes experts from a variety of disciplines : ethicists , consumer advocates , practicing clinicians , methodologists , and others . the consultants present a brief overview of the peer - reviewed literature to the panel and then recommend that the technology either meets or does not meet the 5 criteria for approval . if the recommendation can not be accepted as stated , the panel then entertains and votes on alternatives . approximately 25% of new technologies are approved ( i.e. , found to meet all 5 ctaf criteria for efficacy and safety ) . there is no formal appeal process , but technologies will be assessed again when more data become available and frequently are approved at that time . ctaf does not have the ability to formally track the costs or impact of new technology it has reviewed . a general limitation of technology assessment is the lack of high - quality data derived from randomized clinical trials . there are often no rcts to provide an ideal level of evidence for decision making . as described above , the 510(k ) process permits most devices to be marketed in the absence of high - quality studies . moreover , manufacturers , researchers , clinicians , and other stakeholders frequently have little incentive to conduct the kind of studies needed to answer the most important or relevant clinical questions . the device industry and professional societies often exert significant influence on which devices are evaluated and how they are used . however critical , assessment of the evidence by these groups may be of limited value because of real or potential conflicts of interest . the ties to industry funding also vary among the assessment groups listed in tables 1 , 2 , and 3 , and in some cases , assessments are provided on a for - profit basis . in short , no immunity from conflicts of interest is conferred upon any of the stakeholders in this process . discrepancies between assessment groups conclusions can and do occur due in part to different values used to weight the evidence and different methodologies ( e.g. , ctaf relies solely on published peer - reviewed literature only for their reviews , whereas another group may include unpublished data ) . perhaps more problematic is the fact that the available evidence - based technology assessments remain poorly disseminated . all stakeholders physicians , patients , policy makers , fda advisory committee members , and manufacturers stand to benefit from better access to assessments like those provided by groups such as tec and ctaf , and from well - designed multicenter rcts that can inform practice . as a result of the proliferation , complexity , and magnitude of impact of new and emerging medical devices , there is an urgent need to monitor new technology ever more closely in terms of effectiveness and safety . in spite of this need , significant gaps persist in the scientific literature , in the medical device approval process , and in the realm of postmarketing surveillance . while most pharmaceuticals obtain fda approval only after analysis of randomized clinical trials , relatively few new medical devices are subject to comparable scrutiny . to improve health outcomes , we must enhance our understanding of new medical devices and , by not simply deferring to the fda , support independent , evidence - based technology assessments . however , given the current process that allows for the vast majority of new devices to enter the market under a 510(k ) , once a device is introduced and used , the ability to eliminate inappropriate use , even with rigorous ta , is nearly impossible . in spite of this limitation , technology assessments by independent organizations after fda approval can help identify those medical technologies that are truly beneficial and safe . furthermore , such assessments may motivate research designed to answer remaining questions about emerging technologies and can serve to educate the public and health professionals about the potential promise and pitfalls of new technology .

introductionthe global medical technology industry brings thousands of devices to market every year . however , significant gaps persist in the scientific literature , in the medical device approval process , and in the realm of postmarketing surveillance . although thousands of drugs obtain approval only after review in randomized controlled trials , relatively few new medical devices are subject to comparable scrutiny.objectiveto improve health outcomes , we must enhance our scrutiny of medical devices , and , without simply deferring to the food and drug administration , we must ask ourselves : who is responsible for evaluating the safety and effectiveness of medical devices?conclusionstechnology assessments by independent organizations are a part of the solution to this challenge and may motivate further research focused on patient outcomes .

INTRODUCTION THE DRUG/DEVICE SPLIT: A HISTORICAL ARTIFACT? FDA APPROVAL: PROBLEMS AND PROSPECTS EVIDENCE-BASED MEDICINE AND TECHNOLOGY ASSESSMENT BEYOND THE FDA: APPLYING EBM TO MEDICAL DEVICES Private Payers and Technology Assessment Professional Societies A Case Study: The California Technology Assessment Forum LIMITATIONS OF EVIDENCE-BASED TECHNOLOGY ASSESSMENT CONCLUSION

new technology has been defined as anything that does nt quite work yet.1 while in some instances accurate , this definition would justifiably elicit public outcry if applied to the latest medical technology . and yet , as will be described below , many new medical devices ( i.e. , medical technology ) make it to market without the kind of rigorous evidence expected for drug approval . in this paper , we review the existing process of medical device approval , and we discuss how independent , systematic assessments of newly approved medical devices can evaluate the extent and quality of the evidence for medical devices , thereby supporting the safe and effective use of emerging medical technology . medical technology , regulated as devices , includes items as mundane as bedpans and latex gloves , and as complex as magnetic resonance imaging scanners and hybrid devices such as drug - eluting stents . the explosive growth of medical technology has paved the way for fields as disparate as organ transplantation and preimplantation genetic diagnosis.2 by the late 1990s , the us food and drug administration ( fda ) had approved about 500,000 medical device models produced by approximately 23,000 different manufacturers . it has been estimated that 4% of the population in the usa has at least 1 implanted medical device.3,4 in spite of the ubiquity and impact of emerging medical technology , few physicians , and even fewer patients , understand the process by which new medical devices are evaluated and approved by the fda . , rofecoxib ) , the process for evaluating pharmaceuticals before their release into the market is much more thorough than the analogous process for new medical devices.59 whereas all new drugs must pass rigorous premarketing approval testing with randomized clinical trials , such testing is required for relatively few new devices . devices are defined according to the federal food , drug and cosmetic act , section 201 , as an instrument , apparatus , implement , machine , contrivance , implant , in vitro reagent , or other similar article that is intended for use in the diagnosis of disease or other conditions , or in the cure , mitigation , treatment , or prevention of disease.3,10 this drug / device split in testing extends throughout the health care system to health plans and hospitals , as formulary committees for drug coverage have few device coverage correlates . the somewhat artificial distinction between devices and drugs in contrast , in the uk , the national institute for health and clinical excellence ( nice ) evaluates both drugs and devices according to the same protocol . recently , there has been a call to address the issue in the usa with a center for comparative effectiveness reviews.11 within the fda , the center for devices and radiological health ( cdrh ) has primary responsibility for the premarket assessment of new medical technology . among the thousands of device applications submitted annually , fewer than 100those considered to be novel and high - risk undergo a premarket approval application ( pma ) analogous to the rigorous scrutiny required for new drugs.12 the bulk of new device approval applications are instead initiated via a premarket notification application [ 510(k ) ] , an fda process based on the assumption that most devices are essentially equivalent to those already approved . for example , via the 510(k ) pathway , the manufacturer of an approved laser may not be required to provide safety and efficacy data to employ a second - generation laser for treating conditions beyond those listed in the initial pma approval.13 computed tomographic ( ct ) colonography ( virtual colonoscopy ) , which received 510(k ) fda clearance in may of 2003 , exemplifies an application of ct for which neither validity nor clinical benefit had to be demonstrated before marketing and clinical application.14,15 this pathway to approval does not usually require clinical data derived from randomized trials regarding the effectiveness or safety of a device for a given use or population of patients.3,1618 for the fiscal year 2005 , the office of device evaluation reported receiving 3,130 510(k ) applications ( ca . 1 for an overview of the fda approval process for medical devices).3figure 1overview of the medical device approval process.3 overview of the medical device approval process.3 there are concerns about inadequate scrutiny of devices at the fda , resulting in part from limited resources . in fact , the fda issued a report stating that medical device program resources have been reduced in recent years , and there have been indications that review performance has begun to decline.19 the institute of medicine ( iom ) has described all of the fda evaluation programs as severely underfunded , which has led to a potential increase in public risk and growing reliance on industry - paid user fees . these fees will account in 2008 for more than 40% of the drug regulation resources and almost a quarter of the resources needed for the review of medical devices.9,1921 some have expressed concern that user fees render the fda more accountable to the very drug and device manufacturers it is supposed to be regulating and that the manufacturers are replacing the public as the fda s primary clients.21 in addition , the fda has limited ability and authority to conduct postmarketing surveillance for both devices and pharmaceuticals ; it falls on the manufacturers and academic and clinical investigators to initiate the bulk of recalls and failure reports.3,16,2225 this situation leaves ample room for those with potential and real financial conflicts of interest to exert undue influence on the regulatory process . in the recent controversy over the long - term safety of drug - eluting stents , it was revealed that up to 55% of these devices are implanted for such off - label use.28,29 the stent uproar has been said to represent both a success and a failure of the fda medical - device regulatory system : while a panel was rapidly assembled to respond to concerns , much still remains unknown about the long - term safety of drug - eluting stents , and millions of these devices have already been implanted.25 recognizing that all health care sectors need to cooperate to critically assess the available evidence to ensure the safety and effectiveness of medical care , the institute of medicine ( iom ) addressed the quality chasm by initiating a roundtable on evidence - based medicine based on the findings of numerous studies showing that ... far too much of our health care spending is devoted to activities that do not improve health , and far too little investment is devoted to better understanding the relative advantages among various intervention choices . ... far too much of our health care spending is devoted to activities that do not improve health , and far too little investment is devoted to better understanding the relative advantages among various intervention choices . one means of addressing the problem of our lack of information about the relative efficacy and safety of various medical devices is to use an evidence - based medicine ( ebm ) approach that encourages the uniform application of consistent scientific methodology to inform and tailor care.3234 ebm - based assessments of new medical technology have the potential to enhance safety and efficacy by advocating that clinical practice be informed by analyses of well - designed , randomized , controlled trials optimally conducted at more than 1 center ; these features of ebm may increase transparency and reduce opportunities for bias and conflicts of interest . however , in some circumstances well - designed observational studies may suffice for evaluation of diagnostic tests.35 medical technology assessment may amplify conflicts of interest related to investments , reimbursement for clinical services , research support , political and lobbying influences , and other factors affecting all stakeholders : manufacturers , stockholders , physicians , patients , insurance carriers , and even the fda itself . fortunately , several existing medical technology assessment groups provide complementary critical analyses of the efficacy and safety of new medical devices ( tables 1 , 2 , and 3 ) . among the government - sponsored entities other than the fda that have contributed to the research and dissemination of technology assessments , the most prominent is the centers for medicare and medicaid services ( cms ) , which often initiates coverage of medical devices with an evidence - development process ultimately aimed at synthesizing large amounts of data from various clinical experiences.36 other groups include the iom , the national institutes of health , the us preventive services task force , and the agency for healthcare research and quality ( ahrq ) , which itself includes a network for developing evidence to inform decisions about effectiveness ( decide ) , centers for education and research on therapeutics ( certs ) , and several evidence - based practice centers ( epcs ) funded by the ahrq that not only review relative effectiveness and safety , but also identify areas requiring further study . table 1major evidence - based sources of information on medical technologycenterwebsiteservicescommentsfood and drug administration center for devices and radiological health databases ( fda cdrh)http://www.fda.gov / cdrh / databases.htmlfull listing of medical devices in commercial distribution by both domestic and foreign manufacturers , as well as medical devices at various stages of premarket approval , and those devices which have been recalled from the market . the cdrh also assures that non - medical radiation - emitting products such as cell phones , lasers , and tv sets meet radiation safety standardsaddresses topics such as fda approval , use and purchase of specific medical devices and diagnostic tests , manufacturer contact information , device instructions , post - approval alterations of products , and information about devices applied in the treatment and diagnosis of specific diseasescenters for medicare and medicaid ( cms)http://www.cms.hhs.gov / mcd / index_list.asp?list_type = techinformation on medicare coverage decisions including the rationale . reviews are referenced in the literature , but not as primary documents for medical technology evaluations ; thus do not directly influence policyrequires a subscription to access the database , though reviews are often available for downloadtable 2selection of technology assessment entitiescenterwebsiteservicescommentsagency for healthcare research and quality ( ahrq)http://www.ahrq.govfocus on evidence , high standards , and technology s impact on health outcomes . stated goal is to educate the public , but assessments are often highly technicalhayeshttp://www.hayesinc.comindependent , professional reviews ; consultation about specific health technology topicsmust subscribe in order to read assessmentsinstitute of clinical systems improvement ( icsi)http://www.icsi.orgindependent non - profit organization funded by all 6 of the health payers in minnesotafocus on health care services for people who live and work in the state of minnesota and in adjacent areas of surrounding statesecri institutehttp://www.ecri.org / pages / default.aspxnon - profit health research services agency that also operates an international medical device problem reporting system and a journal , health devices . ecri does not serve as an alerting service , but weekly alerts of medical device hazards and recalls are available through membershiphttp://www.mdsr.ecri.org / information / about.aspxtable 3selection of professional societiescenterweb siteservicescommentsamerican society for bariatric surgery ( asbs)http://www.asbs.org / specialist panels of individuals with experience in a particular field are asked to weigh in on the approval of a new device in their field . members and specialist sites also provide information about post - approval application of new devicesfinancial conflicts of interest and/or other professional bias must be taken into consideration , as such conflicts could reduce objectivityamerican college of cardiology ( acc)http://www.acc.org / american rhinologic society ( ars)http://www.american - rhinologic.org / index.phtmlamerican society for therapeutic radiology and oncology ( astro)http://www.astro.org / american college of physicians ( acp)http://www.acponline.orgamerican academy of family physicians ( aafp)http://www.aafp.orgamerican academy of pediatrics ( aap)http://www.aap.orgamerican osteopathic association ( aoa)http://www.do - online.orgamerican thoracic society ( ats)http://www.thoracic.org major evidence - based sources of information on medical technology selection of technology assessment entities selection of professional societies private payers rely on a mix of both proprietary and publicly available technology assessments for their policy development ; some have their own proprietary technology assessment process or purchase proprietary technology assessments produced by independent , often for - profit , companies . payers typically convene a committee to consider either privately or publicly the findings of an assessment ; consumers , specialists , and practicing physicians may serve as contributors to the assessment itself , as members of an assessment committee , or as majority or minority voting members on a technology assessment . this challenge was officially echoed in a position statement by the american rhinologic society ( ars ) , claiming that in spite of fda approval , ... the scientific literature has no data on the technology for long - term safety , indications , efficacy and outcomes.4547 what the ars did not say is that some of their members might experience a decline in income if the balloon dilation procedure were established as the standard of care over surgery . the embrace of imrt may have been in part driven by its favorable reimbursement compared to that of standard therapies for prostate cancer.48 the safety and effectiveness of medical devices are not yet comprehensively evaluated by the fda , other federal entities , payers , or professional societies . technology assessments by objective , independent , not - for - profit entities are necessary so that deficiencies in the clinical evidence and potential threats to patient safety are recognized in a manner that counterbalances market forces driving diffusion and demand . the california technology assessment forum ( ctaf ) is an independent , nonprofit entity whose mission is to provide transparent , objective reviews of new medical devices to better educate the public , clinicians , and policymakers ( see http://www.ctaf.org ) . potential topics may be brought to the attention of the committee by all potential stakeholders : the health plan , industry , professional societies , consumer groups , or ctaf staff . three times per year , ctaf invites manufacturers , patients , clinicians , and payers to openly debate the safety and effectiveness of 4 to 5 new devices ; these discussions typically include input ranging from anecdotal patient experience to expert testimony from the principal investigators of the pivotal randomized controlled trials . ctaf reviews are systematic analyses of the peer - reviewed literature that synthesize the results of all published clinical trials , case series , and randomized clinical trials , with an emphasis on comparative trials . the medline database , cochrane clinical trials database , cochrane reviews database , and the database of abstracts of reviews of effects ( dare ) are searched using relevant key words from 1966 up to the present . , cms ) , health care organizations , academic centers , and the community are also invited to attend and participate in the discussion . , level 1 , randomized , controlled trials powered to demonstrate clinically significant outcomes ; level 5 , case series without controlsta 3the technology must improve net health outcomesdiagnostic tests must change management in ways that benefit patientsta 4the technology must be as beneficial as any established alternativesit remains unclear how to evaluate diagnostic / prognostic tests lacking a gold - standard referenceta 5the improvement must be attainable outside the investigational settingby definition , trials are conducted within an investigational setting , and therefore this criterion implies a speculation about potential , and not actually studied , applications california technology assessment forum assessment criteria the voting panel includes experts from a variety of disciplines : ethicists , consumer advocates , practicing clinicians , methodologists , and others . the consultants present a brief overview of the peer - reviewed literature to the panel and then recommend that the technology either meets or does not meet the 5 criteria for approval . tec assessments are available to the public at their website and are distributed to all of the blues plans as a common starting point for medical policy coverage development . payers typically convene a committee to consider either privately or publicly the findings of an assessment ; consumers , specialists , and practicing physicians may serve as contributors to the assessment itself , as members of an assessment committee , or as majority or minority voting members on a technology assessment . this challenge was officially echoed in a position statement by the american rhinologic society ( ars ) , claiming that in spite of fda approval , ... the scientific literature has no data on the technology for long - term safety , indications , efficacy and outcomes.4547 what the ars did not say is that some of their members might experience a decline in income if the balloon dilation procedure were established as the standard of care over surgery . the embrace of imrt may have been in part driven by its favorable reimbursement compared to that of standard therapies for prostate cancer.48 the safety and effectiveness of medical devices are not yet comprehensively evaluated by the fda , other federal entities , payers , or professional societies . the california technology assessment forum ( ctaf ) is an independent , nonprofit entity whose mission is to provide transparent , objective reviews of new medical devices to better educate the public , clinicians , and policymakers ( see http://www.ctaf.org ) . potential topics may be brought to the attention of the committee by all potential stakeholders : the health plan , industry , professional societies , consumer groups , or ctaf staff . three times per year , ctaf invites manufacturers , patients , clinicians , and payers to openly debate the safety and effectiveness of 4 to 5 new devices ; these discussions typically include input ranging from anecdotal patient experience to expert testimony from the principal investigators of the pivotal randomized controlled trials . ctaf reviews are systematic analyses of the peer - reviewed literature that synthesize the results of all published clinical trials , case series , and randomized clinical trials , with an emphasis on comparative trials . the technology assessment should specify critical patient - centered outcomes , as well as identify which data are available that will ideally evaluate these outcomes , and where there is insufficient data . table 4california technology assessment forum assessment criterianumberdescriptioncommentsta 1the technology must have final approval from the appropriate government regulatory bodiesapproval via 510(k ) process is streamlined for new devices that are similar to older devicesta 2the scientific evidence must permit conclusions concerning the effectiveness of the technology regarding health outcomesrange : levels 15 , e.g. , level 1 , randomized , controlled trials powered to demonstrate clinically significant outcomes ; level 5 , case series without controlsta 3the technology must improve net health outcomesdiagnostic tests must change management in ways that benefit patientsta 4the technology must be as beneficial as any established alternativesit remains unclear how to evaluate diagnostic / prognostic tests lacking a gold - standard referenceta 5the improvement must be attainable outside the investigational settingby definition , trials are conducted within an investigational setting , and therefore this criterion implies a speculation about potential , and not actually studied , applications california technology assessment forum assessment criteria the voting panel includes experts from a variety of disciplines : ethicists , consumer advocates , practicing clinicians , methodologists , and others . the consultants present a brief overview of the peer - reviewed literature to the panel and then recommend that the technology either meets or does not meet the 5 criteria for approval . the ties to industry funding also vary among the assessment groups listed in tables 1 , 2 , and 3 , and in some cases , assessments are provided on a for - profit basis . as a result of the proliferation , complexity , and magnitude of impact of new and emerging medical devices , there is an urgent need to monitor new technology ever more closely in terms of effectiveness and safety . in spite of this need , significant gaps persist in the scientific literature , in the medical device approval process , and in the realm of postmarketing surveillance . while most pharmaceuticals obtain fda approval only after analysis of randomized clinical trials , relatively few new medical devices are subject to comparable scrutiny . to improve health outcomes , we must enhance our understanding of new medical devices and , by not simply deferring to the fda , support independent , evidence - based technology assessments . in spite of this limitation , technology assessments by independent organizations after fda approval can help identify those medical technologies that are truly beneficial and safe .

evolutionary processes , such as genetic drift and natural selection , can cause organisms developmental processes to evolve . the evolution of such processes can promote morphological and physiological diversity , as well as help an organism adapt to new environmental challenges and ecological niches ( hall , 1999 ) . to understand in detail how developmental processes evolve , we first must identify the selection pressures and molecular mechanisms that elicit these changes e.g . this multidisciplinary approach is difficult to achieve for most organisms because the environmental conditions that originally led to their developmental changes and thereby afforded specific selection advantages are often ambiguous . a few recent studies have begun to reveal the advantages associated with developmental traits , including a feeding advantage for animals with certain jaw morphs or different tooth shapes that depend on an animals ' food sources ( wainwright et al . , 1995 ; albertson et al . , 2001 ; kocher , 2004 ; alfaro et al . , 2005 ; laffont et al . , 2009 ; parsons et al . , 2009 ) ; increased mechanosensors to compensate for animals with poor visual cues ( yoshizawa et al . , 2010 ) ; as well as conspicuous ( hert , 1989 ; couldridge , 2002 ) and cryptic pigmentation patterns ( klingenberg , 2010 ; linnen et al . , 2013 ) . yet , these scientific accomplishments also emphasize the vast knowledge gap regarding how animals evolve adaptive traits as their genomes and developmental processes shift . if we can identify an animal that has evolved under clear and simple selection pressures , however , it will be possible to more directly test the advantages of the developmental changes they have evolved to undergo . the cave ecosystem is characterized by perpetual darkness , the absence of primary productivity , and sparse food resources ( culver , 1982 ; culver et al . , 2009 ) . these conditions exert significant pressure on cavedwelling animals such that caveadapted animals from most major phyla exhibit a remarkable convergence in morphological and physiological changes related to cave life , including features that are both constructive ( longer appendages , novel behaviors , elaborate nonvisual sensory systems ) and regressive ( reduced vision and pigmentation ) ( culver , 1982 ; culver et al . , 2009 ) . some aquatic cave populations , including teleost , have been isolated from their surfacedwelling relatives for such a long time that they have accumulated cavespecific mutations ( for examples , see protas et al . , 2006 ; gross et al . , 2009 ; elipot et al . , 2014 ) . in contrast , it is relatively difficult to identify the original mutations and selection pressures influencing surfacedwelling animals because they frequently hybridize with other populations and live in complex fauna . in this respect , cave animals are valuable models that we can use to analyze adaptive changes that occur under clearly defined selection pressures . behavior is a convenient window through which we can observe the selective advantages associated with morphological and physiological traits . for example , better sensitivity of a sensory organ can improve behavioral responses in predator avoidance , prey hunting , or mating , which lead to enhanced survivorship or fecundity . natural selection then screens animals with the best combination of traits , depending on their fitness ( mayr , 1963 ; bateson , 1988 ; wcislo , 1989 ; westeberhard , 1989 ; gittleman et al . , 1996 ; wimberger et al . , 1996 this review discusses recent advances in our understanding of behavioral evolution and related morphological and physiological traits in an animal that lives in a habitat with clear selection pressures : the mexican cavefish , astyanax mexicanus , an established animal model for studying evolution and development ( mitchell et al . , 1977 ; wilkens , 1988 ; jeffery , 2001 , 2008 , 2009 ; protas et al . 2013 ) . within the past few million years , at least five independent colonizations by two different migrating waves of eyedsurfacefish ancestors have established 29 geographically isolated astyanax cavefish populations in northeastern mexico ( ornelasgarca et al . , 2008 ; bradic et al . , 2012 , 2013 ; strecker et al . , 2012 ; coghill et al . , 2014 after an initial radiation underground , the founder cavefish populations became isolated and evolved independently . food sources are limited in these caves , and likely consist of small soil crustaceans , microorganisms , and organic matter present in the water that drops from the cave ceiling ( culver et al . , 2009 ) . depending on the cave , additional nutrient sources for cavefish may come from the guano dropped by bats living at the cave ceiling and organic matter brought in by seasonal flooding ( culver et al . when the habitat of these fish changed from the surface to the cave , the cavefish ancestor evolved regressed eyes , reduced pigmentation or albinism , enhanced oralpharyngeal morphologies , and expanded nervous systems ( wilkens , 1988 ; yamamoto et al . , 2000 , 2004 , 2009 ; jeffery , 2005 ; protas et al . , 2006 ; alunni et al . , 2007 ; franzodendaal et al . , 2007 ; 2013 ) . despite their 26 million year separation from a. mexicanus surface cohorts , the cavefish and surface fish cohorts remain interfertile , allowing us to study the evolution of behavioral , morphological and physiological traits by genetic analysis . recent work is revealing the genetic architectures that underlie the behavioral evolution of cavefish species , including the examination of the following behaviors : vibrationattraction behavior ( vab ) ( eigenmann , 1909 ; hill , 1969 ; parzefall , 1983 ; yoshizawa et al . , 2010 , 2012a , 2012b ) ; sleep loss ( dubou et al . , 2011 , 2012 ) ; reduced aggression ( parzefall , 2001 ; elipot et al . , 2013 ) ; loss of schooling and shoaling ( parzefall , 2001 ; kowalko et al . , 2013a ) ; stabilized feeding posture ( schemmel , 1980 ; kowalko et al . , 2013b ) ; enhanced chemosensory ability ( protas et al . , 2008 ; bibliowicz et al . , , 2011 ; dubou et al . , 2012 , 2011 ; beale et al . , 2013 ) . among these behaviors , vab is the moststudied , positively selected behavior regarding how it benefits cavefish in their environment , its sensory and genetic basis , and its developmental process . here , i review the behaviors that have been evolutionarily advantageous and most informative in deciphering how a. mexicanus populations have adapted to their caves . cavefish exhibit vab ; that is , they swim toward an oscillating object , either in a natural cave pool or in a laboratory ( fig . vab represents a potential foraging behavior that has evolved repeatedly in at least three different astyanax cavefish populations ( fig . 1b ) ( parzefall , 1983 ; abdellatif et al . , 1990 ; yoshizawa et al . , 2010 ) . considering these populations likely evolved separately under similar ecological conditions ( mitchell et al . , 1977 ; borowsky , 2008 ; ornelasgarca et al . , 2008 ; wilkens , 2010 ; bradic et al . , 2012 ; gross , 2012 ; strecker et al . , 2012 ; coghill et al . , 2014 ) , the convergence of these populations towards vab suggests the distinct advantage this behavior would have in the cave environment . yet , the extent of vab is variable within and among populations , and some individuals of one of the oldest cavefish populations , pachn , even lack this behavior ( fig . the converse is true for surface fish : while most surface fish lack vab , a few individuals exhibit low levels of this behavior ( fig . ( a ) swimming path ( purple lines ) of surface fish ( left ) and pachon cavefish ( right ) during a 3min assay period . dotted lines represent the 2cm diameter quantification area surrounding the glass rod ( dark spot in the center of the chamber ) . three separated cave populations showed vab ( above the threshold level of 4 approaches ) , but no vab was apparent in either surfacefish population . onetailed mannwhitney tests with bonferroni correction for multiple comparisons were performed between a group of ro choy and ro tampan surface fish , and each cavefish population . * , p < 0.05 ; * * , p < 0.01 . ro choy surface fish , n = 4 ; ro tampan surface fish , n = 13 ; pachn cavefish , n = 8 ; los sabinos cavefish , n = 9 ; and piedras cavefish , n = 4 . morphological / physiological bases and ontogeny of cavefish behaviors sf , surface fish ; cf , cavefish ; mpf , months post fertilization ; dpf , days post fertilization ; ? n = 54 ( gray area ) ; cavefish , n = 52 ( orange area ) . vertical dashed line represents the cutoff value of 4 approaches used to classifying fish with ( > 4 approaches ) and without ( < 4 approaches ) vab using a stimulus of 50 hz . bars show the proportion of strikes at prey between pairs of surface fish ( black fish cartoons ) and cavefish ( orange fish cartoons ) with or without vab during a 1min assay period in darkness ( left bars ) and in light ( right bars ) . a total of eight pairs of cavefish versus surface fish ( ba ) , and five pairs of surface fish with versus without vab ( bb ) in the dark and light are shown . values are mean ratio of strikes 95% confidence intervals of the mean . * , p < 0.05 ; * * p < 0.01 . for details about the method , please see yoshizawa et al . taking advantage of this variation within populations , the adaptive significance of vab was tested in competitive preycapture experiments wherein pairs of fish with and without vab were fed small amount of vibrating prey : brine shrimp ( fig . 2b ) . in darkness , cavefish were significantly better at capturing the brine shrimp than surface fish ( fig . the key finding , however , was that surface fish with vab had significantly more brineshrimp captures in the dark than surface fish without vab , a difference that disappeared in the light ( fig . thus , an individual 's ability to utilize vab plays a role in foraging and is likely advantageous for survival in dark caves . in wild populations of surface fish , vab is presumably deleterious because fish with vab may swim toward predators , such as the nocturnal prawn , at night ( wilson et al . , 2004 in contrast , vab is adaptive in cavefish because it increases foraging in an environment with limited food availability , minimal light , and macroscopic predators ( yoshizawa et al . , 2010 , 2011 ) . consequently , the vab already present as a standing variation in ancestral surface populations may have been subject to positive selection once the cavefish ancestors started colonizing caves . probing a little deeper reveals hints at how these cave populations have honed vab to survive . cavefish vab occurs at a relatively low frequency range ( about 550 hz ) , with a peak frequency of 35 hz ( yoshizawa et al . , these values are within the range that cavefish can detect with their mechanosensory lateral line ( 2080 hz ) ( coombs et al . interestingly , none of the surface fish , even those with vab , displayed such behavioral tuning ( yoshizawa et al . furthermore , because many crustaceans produce water fluctuations at 3040 hz while swimming ( lang , 1980 ) , the tuning of cavefish 's vab at 35 hz is a novel trait that is likely adaptive in the cave ecosystem . the cavefish 's optimized frequency detection system suggests that the lateralline sensory organ that cavefish use to detect water flow is likely a vibrationsensory receptor that has been enhanced in cavefish compared to surface fish ( schemmel , 1967 ; mnz , 1989 ) . one type of lateralline sensory organ is the superficial neuromast ( sn ) , which is composed of ciliated hair cells and a gelatinous cupular matrix ( fig . the sns of cavefish are larger and more numerous than those of surface fish , and are primarily responsible for vab , as determined by sn ablation studies and genetic analysis ( schemmel , 1967 ; teyke , 1990 ; yoshizawa et al . , 2010 ) . although sns appear throughout the body and are particularly abundant on the cavefish 's head , those sns located within the orbit of the cavefish 's degenerated eye seem to play a particularly important role in vab ( yoshizawa et al . , 2012b ) . this suggests that the extra cranial space created with the loss of the cavefish 's eyes is an important event that promoted this novel behavior . as there was no difference in the number of eyeorbit sns ( eo sns ) among surface fish in fact , no eo sn was observed in surface fish their appearance in cavefish did not arise through the selection of a standing phenotypic variation ( yoshizawa et al . , 2012b ) ; instead , there must be a distinct evolutionary path linking vab and eo sn evolution . surface fish , n = 10 at 2 months postfertilization ( mpf ) , = 9 at 3 mpf , = 10 at 4 mpf , and = 12 at 1yearold ; pachn cavefish , n = 9 at 2 mpf , = 8 at 3 mpf , = 9 at 4 mpf , and = 11 at 1yearold ; tinaja cavefish , n = 7 at 1yearold . pachn cavefish significantly increased vab and eo sn quantity , whereas tinaja cavefish were indistinguishable from surface fish in both vab level and eo sn number at 1 year old ( oneway anova followed by plannedcontrast analysis at 1 year old ) . * * , p < 0.01 ; * * * , p < 0.001 ; n.s . , not significant . ( c ) example fluorescence images of daspeivital staining of surface fish and two cavefish . thick , white dotted lines indicate the infraorbital canal , and white arrowheads indicate eo sns that were counted in ( b ) . thin , white dotted line encloses the neuromasts in an adjacent region on the cranium , the third infraorbital bone ( previously denoted as the third suborbital bone , so3 ) ( yoshizawa et al . cavefish eyes start degenerating around 36 hr postfertilization , but no significant increases in any sns ( including eo sns ) are detectible , relative to surface fish , until 2 months postfertilization ( yoshizawa et al . , 2010 ) . many aspects of eye degeneration are controlled by increased hedgehog signaling in cavefish ( yamamoto et al . , 2004 ; jeffery , 2005 ) , so a surface fish was engineered to overexpress shh by mrna injection so that its eyes would also degenerate to test the relationship between eye retention and eo sn development ; these shhoverexpressing fish did not develop eo sn ( yoshizawa et al . , 2012b ) . therefore , neither eye degeneration alone nor excess hedgehog signaling in early development induces eo sn formation or enlargement ; note that tests that ask if hedgehog signaling plays a role later in development , when eo sns first appear at 2 months old , have not yet been conducted . although there is no shared developmental process between eye degeneration and eo sn formation , ontogenic analysis and a comparison using different cave populations could help establish the relationship between vab and eo sn evolution . the first approach was to determine how the timing of vab onset correlates with the development of eo sns in surface fish versus pachn cavefish by studying fish at 2 , 3 , and 4 months postfertilization and in the youngadult stage ( 1yearold ) . cavefish eo sn quantity gradually increases between 2 months and the first year of age ( fig 3 ) ; this temporally correlates with the appearance and enhancement of vab , which is first detectible between 2 and 3 months postfertilization ( fig . interestingly , another cavefish population , tinaja , presents with a weak level of vab , similar to the amount that some individual surface fish display , and does not have eo sns even though their eyes degenerate ( fig . this difference further supports the correlation between cavefish vab and eo sn evolution , and also reinforces that eo sns are not directly attributable to eye degeneration . while eo sn ontogeny corresponds with vab cavefish , there is no correlation between vab and the total number of sns in an individual . while pachn and tinaja cavefish have significantly more sns in their infraorbital region than surface fish do ( fig . 3c ) , the development of these organs is not genetically correlated with the level of vab , and their ablation does not detectably affect vab ( yoshizawa et al . , 2012b ) . these observations further emphasize the model that the eo sn , as a minor group of sensory organs , plays an important role in facilitating vab whereas other sns in the infraorbital region may contribute to other sensations , such as hydrodynamic imaging ( see below , and also hassan , 1989 ; montgomery et al . , 2001 ; windsor et al . , 2008a , 2010a ; coombs et al . , 2010 ) . development of the eo sn may therefore have evolved as a consequence of positive selection for the enhancement of an adaptive behavior , indicating that studying behavioral traits can help reveal distinct roles for sns residing in different cranial areas . considering the importance of the eo sns in vab , and thus the cavefish 's ability to forage , it is surprising that vab and eo sns emerge so late in development . such timing may occur for two reasons : ( 1 ) the developmental process might be constrained or ( 2 ) their foraging preference might change with age . to address the first possibility , wada et al . the sns first form at the edge of a developing intramembranous bone in the cranial region ( the operculum , at 45 days postfertilization ) . in astyanax , however , the second and third infraorbital ( suborbital ) bones , which underlie the infraorbital sns and eo sns , develop in cavefish around 23 months postfertilization ( yamamoto et al . , 2003 ) . therefore , the number of sns in cavefish may only be able to increase after the infraorbital bone forms , meaning that the development of eo sn and thereby vab is developmentally constrained until the infraorbital bone forms at 23 months postfertilization . the second reason for the late development of vab and eo sn that cavefish foraging preferences change over time derives from the hypothesis that cavefish larvae and adults may have different foraging preferences , possibly to reduce competition with one another . cavefish adults quickly respond and move toward a water surface that is disturbed by falling water droplets or bat guano ( likely via vab ) , but they also scavenge for food , perhaps relying on chemosensory inputs originally adapted for bottom feeding . in contrast , relatively small , younger fish scavenge for food exclusively at the bottom , again possibly using chemosensation ( parzefall , 1983 ; personal and s. rtaux 's observation at the cave los sabinos ) . in the laboratory , however , large cavefish occasionally eat smaller cavefish , suggesting that small , younger fish can not compete with mature adults . therefore , the late development of eo sn and vab may offer small , young fish a better chance to survive by avoiding the hazards of larger , hungry conspecifics that are using vab to forage just beneath the water 's surface . while it is difficult to determine which of these possibilities developmental constraints or an ecological advantage is responsible for the late development of vab and eo sns , genetic experiments have helped . by overexpressing or knocking down the gene(s ) responsible for eo sn development using available transgenic and/or genome editing methods ( e.g. , talen and crispr technologies ) ( gaj et al . , 2013 ; 2014 ; ma et al . , 2015 ) , and performing generegulatorynetwork analysis ( gavinsmyth et al . , 2013 ) , we will gain insight to the relationship between eo sn development and the timing of dermalbone formation . deciphering which gene(s ) are responsible for vab will through in situ hybridization and/or immunohistochemistry techniques help reveal if the novel sensory tuning occurs at the level of first projection , the octavolateralis nucleus , or higher , such as at the torus semicircularis area . it is unlikely that sensory tuning is achieved at the neuromast level , however , since the sensitivity estimation based on the morphometrics of neuromasts does not peak at 35 hz ( yoshizawa et al . nevertheless , the foraging advantage associated with vab gives us a new way to resolve the driving forces for the evolution of other morphological traits , including cranial sn , intramembranous bone , and also neural connections in the central nervous system . the pleiotropic effects of enhanced hedgehog signaling promote eye degeneration as well as increases in jaw size and number of taste buds in astyanax cavefish ( table 1 ) ( yamamoto et al . , 2009 , 2004 ; jeffery , 2005 ) . given the necessary redistribution of attention to the nonvisual senses , selection for these other constructive traits , which are fostered by hedgehog signaling , during cavefish evolution no one has tested if these gustatory traits ( larger jaws , more taste buds ) improve cavefish 's foraging abilities . nevertheless , an enlarged jaw is likely beneficial in the darkness , possibly as an adaptation to bottom feeding , and carrying more taste buds likely improves foraging because they could help navigate to food in the dark . to understand the adaptive significance of these morphological and sensory shifts , however , behavioral analyses showing that these gustatory traits are advantageous would support the hypothesis that oralpharyngeal shifts can promote eye reduction through pleiotropy . if having a large jaw and more taste buds does not enhance foraging efficiency , however , these traits would constitute another example of not all evolved traits are adaptive hedgehog signaling is also required for other critical developmental processes , beyond cavefish eye degeneration and a few gustatory traits . during development , enhanced hedgehog signaling increases the number of migratory cells that enter the olfactory bulbs ( menuet et al . , 2007 ) and serotonergic neurons at the hypothalamus ( elipot , 2013 ) . while the former may represent a positive behavioral response linked to the chemical stimulus of food ( table 1 ) ( bibliowicz et al . , 2013 ) , the latter is an example of a change in behavioral strategy . for instance , having an enhanced serotonergic system in the anterior hypothalamus and the raphe nucleus redirects attacking behavior towards foraging behavior . attacking behavior is frequently used to establish a hierarchical relationship between individuals in surface dwelling fish ( magurran , 1993 ; elipot et al . , 2013 ) , but this does not occur in cave populations , possibly because a. mexicanus cavefish lack schooling behavior ( elipot et al . , 2013 ; kowalko et al . , 2013b ; see below ) . indeed , cavefish have higher serotonin levels and more serotonergic neurons at the hypothalamic anterior paraventricular nucleus and hindbrain raphe than their surface counterparts ; these anatomical and endocrine changes are believed to have shifted cavefish behavior from attacking to foraging ( elipot et al . , 2013 ) . another study reported that cavefish have higher serotonin levels in the brain because of mutations in the serotonin degradation enzyme , monoamine oxidase ( mao)which again promotes foraging behavior ( table 1 ) ( elipot et al . , 2014 ) . the cavefish 's heightened foraging is likely adaptive to the cave environment , where there is little food and few predators : fish eat on a first come , first served basis . in fact , enhanced foraging behavior has converged in independentlyevolved cave populations ( elipot et al . yet even though cavefish have higher numbers of serotonergic neurons in their hypothalamus by 1 week of age ( elipot et al . , 2013 ) , there is no obvious difference in attacking behavior between cavefish and surface fish at that age ( personal observation ) . this may be because fish at this young age are too immature to establish hierarchical positions , or because some unknown benefit exists for having an enhanced serotonergic system in the larval stage . overall , if we could better understand the difference in the aggression neural circuits between cavefish and surface fish , and their relationship with the number of serotoninergic cells in 1weekold juveniles , we would gain muchneeded insight to the evolution of cavefish 's foraging behavior . in general , this behavioral shift in cavefish from attacking to foraging provides the first example of a likely behavioral advantage underlying their enhanced hedgehog signaling , whose tradeoff is eye development ( c.f . many behaviors have evolved in the company of morphological changes , yet behavior can also evolve without obvious anatomical adaptations e.g . , feeding posture . when in the dark , surface fish feed at a steep angle of 90 relative to a substrate . in contrast , multiple cave populations feed at a much shallower angle of 45 ( table 1 ) ( schemmel , 1980 ) . although no one has yet determined how lowangle feeding is advantageous in the dark ( though this could be demonstrated in a competition assay ) , three a. mexicanus cave populations ( pachn , tinaja , and los sabinos ) ( kowalko et al . , 2013b and personal observation ) and other benthic feeders ( for example , see ferrygraham et al . , 2002 ) all perform lowangle feeding , suggesting that this behavior may be advantageous for foraging at the bottom of caves . since jaw morphology is frequently associated with feeding ( wainwright et al . , 1995 ; albertson et al . , 2001 ; kocher , 2004 ; alfaro et al . , 2005 ; parsons et al . , 2009 ) , it was surprising when a set of studies revealed no detectable correlation between feeding angle and eight craniofacial phenotypes in an f2 intercross derived from a surface fish mated to a cavefish ( kowalko et al . the genetics therefore suggests that a cavefish 's low feeding angle represents an exclusively behavioral adaptation potentially through changes in motor control that required no accompanying morphological changes . the convergent evolution of a cavefish 's feeding posture has also been supported by genomic evidence ( kowalko et al . , 2013a ) . using quantitative trait loci ( qtl ) mapping of feeding posture ( a potentially adaptive behavior ) and eye degeneration in two independently evolved cavefish populations , kowalko et al reported distinct qtl results for different genes in different populations , suggesting that many cavefish traits evolved by de novo mutation rather than by selection from standing genetic variation . at the singlefeedingangle qtl detected in one cavefish population ( tinaja ) , the cave allele actually worked to increase the feeding angle , which was unexpected and indicates there are still undetected qtls ( table 1 ) ( kowalko et al . , 2013b ) . importantly , a different cave population ( pachn ) showed two feedingposture qtls , and at both of them the cave alleles worked to reduce the feeding angle , suggesting that different genes were utilized during the evolution of feeding behavior among independent cave populations ( kowalko et al . , 2013a ) . surface fish readily stabilize their feeding angle at a 45 angle in lighted conditions , so cavefish may have evolved their posture control without visual cues , instead relying on or modifying the vestibular posture control centers . this is supported by observations at the larval stage , when cavefish and surface fish feed similarly . instead of feeding at either 45 or 90 , both larval types bob along the bottom of the tank and attack sunken food , trying out different feeding angles during each attempt . since this larval feeding style is somewhat similar to adult surface fish , it would be interesting to investigate the ontogeny of neural wiring in the basal ganglia , cerebellum , pontine , and vestibular systems which together control posture and how this wiring is associated with feeding angle in cavefish . in addition , future competition assays should test if a lower feeding angle is advantageous in the dark , which will provide insight into the selection pressure for this behavior . cavefish exhibit vab ; that is , they swim toward an oscillating object , either in a natural cave pool or in a laboratory ( fig . vab represents a potential foraging behavior that has evolved repeatedly in at least three different astyanax cavefish populations ( fig . 1b ) ( parzefall , 1983 ; abdellatif et al . , 1990 ; yoshizawa et al . , 2010 ) . considering these populations likely evolved separately under similar ecological conditions ( mitchell et al . , 1977 ; borowsky , 2008 ; ornelasgarca et al . , 2008 ; wilkens , 2010 ; bradic et al . , 2012 ; gross , 2012 ; strecker et al . , 2012 ; coghill et al . , 2014 ) , the convergence of these populations towards vab suggests the distinct advantage this behavior would have in the cave environment . yet , the extent of vab is variable within and among populations , and some individuals of one of the oldest cavefish populations , pachn , even lack this behavior ( fig . the converse is true for surface fish : while most surface fish lack vab , a few individuals exhibit low levels of this behavior ( fig . ( a ) swimming path ( purple lines ) of surface fish ( left ) and pachon cavefish ( right ) during a 3min assay period . dotted lines represent the 2cm diameter quantification area surrounding the glass rod ( dark spot in the center of the chamber ) . three separated cave populations showed vab ( above the threshold level of 4 approaches ) , but no vab was apparent in either surfacefish population . onetailed mannwhitney tests with bonferroni correction for multiple comparisons were performed between a group of ro choy and ro tampan surface fish , and each cavefish population . * , p < 0.05 ; * * , p < 0.01 . ro choy surface fish , n = 4 ; ro tampan surface fish , n = 13 ; pachn cavefish , n = 8 ; los sabinos cavefish , n = 9 ; and piedras cavefish , n = 4 . morphological / physiological bases and ontogeny of cavefish behaviors sf , surface fish ; cf , cavefish ; mpf , months post fertilization ; dpf , days post fertilization ; ? n = 54 ( gray area ) ; cavefish , n = 52 ( orange area ) . vertical dashed line represents the cutoff value of 4 approaches used to classifying fish with ( > 4 approaches ) and without ( < 4 approaches ) vab using a stimulus of 50 hz . bars show the proportion of strikes at prey between pairs of surface fish ( black fish cartoons ) and cavefish ( orange fish cartoons ) with or without vab during a 1min assay period in darkness ( left bars ) and in light ( right bars ) . a total of eight pairs of cavefish versus surface fish ( ba ) , and five pairs of surface fish with versus without vab ( bb ) in the dark and light are shown . values are mean ratio of strikes 95% confidence intervals of the mean . * , p < 0.05 ; * * p < 0.01 . for details about the method , please see yoshizawa et al . taking advantage of this variation within populations , the adaptive significance of vab was tested in competitive preycapture experiments wherein pairs of fish with and without vab were fed small amount of vibrating prey : brine shrimp ( fig . 2b ) . in darkness , cavefish were significantly better at capturing the brine shrimp than surface fish ( fig . the key finding , however , was that surface fish with vab had significantly more brineshrimp captures in the dark than surface fish without vab , a difference that disappeared in the light ( fig . thus , an individual 's ability to utilize vab plays a role in foraging and is likely advantageous for survival in dark caves . in wild populations of surface fish , vab is presumably deleterious because fish with vab may swim toward predators , such as the nocturnal prawn , at night ( wilson et al . , 2004 in contrast , vab is adaptive in cavefish because it increases foraging in an environment with limited food availability , minimal light , and macroscopic predators ( yoshizawa et al . , 2010 , 2011 ) . consequently , the vab already present as a standing variation in ancestral surface populations may have been subject to positive selection once the cavefish ancestors started colonizing caves . probing a little deeper reveals hints at how these cave populations have honed vab to survive . cavefish vab occurs at a relatively low frequency range ( about 550 hz ) , with a peak frequency of 35 hz ( yoshizawa et al . , these values are within the range that cavefish can detect with their mechanosensory lateral line ( 2080 hz ) ( coombs et al . interestingly , none of the surface fish , even those with vab , displayed such behavioral tuning ( yoshizawa et al . furthermore , because many crustaceans produce water fluctuations at 3040 hz while swimming ( lang , 1980 ) , the tuning of cavefish 's vab at 35 hz is a novel trait that is likely adaptive in the cave ecosystem . the cavefish 's optimized frequency detection system suggests that the lateralline sensory organ that cavefish use to detect water flow is likely a vibrationsensory receptor that has been enhanced in cavefish compared to surface fish ( schemmel , 1967 ; mnz , 1989 ) . one type of lateralline sensory organ is the superficial neuromast ( sn ) , which is composed of ciliated hair cells and a gelatinous cupular matrix ( fig . the sns of cavefish are larger and more numerous than those of surface fish , and are primarily responsible for vab , as determined by sn ablation studies and genetic analysis ( schemmel , 1967 ; teyke , 1990 ; yoshizawa et al . , 2010 ) . although sns appear throughout the body and are particularly abundant on the cavefish 's head , those sns located within the orbit of the cavefish 's degenerated eye seem to play a particularly important role in vab ( yoshizawa et al . , 2012b ) . this suggests that the extra cranial space created with the loss of the cavefish 's eyes is an important event that promoted this novel behavior . as there was no difference in the number of eyeorbit sns ( eo sns ) among surface fish in fact , no eo sn was observed in surface fish their appearance in cavefish did not arise through the selection of a standing phenotypic variation ( yoshizawa et al . , 2012b ) ; instead , there must be a distinct evolutionary path linking vab and eo sn evolution . surface fish , n = 10 at 2 months postfertilization ( mpf ) , = 9 at 3 mpf , = 10 at 4 mpf , and = 12 at 1yearold ; pachn cavefish , n = 9 at 2 mpf , = 8 at 3 mpf , = 9 at 4 mpf , and = 11 at 1yearold ; tinaja cavefish , n = 7 at 1yearold . pachn cavefish significantly increased vab and eo sn quantity , whereas tinaja cavefish were indistinguishable from surface fish in both vab level and eo sn number at 1 year old ( oneway anova followed by plannedcontrast analysis at 1 year old ) . * * , p < 0.01 ; * * * , p < 0.001 ; n.s . , not significant . ( c ) example fluorescence images of daspeivital staining of surface fish and two cavefish . thick , white dotted lines indicate the infraorbital canal , and white arrowheads indicate eo sns that were counted in ( b ) . thin , white dotted line encloses the neuromasts in an adjacent region on the cranium , the third infraorbital bone ( previously denoted as the third suborbital bone , so3 ) ( yoshizawa et al . cavefish eyes start degenerating around 36 hr postfertilization , but no significant increases in any sns ( including eo sns ) are detectible , relative to surface fish , until 2 months postfertilization ( yoshizawa et al . , 2010 ) . many aspects of eye degeneration are controlled by increased hedgehog signaling in cavefish ( yamamoto et al . , 2004 ; jeffery , 2005 ) , so a surface fish was engineered to overexpress shh by mrna injection so that its eyes would also degenerate to test the relationship between eye retention and eo sn development ; these shhoverexpressing fish did not develop eo sn ( yoshizawa et al . , 2012b ) . therefore , neither eye degeneration alone nor excess hedgehog signaling in early development induces eo sn formation or enlargement ; note that tests that ask if hedgehog signaling plays a role later in development , when eo sns first appear at 2 months old , have not yet been conducted . although there is no shared developmental process between eye degeneration and eo sn formation , ontogenic analysis and a comparison using different cave populations could help establish the relationship between vab and eo sn evolution . the first approach was to determine how the timing of vab onset correlates with the development of eo sns in surface fish versus pachn cavefish by studying fish at 2 , 3 , and 4 months postfertilization and in the youngadult stage ( 1yearold ) . cavefish eo sn quantity gradually increases between 2 months and the first year of age ( fig 3 ) ; this temporally correlates with the appearance and enhancement of vab , which is first detectible between 2 and 3 months postfertilization ( fig . interestingly , another cavefish population , tinaja , presents with a weak level of vab , similar to the amount that some individual surface fish display , and does not have eo sns even though their eyes degenerate ( fig . this difference further supports the correlation between cavefish vab and eo sn evolution , and also reinforces that eo sns are not directly attributable to eye degeneration . while eo sn ontogeny corresponds with vab cavefish , there is no correlation between vab and the total number of sns in an individual . while pachn and tinaja cavefish have significantly more sns in their infraorbital region than surface fish do ( fig . 3c ) , the development of these organs is not genetically correlated with the level of vab , and their ablation does not detectably affect vab ( yoshizawa et al . , 2012b ) . these observations further emphasize the model that the eo sn , as a minor group of sensory organs , plays an important role in facilitating vab whereas other sns in the infraorbital region may contribute to other sensations , such as hydrodynamic imaging ( see below , and also hassan , 1989 ; montgomery et al . , 2001 ; windsor et al . , 2008a , 2010a ; coombs et al . , 2010 ) . development of the eo sn may therefore have evolved as a consequence of positive selection for the enhancement of an adaptive behavior , indicating that studying behavioral traits can help reveal distinct roles for sns residing in different cranial areas . considering the importance of the eo sns in vab , and thus the cavefish 's ability to forage , it is surprising that vab and eo sns emerge so late in development . such timing may occur for two reasons : ( 1 ) the developmental process might be constrained or ( 2 ) their foraging preference might change with age . to address the first possibility , wada et al . the sns first form at the edge of a developing intramembranous bone in the cranial region ( the operculum , at 45 days postfertilization ) . in astyanax , however , the second and third infraorbital ( suborbital ) bones , which underlie the infraorbital sns and eo sns , develop in cavefish around 23 months postfertilization ( yamamoto et al . , 2003 ) . therefore , the number of sns in cavefish may only be able to increase after the infraorbital bone forms , meaning that the development of eo sn and thereby vab is developmentally constrained until the infraorbital bone forms at 23 months postfertilization . the second reason for the late development of vab and eo sn that cavefish foraging preferences change over time derives from the hypothesis that cavefish larvae and adults may have different foraging preferences , possibly to reduce competition with one another . cavefish adults quickly respond and move toward a water surface that is disturbed by falling water droplets or bat guano ( likely via vab ) , but they also scavenge for food , perhaps relying on chemosensory inputs originally adapted for bottom feeding . in contrast , relatively small , younger fish scavenge for food exclusively at the bottom , again possibly using chemosensation ( parzefall , 1983 ; personal and s. rtaux 's observation at the cave los sabinos ) . in the laboratory , however , large cavefish occasionally eat smaller cavefish , suggesting that small , younger fish can not compete with mature adults . therefore , the late development of eo sn and vab may offer small , young fish a better chance to survive by avoiding the hazards of larger , hungry conspecifics that are using vab to forage just beneath the water 's surface . while it is difficult to determine which of these possibilities developmental constraints or an ecological advantage is responsible for the late development of vab and eo sns , genetic experiments have helped . by overexpressing or knocking down the gene(s ) responsible for eo sn development using available transgenic and/or genome editing methods ( e.g. , talen and crispr technologies ) ( gaj et al . , 2013 ; 2014 ; ma et al . , 2015 ) , and performing generegulatorynetwork analysis ( gavinsmyth et al . , 2013 ) , we will gain insight to the relationship between eo sn development and the timing of dermalbone formation . deciphering which gene(s ) are responsible for vab will through in situ hybridization and/or immunohistochemistry techniques help reveal if the novel sensory tuning occurs at the level of first projection , the octavolateralis nucleus , or higher , such as at the torus semicircularis area . it is unlikely that sensory tuning is achieved at the neuromast level , however , since the sensitivity estimation based on the morphometrics of neuromasts does not peak at 35 hz ( yoshizawa et al . nevertheless , the foraging advantage associated with vab gives us a new way to resolve the driving forces for the evolution of other morphological traits , including cranial sn , intramembranous bone , and also neural connections in the central nervous system . the pleiotropic effects of enhanced hedgehog signaling promote eye degeneration as well as increases in jaw size and number of taste buds in astyanax cavefish ( table 1 ) ( yamamoto et al . , 2009 , 2004 ; jeffery , 2005 ) . given the necessary redistribution of attention to the nonvisual senses , selection for these other constructive traits , which are fostered by hedgehog signaling , during cavefish evolution no one has tested if these gustatory traits ( larger jaws , more taste buds ) improve cavefish 's foraging abilities . nevertheless , an enlarged jaw is likely beneficial in the darkness , possibly as an adaptation to bottom feeding , and carrying more taste buds likely improves foraging because they could help navigate to food in the dark . to understand the adaptive significance of these morphological and sensory shifts , however for example , behavioral analyses showing that these gustatory traits are advantageous would support the hypothesis that oralpharyngeal shifts can promote eye reduction through pleiotropy . if having a large jaw and more taste buds does not enhance foraging efficiency , however , these traits would constitute another example of not all evolved traits are adaptive hedgehog signaling is also required for other critical developmental processes , beyond cavefish eye degeneration and a few gustatory traits . during development , enhanced hedgehog signaling increases the number of migratory cells that enter the olfactory bulbs ( menuet et al . , 2007 ) and serotonergic neurons at the hypothalamus ( elipot , 2013 ) . while the former may represent a positive behavioral response linked to the chemical stimulus of food ( table 1 ) ( bibliowicz et al . , 2013 ) , the latter is an example of a change in behavioral strategy . for instance , having an enhanced serotonergic system in the anterior hypothalamus and the raphe nucleus redirects attacking behavior towards foraging behavior . attacking behavior is frequently used to establish a hierarchical relationship between individuals in surface dwelling fish ( magurran , 1993 ; elipot et al . , 2013 ) , but this does not occur in cave populations , possibly because a. mexicanus cavefish lack schooling behavior ( elipot et al . , 2013 ; kowalko et al . , 2013b ; see below ) . indeed , cavefish have higher serotonin levels and more serotonergic neurons at the hypothalamic anterior paraventricular nucleus and hindbrain raphe than their surface counterparts ; these anatomical and endocrine changes are believed to have shifted cavefish behavior from attacking to foraging ( elipot et al . , 2013 ) . another study reported that cavefish have higher serotonin levels in the brain because of mutations in the serotonin degradation enzyme , monoamine oxidase ( mao)which again promotes foraging behavior ( table 1 ) ( elipot et al . , 2014 ) . the cavefish 's heightened foraging is likely adaptive to the cave environment , where there is little food and few predators : fish eat on a first come , first served basis . in fact , enhanced foraging behavior has converged in independentlyevolved cave populations ( elipot et al . , 2013 ) . yet even though cavefish have higher numbers of serotonergic neurons in their hypothalamus by 1 week of age ( elipot et al . , 2013 ) , there is no obvious difference in attacking behavior between cavefish and surface fish at that age ( personal observation ) . this may be because fish at this young age are too immature to establish hierarchical positions , or because some unknown benefit exists for having an enhanced serotonergic system in the larval stage . overall , if we could better understand the difference in the aggression neural circuits between cavefish and surface fish , and their relationship with the number of serotoninergic cells in 1weekold juveniles , we would gain muchneeded insight to the evolution of cavefish 's foraging behavior . in general , this behavioral shift in cavefish from attacking to foraging provides the first example of a likely behavioral advantage underlying their enhanced hedgehog signaling , whose tradeoff is eye development ( c.f . many behaviors have evolved in the company of morphological changes , yet behavior can also evolve without obvious anatomical adaptations e.g . , feeding posture . when in the dark , surface fish feed at a steep angle of 90 relative to a substrate . in contrast , multiple cave populations feed at a much shallower angle of 45 ( table 1 ) ( schemmel , 1980 ) . although no one has yet determined how lowangle feeding is advantageous in the dark ( though this could be demonstrated in a competition assay ) , three a. mexicanus cave populations ( pachn , tinaja , and los sabinos ) ( kowalko et al . , 2013b and personal observation ) and other benthic feeders ( for example , see ferrygraham et al . , 2002 ) all perform lowangle feeding , suggesting that this behavior may be advantageous for foraging at the bottom of caves . since jaw morphology is frequently associated with feeding ( wainwright et al . , 1995 ; albertson et al . , 2001 ; kocher , 2004 ; alfaro et al . , 2005 ; parsons et al . , 2009 ) , it was surprising when a set of studies revealed no detectable correlation between feeding angle and eight craniofacial phenotypes in an f2 intercross derived from a surface fish mated to a cavefish ( kowalko et al . , 2013b ) . the genetics therefore suggests that a cavefish 's low feeding angle represents an exclusively behavioral adaptation potentially through changes in motor control that required no accompanying morphological changes . the convergent evolution of a cavefish 's feeding posture has also been supported by genomic evidence ( kowalko et al . using quantitative trait loci ( qtl ) mapping of feeding posture ( a potentially adaptive behavior ) and eye degeneration in two independently evolved cavefish populations , kowalko et al reported distinct qtl results for different genes in different populations , suggesting that many cavefish traits evolved by de novo mutation rather than by selection from standing genetic variation . at the singlefeedingangle qtl detected in one cavefish population ( tinaja ) , the cave allele actually worked to increase the feeding angle , which was unexpected and indicates there are still undetected qtls ( table 1 ) ( kowalko et al . , 2013b ) . importantly , a different cave population ( pachn ) showed two feedingposture qtls , and at both of them the cave alleles worked to reduce the feeding angle , suggesting that different genes were utilized during the evolution of feeding behavior among independent cave populations ( kowalko et al . , 2013a ) . surface fish readily stabilize their feeding angle at a 45 angle in lighted conditions , so cavefish may have evolved their posture control without visual cues , instead relying on or modifying the vestibular posture control centers . this is supported by observations at the larval stage , when cavefish and surface fish feed similarly . instead of feeding at either 45 or 90 , both larval types bob along the bottom of the tank and attack sunken food , trying out different feeding angles during each attempt . since this larval feeding style is somewhat similar to adult surface fish , it would be interesting to investigate the ontogeny of neural wiring in the basal ganglia , cerebellum , pontine , and vestibular systems which together control posture and how this wiring is associated with feeding angle in cavefish . in addition , future competition assays should test if a lower feeding angle is advantageous in the dark , which will provide insight into the selection pressure for this behavior . foraging is not the only behavior to have evolved in cavefish ; they also show differences from surface fish in their social interactions , one of which is schooling . schooling has multiple benefits , including helping fish avoid predators and foraging , but these benefits may not be valid in caves , which have limited food and few predators . indeed , the loss of schooling depends on the loss of visual sensing and on a nonvisuallyrelated genetic factor ( kowalko et al . , 2013a ) . yet , the same genetic factor encoded at this allele actually promotes schooling in cavefish compared to surface fish ( table 1 ) ( kowalko et al . , 2013a ) . such a finding suggests that the absence of cavefish schooling is mainly due to eye loss , which is supported by ablation of the lateralline sensory system . although it was once suggested that the lateral line controls schooling behavior ( partridge et al . , 1980 ) , ablating the lateral line did n't affect schooling in either surface fish or cavefish ( kowalko et al . , 2013a ) . therefore loss of schooling behavior may simply be a consequence of eye loss which could be beneficial , as opposed to deleterious , because being solitary may provide a better chance to find the rare food sources . a test of foodfinding ability using eyed , schooling individuals and eyed , nonschooling individuals ( 2013a ) isolated among f2 hybrids from a surface fish cavefish cross should reveal an advantage for the loss of schooling under sparsefood condition . female surface fish prefer to mate with large males ; in dark environments , though , this preference disappears , suggesting that mate preference depends on visual cues ( table 1 ) ( plath et al . , 2006 ) . yet even in the dark , two out of six cavefish populations evolved a preference for large males ( micos and yerbaniz ; others are molino , pachn , piedras , and curva cavefish ) , suggesting the evolution of a nonvisual mate preference ( plath et al . , 2006 ) . such a preference could be a consequence of adaptations and reliance on the mechanosensory lateralline system , which can sense the vortices produced by fish ( bleckmann et al . , 1991 ) . it would therefore be interesting to investigate if the frequency stimulus of vortices produced by large males ( 100 hz ) ( bleckmann et al . , 1991 ) attracts females and stimulates their release of gonadotropin releasing hormone ( gnrh ) to initiate reproductive behavior ( hofmann , 2006 ) . such an investigation could then explore how the reproduction process adapted after cavefish were no longer able to use visual sensory inputs . another behavior cavefish exhibit is wallfollowing , which is thought to function in space recognition and avoiding collisions with cave walls ( table 1 ) ( hassan 1989 ; de perera , 2004 ; windsor et al . , 2008 , 2010a , 2010b ; coombs et al . , 2010 ) . this behavior has evolved in all astyanax cavefish populations , and likely helps them navigate in the absence of visual cues . as mentioned , the cavefish 's lateralline system senses hydrodynamic changes in the flow field of water caused by objects in the cave ( hassan 1989 , 1992 ; coombs et al . , 2010 ) . the ontogeny of this behavior is not clear , although it seems to appear by 34 months postfertilization ( personal observations ) . because numbers of infraorbital sns significantly increase after 2 months postfertilization ( yoshizawa et al . furthermore , wallfollowing behavior may be tightly associated with the ability to recognize and navigate spaces in the absence of visual cues ( de perera , 2004 ; holbrook et al . , 2009 ) . the part of the nervous system most likely involved in space recognition is at a region homologous to the hippocampus , in the lateral part of the dorsalis of the telencephalon , where spatial memories form ( lo et al . , 2002 ) . it would therefore be interesting to find out how cavefish establish spatial memory that is only based on temporal information from hydrodynamic stimuli . for the most part , cave animals adeptly confront the challenges of sparse food and perpetual darkness . consequently , many researchers have associated cave animals ' traits with these selection pressures without actually testing the advantages of these evolved traits , thereby leading to misunderstanding or an overemphasis of the significance of the evolution of these traits . for example , the significant increase of infraorbital sn was first predicted as the receptor for adaptive vab , but the minor sensory population of eo sns turned out to be the major receptors for vab ( yoshizawa et al . , 2010 , 2012b ) . this case study reminds us to consider a classic criticism of the adaptive program : not all evolved traits are adaptive ( gould et al . , 1979 ) . the a. mexicanus cavefish system makes it easy to avoid such pitfalls by permitting laboratory studies in simulated cave environments , which are easy to replicate with dark and fooddeprived conditions . some interesting , classic evolutionary questions could be answered by surveying behavioral and morphological phenotypes in the a. mexicanus system , such as : do behavioral adaptations emerge before or after morphological changes ? since behavior can be drastically modified by a simple change such as a hormone concentration in the central nervous system ( kobayashi et al . , 1999)the evolution of behaviors has been assumed to precede morphological and sensory evolution ( reviewed in westeberhard , 1989 ) . yet , multiple studies in the cockroach , moth , silkworm , and mouse suggest that changes in the expression level of some sensory receptors is enough to trigger adaptive shifts of behaviors ( jacobs et al . , 2007 ; sakurai et al . , 2011 ; leary et al . , 2012 ; wadakatsumata et al a few behavioral traits , including vab and stabilized feeding angle , may have preceded morphological or sensory evolution because there are standing phenotypic variations of these behaviors among the cavefish and their related surfacedwelling cohorts . thus , during the initial steps of the adaptation process , individual cavefish ancestors that expressed these caveassociated behaviors were at a selective advantage because they did not require extreme morphological / sensory changes to adjust to the cave enviroment ( yoshizawa et al . , 2010 , 2011 , 2012 ; in contrast , cavefish behaviors that require eye regression , including loss of schooling ( kowalko et al . , 2013 ) and wallfollowing ( personal observation of eyeablated surface fish ) , may have emerged after the morphological loss of the eye . furthermore , loss of pigment , another morphological trait , may have also induced behavioral changes , including higher locomotor activity and/or sleep loss , because a null mutation of the oculocutaneous albinism ii ( oca2 ) gene increases the production of dopamine and noradrenalin , two neurotransmitters that affect sleep and locomotor behaviors ( dubou et al . in addition , wellknown pleiotropic hedgehog signaling controls both morphology / sensory system ( eye regression , widening jaw , increase of taste buds ) and behavior ( behavioral shift from aggression to foraging via increase of serotonergic neurons in the hypothalamus ) ( yamamoto et al . thus , traits controlled by pleiotropic genes could have evolved concurrently as a result of the mutation of these genes . in summary , the cavefish serves as an excellent model to study the evolution of multiple morphological and behavioral traits because it has provided evidence that morphological and behavioral traits evolved through complex manners . the latest a. mexicanus behaviors studied include : enhanced prey capture ability in 25dayold cavefish in the dark ( espinasa et al . , 2014 ) ; feeding control via appetiterelated hormones ( penney et al . , 2014 ) ; and loss of circadian rhythm and sleep ( dubou et al . , 2011 , 2012 ; beale et al . , 2013 ; moran et al . , 2014 the next frontier in cavefish research will be to identify more of the genes and mutations involved in the adaptation to the cave environment , thereby establishing a field where genetics , ontogeny , neuroscience , phylogeny , and ecology are integrated . the recent development of powerful tools has led to a wealth of important information we can use to unravel evolutionary mysteries , including available genome sequences ( astmex102 from the ensembl genome browser , at www.ensembl.org ) ; available embryology techniques that can modify gene expression ( yamamoto et al . , 2000 , 2004 , 2009 ; gross et al . , 2009 ; bilandija et al . , 2013 ) ; defined embryonic and larval stages ( hinaux et al . , 2011 ) ; transcriptome datasets ( gross et al . , 2013 ; hinaux et al . , 2013 ) ; and defined evolutionary relationships among populations ( ornelasgarca et al . , 2008 ; bradic et al . , 2012 ; gross , 2012 ; bradic et al . , 2013 ) . further advances in transgenic capabilities ( elipot et al . , 2014 ) and genomic engineering methods , such as talen and crispr technologies ( gaj et al . , 2013 ; hwang et al . , 2013 ) , allow us to perform more directed genetic studies . such technology is helping to fill the technical gap between the a. mexicanus system with its clear selection pressures and many caveadapted traits and other model animal systems with their historical knowledge base . with new information and better experimental techniques , we can further exploit a. mexicanus as an evolutionary vertebrate model , which will ultimately allow us to comprehensively understand the evolutionary processes through which genomic and developmental shifts produce enhanced or coopted adaptive traits .

summarymany developmental processes have evolved through natural selection , yet in only a few cases do we understand if and how a change of developmental process produces a benefit . for example , many studies in evolutionary biology have investigated the developmental mechanisms that lead to novel structures in an animal , but only a few have addressed if these structures actually benefit the animal at the behavioral level of prey hunting and mating . as such , this review discusses an animal 's behavior as the integrated functional output of its evolved morphological and physiological traits . specifically , we focus on recent findings about the blind mexican cavefish , astyanax mexicanus , for which clear relationships exist between its physical traits and ecosystem . this species includes two morphotypes : an eyed surface dweller versus many conspecific types of blind cave dwellers , some of which evolved independently ; all of the blind subtypes derived from eyed surface dwellers . the blind cavefish evolved under clear selection pressures : food is sparse and darkness is perpetual . simulating the major aspects of a cave ecosystem in the laboratory is relatively easy , so we can use this species to begin resolving the relationships between evolved traits and selection pressures relationships which are more complex for other animals models . this review discusses the recent advances in cavefish research that have helped us establish some key relationships between morphological evolution and environmental shifts . mol . reprod . dev . 82 : 268280 , 2015 . 2015 wiley periodicals , inc .

INTRODUCTION PREY DETECTION AND FORAGING BEHAVIOR VAB: Adaptive CaveAssociated Foraging Behavior Evolution of ForagingRelated Traits Through the Pleiotropy of Hedgehog Signaling Evolution of Foraging Behavior Without Obvious Morphological Shifts SOCIAL INTERACTION NAVIGATION WITHOUT VISUAL INFORMATION ON THE EVOLUTION OF BEHAVIOR

evolutionary processes , such as genetic drift and natural selection , can cause organisms developmental processes to evolve . the evolution of such processes can promote morphological and physiological diversity , as well as help an organism adapt to new environmental challenges and ecological niches ( hall , 1999 ) . to understand in detail how developmental processes evolve , we first must identify the selection pressures and molecular mechanisms that elicit these changes e.g . if we can identify an animal that has evolved under clear and simple selection pressures , however , it will be possible to more directly test the advantages of the developmental changes they have evolved to undergo . these conditions exert significant pressure on cavedwelling animals such that caveadapted animals from most major phyla exhibit a remarkable convergence in morphological and physiological changes related to cave life , including features that are both constructive ( longer appendages , novel behaviors , elaborate nonvisual sensory systems ) and regressive ( reduced vision and pigmentation ) ( culver , 1982 ; culver et al . in contrast , it is relatively difficult to identify the original mutations and selection pressures influencing surfacedwelling animals because they frequently hybridize with other populations and live in complex fauna . in this respect , cave animals are valuable models that we can use to analyze adaptive changes that occur under clearly defined selection pressures . behavior is a convenient window through which we can observe the selective advantages associated with morphological and physiological traits . for example , better sensitivity of a sensory organ can improve behavioral responses in predator avoidance , prey hunting , or mating , which lead to enhanced survivorship or fecundity . natural selection then screens animals with the best combination of traits , depending on their fitness ( mayr , 1963 ; bateson , 1988 ; wcislo , 1989 ; westeberhard , 1989 ; gittleman et al . , 1996 this review discusses recent advances in our understanding of behavioral evolution and related morphological and physiological traits in an animal that lives in a habitat with clear selection pressures : the mexican cavefish , astyanax mexicanus , an established animal model for studying evolution and development ( mitchell et al . food sources are limited in these caves , and likely consist of small soil crustaceans , microorganisms , and organic matter present in the water that drops from the cave ceiling ( culver et al . despite their 26 million year separation from a. mexicanus surface cohorts , the cavefish and surface fish cohorts remain interfertile , allowing us to study the evolution of behavioral , morphological and physiological traits by genetic analysis . recent work is revealing the genetic architectures that underlie the behavioral evolution of cavefish species , including the examination of the following behaviors : vibrationattraction behavior ( vab ) ( eigenmann , 1909 ; hill , 1969 ; parzefall , 1983 ; yoshizawa et al . here , i review the behaviors that have been evolutionarily advantageous and most informative in deciphering how a. mexicanus populations have adapted to their caves . yet , the extent of vab is variable within and among populations , and some individuals of one of the oldest cavefish populations , pachn , even lack this behavior ( fig . the converse is true for surface fish : while most surface fish lack vab , a few individuals exhibit low levels of this behavior ( fig . dotted lines represent the 2cm diameter quantification area surrounding the glass rod ( dark spot in the center of the chamber ) . three separated cave populations showed vab ( above the threshold level of 4 approaches ) , but no vab was apparent in either surfacefish population . n = 54 ( gray area ) ; cavefish , n = 52 ( orange area ) . a total of eight pairs of cavefish versus surface fish ( ba ) , and five pairs of surface fish with versus without vab ( bb ) in the dark and light are shown . for details about the method , please see yoshizawa et al . , 2004 in contrast , vab is adaptive in cavefish because it increases foraging in an environment with limited food availability , minimal light , and macroscopic predators ( yoshizawa et al . furthermore , because many crustaceans produce water fluctuations at 3040 hz while swimming ( lang , 1980 ) , the tuning of cavefish 's vab at 35 hz is a novel trait that is likely adaptive in the cave ecosystem . although sns appear throughout the body and are particularly abundant on the cavefish 's head , those sns located within the orbit of the cavefish 's degenerated eye seem to play a particularly important role in vab ( yoshizawa et al . as there was no difference in the number of eyeorbit sns ( eo sns ) among surface fish in fact , no eo sn was observed in surface fish their appearance in cavefish did not arise through the selection of a standing phenotypic variation ( yoshizawa et al . surface fish , n = 10 at 2 months postfertilization ( mpf ) , = 9 at 3 mpf , = 10 at 4 mpf , and = 12 at 1yearold ; pachn cavefish , n = 9 at 2 mpf , = 8 at 3 mpf , = 9 at 4 mpf , and = 11 at 1yearold ; tinaja cavefish , n = 7 at 1yearold . thin , white dotted line encloses the neuromasts in an adjacent region on the cranium , the third infraorbital bone ( previously denoted as the third suborbital bone , so3 ) ( yoshizawa et al . many aspects of eye degeneration are controlled by increased hedgehog signaling in cavefish ( yamamoto et al . while eo sn ontogeny corresponds with vab cavefish , there is no correlation between vab and the total number of sns in an individual . these observations further emphasize the model that the eo sn , as a minor group of sensory organs , plays an important role in facilitating vab whereas other sns in the infraorbital region may contribute to other sensations , such as hydrodynamic imaging ( see below , and also hassan , 1989 ; montgomery et al . development of the eo sn may therefore have evolved as a consequence of positive selection for the enhancement of an adaptive behavior , indicating that studying behavioral traits can help reveal distinct roles for sns residing in different cranial areas . such timing may occur for two reasons : ( 1 ) the developmental process might be constrained or ( 2 ) their foraging preference might change with age . the sns first form at the edge of a developing intramembranous bone in the cranial region ( the operculum , at 45 days postfertilization ) . in the laboratory , however , large cavefish occasionally eat smaller cavefish , suggesting that small , younger fish can not compete with mature adults . , 2013 ) , we will gain insight to the relationship between eo sn development and the timing of dermalbone formation . deciphering which gene(s ) are responsible for vab will through in situ hybridization and/or immunohistochemistry techniques help reveal if the novel sensory tuning occurs at the level of first projection , the octavolateralis nucleus , or higher , such as at the torus semicircularis area . given the necessary redistribution of attention to the nonvisual senses , selection for these other constructive traits , which are fostered by hedgehog signaling , during cavefish evolution no one has tested if these gustatory traits ( larger jaws , more taste buds ) improve cavefish 's foraging abilities . nevertheless , an enlarged jaw is likely beneficial in the darkness , possibly as an adaptation to bottom feeding , and carrying more taste buds likely improves foraging because they could help navigate to food in the dark . if having a large jaw and more taste buds does not enhance foraging efficiency , however , these traits would constitute another example of not all evolved traits are adaptive hedgehog signaling is also required for other critical developmental processes , beyond cavefish eye degeneration and a few gustatory traits . , 2013 ) , the latter is an example of a change in behavioral strategy . this may be because fish at this young age are too immature to establish hierarchical positions , or because some unknown benefit exists for having an enhanced serotonergic system in the larval stage . overall , if we could better understand the difference in the aggression neural circuits between cavefish and surface fish , and their relationship with the number of serotoninergic cells in 1weekold juveniles , we would gain muchneeded insight to the evolution of cavefish 's foraging behavior . in general , this behavioral shift in cavefish from attacking to foraging provides the first example of a likely behavioral advantage underlying their enhanced hedgehog signaling , whose tradeoff is eye development ( c.f . many behaviors have evolved in the company of morphological changes , yet behavior can also evolve without obvious anatomical adaptations e.g . when in the dark , surface fish feed at a steep angle of 90 relative to a substrate . , 2013b and personal observation ) and other benthic feeders ( for example , see ferrygraham et al . , 2009 ) , it was surprising when a set of studies revealed no detectable correlation between feeding angle and eight craniofacial phenotypes in an f2 intercross derived from a surface fish mated to a cavefish ( kowalko et al . surface fish readily stabilize their feeding angle at a 45 angle in lighted conditions , so cavefish may have evolved their posture control without visual cues , instead relying on or modifying the vestibular posture control centers . this is supported by observations at the larval stage , when cavefish and surface fish feed similarly . since this larval feeding style is somewhat similar to adult surface fish , it would be interesting to investigate the ontogeny of neural wiring in the basal ganglia , cerebellum , pontine , and vestibular systems which together control posture and how this wiring is associated with feeding angle in cavefish . , 2014 ) , the convergence of these populations towards vab suggests the distinct advantage this behavior would have in the cave environment . dotted lines represent the 2cm diameter quantification area surrounding the glass rod ( dark spot in the center of the chamber ) . three separated cave populations showed vab ( above the threshold level of 4 approaches ) , but no vab was apparent in either surfacefish population . a total of eight pairs of cavefish versus surface fish ( ba ) , and five pairs of surface fish with versus without vab ( bb ) in the dark and light are shown . for details about the method , please see yoshizawa et al . , 2004 in contrast , vab is adaptive in cavefish because it increases foraging in an environment with limited food availability , minimal light , and macroscopic predators ( yoshizawa et al . furthermore , because many crustaceans produce water fluctuations at 3040 hz while swimming ( lang , 1980 ) , the tuning of cavefish 's vab at 35 hz is a novel trait that is likely adaptive in the cave ecosystem . as there was no difference in the number of eyeorbit sns ( eo sns ) among surface fish in fact , no eo sn was observed in surface fish their appearance in cavefish did not arise through the selection of a standing phenotypic variation ( yoshizawa et al . thin , white dotted line encloses the neuromasts in an adjacent region on the cranium , the third infraorbital bone ( previously denoted as the third suborbital bone , so3 ) ( yoshizawa et al . many aspects of eye degeneration are controlled by increased hedgehog signaling in cavefish ( yamamoto et al . while eo sn ontogeny corresponds with vab cavefish , there is no correlation between vab and the total number of sns in an individual . 3c ) , the development of these organs is not genetically correlated with the level of vab , and their ablation does not detectably affect vab ( yoshizawa et al . these observations further emphasize the model that the eo sn , as a minor group of sensory organs , plays an important role in facilitating vab whereas other sns in the infraorbital region may contribute to other sensations , such as hydrodynamic imaging ( see below , and also hassan , 1989 ; montgomery et al . development of the eo sn may therefore have evolved as a consequence of positive selection for the enhancement of an adaptive behavior , indicating that studying behavioral traits can help reveal distinct roles for sns residing in different cranial areas . such timing may occur for two reasons : ( 1 ) the developmental process might be constrained or ( 2 ) their foraging preference might change with age . the sns first form at the edge of a developing intramembranous bone in the cranial region ( the operculum , at 45 days postfertilization ) . in the laboratory , however , large cavefish occasionally eat smaller cavefish , suggesting that small , younger fish can not compete with mature adults . , 2015 ) , and performing generegulatorynetwork analysis ( gavinsmyth et al . , 2013 ) , we will gain insight to the relationship between eo sn development and the timing of dermalbone formation . deciphering which gene(s ) are responsible for vab will through in situ hybridization and/or immunohistochemistry techniques help reveal if the novel sensory tuning occurs at the level of first projection , the octavolateralis nucleus , or higher , such as at the torus semicircularis area . it is unlikely that sensory tuning is achieved at the neuromast level , however , since the sensitivity estimation based on the morphometrics of neuromasts does not peak at 35 hz ( yoshizawa et al . given the necessary redistribution of attention to the nonvisual senses , selection for these other constructive traits , which are fostered by hedgehog signaling , during cavefish evolution no one has tested if these gustatory traits ( larger jaws , more taste buds ) improve cavefish 's foraging abilities . to understand the adaptive significance of these morphological and sensory shifts , however for example , behavioral analyses showing that these gustatory traits are advantageous would support the hypothesis that oralpharyngeal shifts can promote eye reduction through pleiotropy . if having a large jaw and more taste buds does not enhance foraging efficiency , however , these traits would constitute another example of not all evolved traits are adaptive hedgehog signaling is also required for other critical developmental processes , beyond cavefish eye degeneration and a few gustatory traits . , 2013 ) , the latter is an example of a change in behavioral strategy . indeed , cavefish have higher serotonin levels and more serotonergic neurons at the hypothalamic anterior paraventricular nucleus and hindbrain raphe than their surface counterparts ; these anatomical and endocrine changes are believed to have shifted cavefish behavior from attacking to foraging ( elipot et al . overall , if we could better understand the difference in the aggression neural circuits between cavefish and surface fish , and their relationship with the number of serotoninergic cells in 1weekold juveniles , we would gain muchneeded insight to the evolution of cavefish 's foraging behavior . in general , this behavioral shift in cavefish from attacking to foraging provides the first example of a likely behavioral advantage underlying their enhanced hedgehog signaling , whose tradeoff is eye development ( c.f . many behaviors have evolved in the company of morphological changes , yet behavior can also evolve without obvious anatomical adaptations e.g . , 2013b and personal observation ) and other benthic feeders ( for example , see ferrygraham et al . , 2009 ) , it was surprising when a set of studies revealed no detectable correlation between feeding angle and eight craniofacial phenotypes in an f2 intercross derived from a surface fish mated to a cavefish ( kowalko et al . surface fish readily stabilize their feeding angle at a 45 angle in lighted conditions , so cavefish may have evolved their posture control without visual cues , instead relying on or modifying the vestibular posture control centers . since this larval feeding style is somewhat similar to adult surface fish , it would be interesting to investigate the ontogeny of neural wiring in the basal ganglia , cerebellum , pontine , and vestibular systems which together control posture and how this wiring is associated with feeding angle in cavefish . foraging is not the only behavior to have evolved in cavefish ; they also show differences from surface fish in their social interactions , one of which is schooling . yet even in the dark , two out of six cavefish populations evolved a preference for large males ( micos and yerbaniz ; others are molino , pachn , piedras , and curva cavefish ) , suggesting the evolution of a nonvisual mate preference ( plath et al . as mentioned , the cavefish 's lateralline system senses hydrodynamic changes in the flow field of water caused by objects in the cave ( hassan 1989 , 1992 ; coombs et al . the part of the nervous system most likely involved in space recognition is at a region homologous to the hippocampus , in the lateral part of the dorsalis of the telencephalon , where spatial memories form ( lo et al . consequently , many researchers have associated cave animals ' traits with these selection pressures without actually testing the advantages of these evolved traits , thereby leading to misunderstanding or an overemphasis of the significance of the evolution of these traits . for example , the significant increase of infraorbital sn was first predicted as the receptor for adaptive vab , but the minor sensory population of eo sns turned out to be the major receptors for vab ( yoshizawa et al . this case study reminds us to consider a classic criticism of the adaptive program : not all evolved traits are adaptive ( gould et al . the a. mexicanus cavefish system makes it easy to avoid such pitfalls by permitting laboratory studies in simulated cave environments , which are easy to replicate with dark and fooddeprived conditions . yet , multiple studies in the cockroach , moth , silkworm , and mouse suggest that changes in the expression level of some sensory receptors is enough to trigger adaptive shifts of behaviors ( jacobs et al . thus , traits controlled by pleiotropic genes could have evolved concurrently as a result of the mutation of these genes . in summary , the cavefish serves as an excellent model to study the evolution of multiple morphological and behavioral traits because it has provided evidence that morphological and behavioral traits evolved through complex manners . , 2014 the next frontier in cavefish research will be to identify more of the genes and mutations involved in the adaptation to the cave environment , thereby establishing a field where genetics , ontogeny , neuroscience , phylogeny , and ecology are integrated . the recent development of powerful tools has led to a wealth of important information we can use to unravel evolutionary mysteries , including available genome sequences ( astmex102 from the ensembl genome browser , at www.ensembl.org ) ; available embryology techniques that can modify gene expression ( yamamoto et al . such technology is helping to fill the technical gap between the a. mexicanus system with its clear selection pressures and many caveadapted traits and other model animal systems with their historical knowledge base . with new information and better experimental techniques , we can further exploit a. mexicanus as an evolutionary vertebrate model , which will ultimately allow us to comprehensively understand the evolutionary processes through which genomic and developmental shifts produce enhanced or coopted adaptive traits .

together , the world health organization 's policy framework for active aging and its guide for the development of age - friendly cities have set a policy and research agenda in which the built environment is conceived to play a crucial role as a facilitator or inhibitor of older people 's capacity to age actively . this theoretical standpoint assumes that built environments which are conducive to the outdoor mobility of older people will help provide them with ongoing opportunities for both participation within society and maintenance of their health through the gaining of physical exercise as they move around their neighborhoods . it remains unclear , however , whether or not older people 's self - assessments of being active are linked to either their levels of participation within society or their engagement in outdoor physical activity , and , to date , there is no convincing evidence of a direct relationship between the built environment and older people 's walking behavior . active aging is defined by the who [ 1 , page 12 ] as the process of optimizing opportunities for health , participation , and security in order to enhance quality of life as people age . this concept presents both opportunities and challenges to policy makers and researchers alike . in terms of policy , active aging has been acknowledged as providing a sound basis for responding to the challenges posed by population aging in industrialized nations by linking a number of key policy domains including employment , pensions , retirement , health , and citizenship [ 3 , page 121 ] . yet within europe , for example , the focus on active aging has so far been largely limited to a crude reduction in terms of working longer [ 4 , page s117 ] . walker [ 3 , page 124 ] noted more than a decade ago that active aging does not amount to a coherent strategy and is sometimes just a slogan used to cover anything that seems to fit under it . more recently , he has stressed that this concept is more of an aspiration for both individuals and collectivities than it is a process that exists in practice [ 4 , page s126 ] . the usefulness of the active aging concept for policy makers aiming to comprehensively address issues associated with the growing aging population will necessarily rest on their capacity to devise a coherent set of strategies that effectively deal with all of the three key determinants of active aging identified by the who participation , health , and security . one key opportunity presented to researchers by the development of the active aging concept is its potential to act as a driver for greater attention on the older person - macroenvironment relationship . indeed , there has been unprecedented research interest in this area since the release of the who 's policy framework ; prior to the early 2000s , it was the microenvironments of older people which predominated as the main focus of investigations into the older person - environment relationship ( see ) . nevertheless , the complex , multifaceted nature of the active aging concept is problematic , especially since it effectively encompasses dimensions covered by a number of earlier concepts . these include successful ageing which walker traces back to the early 1960s ( a concept emphasizing the importance of maintaining activity patterns and values of middle age into old age ) and productive aging which emerged in the 1980s ( mostly focused on removing barriers to older persons engaging in work paid or unpaid and thereby extending their productive capacity beyond retirement age ) . walker [ 3 , page 123 - 124 ] notes that active aging emerged in the 1990s , giving recognition to both the connection between activity and health and the importance of healthy aging . his account of the development of the active aging concept clearly demonstrates that active aging is much broader in scope than all of these earlier concepts . nevertheless , a review of empirical literature recently undertaken by hung and colleagues grouped active aging with successful , productive , positive , and robust aging as cognates of healthy agingdespite the fact that the active aging concept encompasses more dimensions than any of these other concepts ( including continued participation in society ; the maximization of social , mental , and physical health ; the maintenance of dignity , self - efficacy , and human rights ; and the creation of age - friendly physical environments to facilitate autonomy and independence ) ( see [ 3 , 7 ] ) . coherency of the accumulating evidence on active aging may well be undermined by both the overlap between this concept and its predecessors and the treatment of them as interchangeable terms . it is also noteworthy that successful aging has now been a focus of research for around half a century and there is still no widely agreed definition for this term or consistency between studies in the measures used to investigate this subject area , and the first rigorous analysis of measures was only undertaken recently ( see ) . active aging is operationally defined by researchers given the broad scope of this concept . if active aging objectives are to be met , it would seem crucial that laypersons ascribe meanings to this term that are in line with the who 's policy framework and fully grasp the nature and importance of all of the constituents of active aging as laid out in this framework . to date research undertaken in the united kingdom by bowling found this term was most commonly conceived by a sample of older people aged 60 years and over , to involve having / maintaining physical health and functioning ( 43% ) , leisure and social activities ( 34% ) , mental functioning and activity ( 18% ) , and social relationships and contacts ( 15% ) . comparison with academic conceptualizations of active aging showed that there was overlap between lay perceptions of the constituents of active ageing and those used in theoretical models within the literature , although the latter entailed additional components ( productivity , empowerment , human rights , and dignity ) to those identified by laypersons . multiple regression analyses revealed that health status , longstanding illness , and quality of life ( all based on self - report ) explained 41% of the variance in self - ratings of active aging , while sociodemographic and economic variables were not significantly associated with this outcome [ 7 , page 298 ] . comparison of data gathered on the perceived constituents of active aging in this particular study with data obtained from an earlier survey of older people 's perceptions of successful aging , also administered in the united kingdom but with adults aged 50 years and over ( for further details see ) , showed that there was considerable overlap in the meanings ascribed to a later study by bowling compared the perceptions of minority ethnic groups with those of the general population in the united kingdom , revealing that ethnically diverse respondents were less likely to define active aging in terms of good physical health and fitness ( and exercise to promote these two aims ) and also less likely to rate themselves as aging actively than respondents within the general population . from bowling 's research , it would seem that individual factors such as health status and longstanding illness impact on older people 's self - assessments of aging actively , but we currently have little understanding of whether or not there is any relationship between these self - assessments and the actual behaviors of older persons with respect to their actual levels of participation and physical activity outside of the home domain . older people 's participation in the wider community necessarily relies on their capacity for remaining mobile in their out - of - home environments . the accumulated body of evidence suggests that individual factors as well as multiple aspects of the built environment affect older people 's potential out - of - home mobility and thus their capacity to participate in society . driving status [ 11 , 12 ] , quality and availability of public transport [ 13 , 14 ] , and numerous other features of the built environment ( see [ 15 , 16 ] ) are all implicated in this regard . walkable environments are particularly important , given that neighborhood walking increases not only older people 's opportunities for physical exercise but also social interaction . engagement in outdoor physical activity by older people is crucial to them remaining ambulatory and also provides numerous other health benefits . in the united states , lack of physical activity among older people has been described as a major public health issue ; the release of surgeon - general 's report in 1996 indicated that less than one - third of adults aged 65 years and over met health recommendations of moderate intensity exercise ( e.g. , brisk walking ) for 30 minutes , five or more days per week [ 19 , s267 ] . more recent research conducted in new jersey shows a similar pattern , with just 28.6 percent of 50- to 74-year - olds being found to meet exercise recommendations . the proportion of older people who meet exercise guidelines has been shown to be highly variable , however , both within and between different countries . a systematic review of studies investigating physical activity among older adults ( limited to those studies which reported their findings in terms of older people meeting exercise guidelines ) revealed that this proportion ranges between 2.4 and 83.0 percent , with the majority of the 53 studies reviewed being found to report proportions of between 20 and 60 percent . only 6 of these studies ( undertaken in the united states , australia , the united kingdom , brazil , china , canada , new zealand , columbia , south africa , greece , cyprus , sweden , and switzerland ) used an objective measure of physical activity ( i.e. , accelerometer ) , with the remainder being based on self - reported activity . research efforts over the past decade have provided insight into many features of the built environment that encourage or constrain older people 's walking decisions . yet , the extent to which older people 's walking behavior actually differs as a function of the particular built environment in which they live remains unclear . much of the research literature related to the built environment and walking activity is based on random samples , with findings being largely representative of those living in urbanized areas ( i.e. , similar built environments ) . in addition , the vast majority of available studies of walking behavior among older people are based on self - report , making the findings subject to bias ( e.g. , participant recall and selection of socially acceptable responses ) . a recent review of empirical literature that examined mobility or disability among older people and also utilized objective measures of the built environment identified 17 studies published between 1990 and 2010 that met these criteria . of these , 14 studies investigated walking behavior , with all but one being based on self - reports of walking . rosso et al . concluded that although evidence of a direct relationship between the built environment and elderly walking behavior was lacking , of all of the features of the built environment investigated , high density of intersections , street and traffic conditions , proximity to destinations , and green space were the factors most likely to have a direct effect on older people 's out - of - home mobility . nevertheless , a study of the consumption patterns and mode of travel used by older people ( via gps tracking ) for accessing goods and services within two different countries ( suburbs of canada and france ) showed that older people in both locations were highly reliant on travel by motor vehicle , despite substantial differences in the built environment and availability of public transport in the two study sites . this raises questions about the assumption that levels of walking outdoors among the older segment of the population will naturally follow modifications to the built environment that make them more age - friendly , without a shift away from sedentary lifestyles and a change in the propensity of the elderly to rely predominantly on motor vehicles for movement outside of the home domain . older people have identified particular urban design features such as green and open spaces [ 25 , 26 ] as being conducive to walking . other features like poorly maintained pedestrian infrastructure and traffic , however , heighten their fears about personal safety . evidence suggests that these fears about safety are well grounded , given that most falls occur outside of the home domain with the majority taking place on curbs , sidewalks , and streets and that older people are the most likely group to present at hospital for injuries sustained from pedestrian - cyclist collisions . unfortunately , however , fear of moving outdoors also increases older people 's risk of diminished capacity to walk outdoors in the longer term . longitudinal research has shown that older people who fear walking outdoors are 4.6 times more likely than those without this fear to develop difficulties in walking a distance of 0.5 kms . older people 's personal expectations about aging also influence their walking behavior . a pilot test of a behavioral intervention aimed at altering belief in the inevitability of becoming sedentary as a consequence of the aging process demonstrated that older people not only increased their walking after intervention but also experienced multiple other benefits including ease in performing daily life tasks and improvements in mental health - related quality of life , pain , energy , and quality of sleep . meeting the objective of maximising people 's opportunities for ongoing participation within society as they proceed into older age is likely to be a major challenge for policy makers in industrialized nations . cross - country analyses ( of canada , finland , germany , italy , japan , the netherlands , sweden , the united kingdom , and the united states ) indicate that there is a remarkably similar pattern in the age profile of work and passive and active leisure activities across countries . despite considerable variation in hours spent doing paid work between countries for adults in the 4554 age group , intercountry differences seem to vanish by the age of 75 years and over , with time that was previously spent doing paid work being predominantly reallocated to passive rather than active leisure activities . much of older people 's time also appears to be spent at home . research undertaken in berlin with community - based and institutionalized adults aged 70 years and over , for example , indicates that older people spent the majority of their day alone at home ( 80% of awake time ) and less than a fifth of their day outdoors ( 18.7% ) . similarly , more recent australian research indicates that only a small proportion of time is spent away from home ( 90.2% spent at home ) by community - based adults aged 65 years and over , with the mean number of episodes spent away from home in the previous week being 6.3 ( sd = 4.5 ; range 019 ) . in addition , the average daily time spent away from home by older people who have had a stroke and those who have not appears to very similar ( 0.9 versus 1.1 hours , resp . ) . age- and/or health - related issues are implicated , however , in a substantial difference observed in older australians ' engagement in volunteering . twice as many current drivers ( 66% ) as retired drivers ( 30% ) reported involvement in this particular form activity . the current study used a mixed - methods approach to explore areas of interest within the area of active aging that are currently underresearched , including ( 1 ) the connection between older people 's self - perceptions of being active and indicators of their health and participation ( domains which are proposed by the who to be two of the three key determinants of active aging ) ; ( 2 ) older people 's perceptions of characteristics of their respective communities , which are generally conceived to facilitate aging in place ; and ( 3 ) the extent to which older people 's out - of - home physical activity differs across different built environments , based on high quality objective measures of active travel ( walking and biking ) . a convenience sampling method was used to recruit a total sample of 48 adults aged 55 years and over , comprising 4 subsamples of equal numbers ( n = 12 ) of individuals living in ( 1 ) inner city suburbs and ( 2 ) suburbs outside the inner city area of a capital city , ( 3 ) a regional city , and ( 4 ) a rural town in queensland , australia . the first involved residents of inner city suburbs of brisbane ( within 5 kms of the central business district ) who had participated in a previous project ( living in the city ) and also indicated their willingness to be contacted for future research being invited to participate as representatives of high density living environments . the second recruitment method involved key community organisations and groups being approached to assist in identifying potential participants living in brisbane suburbs situated outside the inner city , toowoomba ( a regional city ) , and roma ( a rural town ) , as well as one individual to complete the brisbane inner city subsample . each individual was subsequently contacted via phone and/or email and invited to participate in the study . the total sample comprised 24 males and 24 females with each of the four subsamples being comprised of equal numbers of males and females ( a thirteenth individual from roma who volunteered to participate in the study jointly with her spouse was excluded from the study to ensure both an equal gender ratio and subsample size ) . all participants were ambulatory and the age range for the whole sample was 56 to 93 years ( average age of 72.02 years ; sd = 8.46 ) . table 1 provides a demographic profile of the sample across the four study locations with respect to age , income , marital status , and current housing ( derived from survey data ) . the population density for each of the four study locations the mean age for three of the subsamples was almost identical , but the mean age of the regional city ( rc ) subsample was between 2.5 and 2.7 years higher than the inner city ( ic ) , city suburban ( cs ) , and rural town ( rt ) groups . nearly all of those from the ic group had high incomes , while the majority living in the cs , rc , and rt locations had incomes that fell in the low range . the most common source of income across the whole sample was the old age pension ( 33.3% ) , but superannuation also provided whole or part of the annual incomes of one - third of the sample . only a small proportion of the whole sample was engaged in full- or part - time work ( 15.2% ) . the majority of participants in each subsample were married and also owned the homes in which they resided . all members of the ic group were living in a flat or unit ( interview data revealed that most lived in a unit ) , while the majority of the other three subsamples were living in a house . most of the older people in each of the four study locations owned their own dwelling . quantitative data were gathered from a gps device ( which captured one logged position every one minute and was used to track all out - of - home travel over seven consecutive days ) as well as responses to a brief survey contained in the front section of a travel diary . the gps device , gps charger , and diary were posted to each participant , which they returned to the project site by post or courier at the end of the tracking period . qualitative data were obtained from daily travel diary entries made by each participant ( documenting all out - of - home travel , activities undertaken outside the environment , and the mode of transport used for each trip ) . in a few cases , participants forgot to either recharge their gps device or take the device when they left home on one or two days and so were asked to continue completing their travel diaries and using the gps to ensure that their out - of - home travel was monitored for the required seven days . following the return of the completed diaries , gps device , and charger , the gps data were converted into individual time / space maps for each participant using google earth software . diary entries were used to color - code each trip line shown on the maps to indicate the means of travel used for each trip made ( by car , bus , ferry , train , taxi , and bicycle and on foot ) . these maps were subsequently used to direct discussion and verify correspondence between the gps data and diary entries during in - depth , semistructured interviews held with each participant approximately two weeks after the tracking period . survey items tapped demographic characteristics ( see table 1 ) as well as attributes of the built environment in which participants lived . the latter were assessed from endorsement of statements that represented reasons for them living in their current community and included proximity to destinations ( it is close to shops , etc . , and close to my family / friends ) and safety ( it is a safe area ) . participants ' perceptions of the age - friendliness and disability - friendliness of their communities were captured by responses to two separate items with a shared lead - in question ( do you think your community is : ( 1 ) age - friendly ; and ( 2 ) disability - friendly . available options were the same for each item ( yes , no , do not know , and never thought about it ) . self - perceptions of being healthy and being active were tapped from responses to two single - item , five - level likert scales ( options and coding for these variables are reported in table 3 ) . survey forms also included a question asking about all modes of transport normally used for moving around one 's neighbourhood ( for details of survey item and available options , see footnotes of table 4 ) . travel diary entries were used in conjunction with gps data for determining the number of days when participants ventured out of home , their use of public transport , time spent travelling on foot and by bicycle outside of the home environment , and the total time spent traveling outside home over the seven - day tracking period . in combination , travel diary entries and gps tracking produce high quality data on travel behavior . a new variable was created which measured the total time spent travelling on foot and by bicycle to ensure that all of participants ' travel by active means over the tracking period was captured . the continuous variables for time spent walking and travelling by active means were converted into a six - level measure of 30-minute intervals ( ranging from zero to a maximum of over 2 hours ) for the purpose of correlational analysis because of the wide variation in minutes travelled on foot and walking / biking by those who walked or used active means for a period of more than two hours . travel time captured by gps in off - road open spaces ( such as parks and golf courses ) was included , but any time spent inside buildings ( such as shopping centers ) was excluded from travel calculations . results are reported in tables in the form of cell counts due to the small size of each subsample , with percentages being shown in the totals column of each table . missing values are reported in the tables ( as no response ) and are included in the row totals . pearson correlation analyses were performed to identify relationships between select variables for the entire sample for exploratory purposes to determine relationships between self - perceptions of being active and being healthy and objective measures of participation ( number of days when participants travelled out of their homes ) and travel by active means outside the home environment . comparative analyses of the four study locations were undertaken using a nonparametric approach , the kruskal - wallis test of significance , due to its appropriateness for three or more independent , small , and nonrandom samples . the statistical significance of differences between groups determined by this test is based on ordinal information only , with observations being ranked and the mean rankings of the various groups being compared . the original continuous variable minutes spent walking and minutes spent walking / biking were used for these analyses to ensure true ranking of data ( actual minutes ) and minimization of tied ranks . table 2 shows that the majority ( 64.6% ) of the whole sample believed that their communities were age - friendly but less than half ( 41.4% ) agreed that they were interestingly , a similar proportion ( 41.7% in total ) neither agreed nor disagreed about the disability - friendliness of their communities and either did not know ( 18.8% ) , had never thought about it ( 10.4% ) , or did not respond to the question ( 12.5% ) . more individuals within the rc and rt subsamples rated their communities as being age- and disability - friendly than the two capital city subsamples ( ic and cs ) . participants ' reasons for living in their current community are shown in the lower part of table 2 . one - third of the whole sample identified their respective communities as being safe areas ( 33.3% ) and similar proportions reported proximity as a factor that contributed to their reasons for living in their current communities ( close to family / friends more older people living in the rc location identified being close to family / friends than those in the ic , cs , and rt groups ( 7 versus 3 ) as a reason for living in their current community , while similar numbers selected being close to shops , and so forth . only two older people living in the cs area agreed that safeness of the area was a reason for them living in their communities ( compared to 4 or 5 in the other three groups ) . the patterns of self - ratings of being active and of being healthy were each found to follow a normal distribution with a left skew ( prone to being healthy and active ) , results that are to be expected for a community - based older sample . ( 47.9% ) , and just under one - third of the sample considered themselves to be somewhat active ( 45.8% ) . less than one - fifth rated themselves as unhealthy ( 14.6% ) and none of the participants reported being very unhealthy ( see table 3 ) . no relationship was found to exist between self - ratings of being active and self - rated health status ( r = 0.24 ; p = 0.098 ) . self - perceptions of being active were also found to be unrelated to age ( r = 0.15 ; p = 0.317 ) , but a statistically significant , negative correlation was found between self - rated health status and age ( r = 0.32 ; p = 0.027 ) . only five of the total sample of 48 travelled outside of their home in four days or less over the monitored week . the number of days on which participants ventured out of home was positively associated with their self - ratings of being active ( r = 0.36 ; p = 0.011 ) but was unrelated to both self - ratings of health ( r = 0.11 ; p = 0.468 ) and age ( r = 0.26 ; p = 0.072 ) , and the subsample means and medians for days travelled outside home were remarkably similar ( see table 3 ) . the first part of table 4 summarizes self - report travel - related information derived from survey to provide context to the gps tracking results for time actually spent walking and walking / biking combined . most drove cars but one person drove a motorcycle and another two drove both a car and a motorcycle . another six ( 12.5% ) travelled by motor vehicle as a passenger , and only three reported that they did not travel by motor vehicle ( as either drivers or passengers ) . less than half ( 41.7% ) of the whole sample reported ever using public transport , and all but one of this group were from the ic and cs locations . none of the rc sample reported using public transport even though bus services are available in their location . both the rc and rt locations only have access to train travel for the purpose of traveling outside of their local areas ( rail link extends eastbound to brisbane and westbound to charleville ) , and only one participant from the rt group reported ever using the train . gps data showed that only a quarter ( 25.0% ) of the sample actually used public transport during the tracking period . the actual time spent walking and time spent walking and biking combined by individuals living within the four study locations ( based on gps data ) are shown in the lower section of table 4 . with respect to walking , one - third of the whole sample did not travel on foot out of their home environments at all over the tracking period . considering this group and the next two categories ( 130 mins and 3160 mins ) , more than half ( 56.2% ) of the whole sample travelled out of home on foot for an hour or less during the monitored week . just under one - third ( 31.3% ) travelled on foot for more than two hours over the seven days . no correlation was found between time spent walking and self - perceptions of being active ( r = 0.12 ; p = 0.401 ) and being healthy ( r = 0.12 ; p = 0.414 ) or age ( r = 0.06 ; p = 0.688 ) . in table 4 , it can also be seen that the number of nonwalkers in the lower density suburbs of brisbane was twice that of their counterparts living in the inner city suburbs of brisbane , with the numbers of nonwalkers in the ic group being similar to both the rc and rt groups . considering the actual time spent walking ( in minutes ) by each individual across each of the four groups , however , the kruskal - wallis test indicated there was no significant difference in the mean rankings by study location ( h(3 ) = 0.892 ; p = 0.827 ) . once time spent traveling by bicycle was taken into account , only three individuals in the cs group were found to have spent no time using active means of travel ( on foot or biking ) , but the remainder of the findings were similar to those observed for walking . time spent travelling by active means was found to be unrelated to both self - ratings of being active ( r = 0.16 ; p = 0.279 ) and healthy ( r = 0.18 ; p = 0.216 ) and to age ( r = 0.16 ; p = 0.284 ) . no significant difference between the four study locations was identified in the mean rankings of active travel ( based on actual minutes ) either ( h(3 ) = 0.520 , p = 0.915 ) . a visual representation of the lack of correlation between self - perceptions of being active and use of active means of travel is shown in figure 1 . note , for example , that self - ratings of being active among those who spent no time walking or biking are distributed across four categories , from very inactive to very active . a visual representation of the proportion of all time travelled over the tracking period spent using active forms of transport is shown in figure 2 . the similarity in the overall pattern that emerged for each of the four study locations is clearly evident and the kruskal - wallis test confirmed that there was no significant difference in the mean rankings of the proportion of overall time spent travelling by active means across the four study locations ( h(3 ) = 0.457 , p = 0.928 ) . overall , the findings contribute to a relatively small but growing empirical literature focused on active aging , by way of whole sample analysis and equal - sized subsample comparison . they add value to this body of evidence in five main ways . firstly , the whole sample analyses enabled determinations to be made about the distribution of self - ratings of being active among community - based adults living in australia . like bowling 's study undertaken in the united kingdom ( based on a larger sample than the current study ) , the most common self - rating for being active was the fourth level of a five - level single - item likert scale ( active in this study and fairly actively in bowling 's study ) . however , while the second most common rating in bowling 's study was the fifth - level option very actively , our research found the third - level option somewhat active to be the second most common , followed by very active . this difference may be attributable in part to the wording and response options used by each study but may reflect either true difference in activity levels between older people in the united kingdom and australia or difference in the benchmark used by older people in these two nations when making this form of self - assessment . replication studies of bowling 's [ 7 , 10 ] research are needed in australia and elsewhere in order to further understand older people 's perceptions about the key constituents of active aging from their own perspective if we are to gauge similarities and differences across different cultures and countries . secondly , the current study was able to investigate correlates of self - ratings of being active beyond those examined by previous studies . our finding that age was unrelated to self - ratings of being active is consistent with bowling 's study . our finding that self - rated health status was unrelated to self - perceptions of being active differs however . this inconsistency may rest on the nature of the sample used in the current study , whereby three - quarters were recruited through community organisations ( and thus were actively participating in community activities ) and around two - thirds reported being healthy or very healthy or differences in the measure of health status used in each study ( bowling 's study used presence of a limiting longstanding condition or disability while this study used self - rated health status ) . the negative correlation we found between age and self - rated health is consistent with previous research showing an association between increasing age and more chronic health problems and declining functional ability . thirdly , this study had the capacity to explore whether self - ratings of being active were connected with actual behavior . the fact that these self - ratings were positively correlated with the number of days when time was spent out of home but unrelated to levels of out - of - home physical activity raises the possibility that older people in this study have embraced the idea that active aging is primarily about one 's current participation within society . future research efforts need to be directed at exploring the degree that self - assessments of being active as people age correspond with objective measures of both participation and outdoor physical activity . active aging objectives are unlikely to be met if a gap exists between the meanings ascribed to active aging by older people and their actual behavior with respect to the latter . more than half of the sample ( all ambulatory and mostly healthy ) spent a daily average of between zero and 8.6 minutes walking outside of home over seven days . if this level of outdoor physical activity is representative of these participants ' normal pattern of walking outdoors , their long - term prospects of remaining ambulatory and capable of performing daily life activities could potentially be compromised , with this in turn eventually undermining their capacity for living independently , aging in place , and participating in society . our finding that less than one - third of older people in this study had walked for more than 2 hours over seven days is consistent with findings from studies undertaken in the united states [ 19 , 20 ] , where a similar car culture to australia 's prevails . given the accumulated evidence about barriers to walking ( see [ 16 , 27 ] ) and the difficulties associated with using public transport experienced by older people , as well as this group 's reliance on cars [ 12 , 24 ] , it may be that the developments of community - based programs that encourage walking behavior among older people with a specific focus on recreational walking in spaces where pedestrian infrastructure is in good condition and free of traffic ( e.g. , parks and cyclist - free pedestrian walkways ) are needed . these programs could target individuals as well as groups ( such as senior citizens organizations ) and may require the use of motorized transport ( e.g. , private motor vehicles and public or private bus ) for gaining access to well - maintained , safe walking tracks . this approach could potentially create opportunities for older people to overcome their fears about moving outdoors , increase their number of social interactions , maintain or improve their overall health and well - being through the gaining of regular exercise [ 18 , 31 ] , and also change expectations that an increasingly sedentary life is a natural part of the aging process . fourthly , this study was able to explore whether different built environments ( with varying public transport services and population densities ) were associated with higher or lower levels of active travel . while the lack of difference in time spent walking ( or walking / biking combined ) between locations observed in this study could potentially be due to the size of the groups in the subsample , there is no reason to suspect that this particular finding is simply spurious in nature . we were able to gather a range of information about each location , including older people 's perceptions of the age- and disability - friendliness of their respective communities , their safeness and closeness to amenities and family / friends , and differences in public transport services . interestingly , similar numbers of participants in each location identified being close to shops , and so forth as a reason for living in their current communities . thus this particular feature of the environment was not one that differentiated the inner city subsample from the other three lower density areas , even though higher density areas are often touted to be places that provide greater and easier access to amenities , goods , and services than lower density areas . previous research indicates that it is proximity to particular destinations ( especially shopping malls , retail outlets , and places of employment ) and not population density that is associated with the walking behavior of older people ( see ) . despite differences in public transport services in each study location and in older residents ' perceptions of the age- and disability - friendliness of their respective communities , the emergent pattern of the proportion of time traveled by active means over seven days by older people was remarkably similar this along with the finding from the whole sample analysis that only one - third engaged in active travel for more than two hours , a proportion that is consistent with findings from large population studies of older people 's walking behaviour [ 19 , 20 ] , raises the possibility that variability in the walking behavior of older people has more to do with normal variation within populations ( as a consequence of numerous factors ) than it does to features of the surrounding environment . together with the absence of any convincing evidence that there is a direct relationship between the built environment and older people 's walking behavior and the many barriers identified in the extant empirical literature which deter older people from venturing out of home on foot , our study 's findings give grounds for questioning any taken - for - granted assumption that older people will walk outdoors more frequently if the surrounding environment is made more a universal approach may need to be taken with respect to the provision of community - based walking programs for older people ( discussed earlier ) to ensure they are widely available , irrespective of the nature of the surrounding built environment of their homes , if a substantial increase in the outdoor physical activity of this segment of the population is to be achieved . fifthly , the mixed - methods approach used in this study provided the opportunity to explore connections between self - ratings of being active and objective measures of physical activity outside of the home domain via gps tracking , as well as comparison of physical activity across four different settings that differ by population density and built environment ( including public transport services ) . the capturing of time spent biking is an additional strength of this study , since research that relies on walking behavior alone ignores physical activity undertaken through alternative means . by instructing participants to document all modes of transport used for their out - of - home travel in combination with gps tracking , this research avoided the circumstance where the research process itself leads participants to modify their walking behavior . this is a limitation of studies based on accelerometer - based information , the results of which do not necessarily reflect people 's usual pattern of walking , because participation procedures ( i.e. , wearing an accelerometer ) potentially prompt individuals to walk more frequently than normal for the monitored period . finally , further research based on objective measures of walking ( as well as larger samples recruited from urban and rural settings ) is needed if evidence - based determinations are to be made about the extent to which particular built environments foster greater levels of walking among older people ( as proposed by giles - corti et al . ) . the constraints imposed on the size of this study 's sample by the nature of its methodological approach may be averted by future studies , given that technological advancements in recent years are making this approach increasingly feasible for larger samples . the developments of digital diaries , which are able to be used by participants at the same time and on the same device as gps tracking takes place , hold great promise in their capacity to streamline data collection of movement and activity information , as well as data management and analysis , and may also reduce participant burden ( see draijer et al . ) . whether or not these new devices are useful for older samples ( who may resist making diary entries in a technological device in place of paper and pencil diaries ) remains to be seen however . the high quality of data produced by gps / travel diary methods suggests significant benefits could accrue from the trialing of these new technologies with older samples . if successful , substantial headway could be made in expanding the body of evidence on the relationship between the built environment and older people 's walking behavior gathered from high quality measures of walking . active aging is being promoted as a key policy agenda for dealing with the growing aging populations in industrialized nations , this particular subject area warrants much greater attention than it has attracted to date . it is imperative that older people understand the nature of all the constituents of the active aging concept as outlined by the who if the objectives of its policy framework are to be met . currently , evidence related to the meanings older people ascribe to this concept as well as the particular constituents of active aging that inform this group 's self - ratings of being active is limited . more research is needed to determine if older populations are prone to connect active aging with getting out and about and doing things in the present more than they do behaviors such as walking outdoors as a means to maintain their health into the future . active aging is extremely broad in scope compared to its conceptual predecessors ( successful , it may therefore be necessary for messages communicated to the public under the banner of active aging to clearly articulate and emphasize the need for physical activity as a means to maintain health and prolong older people 's participation within society . furthermore , the flurry of research interest in the relationship between the built environment and older people 's walking behavior that appears to have been sparked by the who 's global age - friendly cities publication has not been matched by similar interest in other areas pertaining to active aging . researchers and policy makers alike need to remain mindful that the built environment is just one of many factors affecting older people 's opportunities to age actively and that modifications to the built environment may be insufficient to the task of overturning older people 's unwillingness to move outdoors on foot through fear of falls and injuries from traffic and poorly maintained pedestrian infrastructure , their expectations about aging , or the perceived comfort and convenience of the motor vehicle .

we examined whether self - ratings of being active among older people living in four different settings ( major city high and lower density suburbs , a regional city , and a rural area ) were associated with out - of - home participation and outdoor physical activity . a mixed - methods approach ( survey , travel diary , and gps tracking over a one - week period ) was used to gather data from 48 individuals aged over 55 years . self - ratings of being active were found to be positively correlated with the number of days older people spent time away from home but unrelated to time traveled by active means ( walking and biking ) . no significant differences in active travel were found between the four study locations , despite differences in their respective built environments . the findings suggest that additional strategies to the creation of age - friendly environments are needed if older people are to increase their levels of outdoor physical activity . active aging promotion campaigns may need to explicitly identify the benefits of walking outdoors to ambulatory older people as a means of maintaining their overall health , functional ability , and participation within society in the long - term and also encourage the development of community - based programs in order to facilitate regular walking for this group .

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

together , the world health organization 's policy framework for active aging and its guide for the development of age - friendly cities have set a policy and research agenda in which the built environment is conceived to play a crucial role as a facilitator or inhibitor of older people 's capacity to age actively . this theoretical standpoint assumes that built environments which are conducive to the outdoor mobility of older people will help provide them with ongoing opportunities for both participation within society and maintenance of their health through the gaining of physical exercise as they move around their neighborhoods . it remains unclear , however , whether or not older people 's self - assessments of being active are linked to either their levels of participation within society or their engagement in outdoor physical activity , and , to date , there is no convincing evidence of a direct relationship between the built environment and older people 's walking behavior . active aging is defined by the who [ 1 , page 12 ] as the process of optimizing opportunities for health , participation , and security in order to enhance quality of life as people age . in terms of policy , active aging has been acknowledged as providing a sound basis for responding to the challenges posed by population aging in industrialized nations by linking a number of key policy domains including employment , pensions , retirement , health , and citizenship [ 3 , page 121 ] . the usefulness of the active aging concept for policy makers aiming to comprehensively address issues associated with the growing aging population will necessarily rest on their capacity to devise a coherent set of strategies that effectively deal with all of the three key determinants of active aging identified by the who participation , health , and security . one key opportunity presented to researchers by the development of the active aging concept is its potential to act as a driver for greater attention on the older person - macroenvironment relationship . nevertheless , a review of empirical literature recently undertaken by hung and colleagues grouped active aging with successful , productive , positive , and robust aging as cognates of healthy agingdespite the fact that the active aging concept encompasses more dimensions than any of these other concepts ( including continued participation in society ; the maximization of social , mental , and physical health ; the maintenance of dignity , self - efficacy , and human rights ; and the creation of age - friendly physical environments to facilitate autonomy and independence ) ( see [ 3 , 7 ] ) . it is also noteworthy that successful aging has now been a focus of research for around half a century and there is still no widely agreed definition for this term or consistency between studies in the measures used to investigate this subject area , and the first rigorous analysis of measures was only undertaken recently ( see ) . multiple regression analyses revealed that health status , longstanding illness , and quality of life ( all based on self - report ) explained 41% of the variance in self - ratings of active aging , while sociodemographic and economic variables were not significantly associated with this outcome [ 7 , page 298 ] . from bowling 's research , it would seem that individual factors such as health status and longstanding illness impact on older people 's self - assessments of aging actively , but we currently have little understanding of whether or not there is any relationship between these self - assessments and the actual behaviors of older persons with respect to their actual levels of participation and physical activity outside of the home domain . older people 's participation in the wider community necessarily relies on their capacity for remaining mobile in their out - of - home environments . the accumulated body of evidence suggests that individual factors as well as multiple aspects of the built environment affect older people 's potential out - of - home mobility and thus their capacity to participate in society . engagement in outdoor physical activity by older people is crucial to them remaining ambulatory and also provides numerous other health benefits . in the united states , lack of physical activity among older people has been described as a major public health issue ; the release of surgeon - general 's report in 1996 indicated that less than one - third of adults aged 65 years and over met health recommendations of moderate intensity exercise ( e.g. a systematic review of studies investigating physical activity among older adults ( limited to those studies which reported their findings in terms of older people meeting exercise guidelines ) revealed that this proportion ranges between 2.4 and 83.0 percent , with the majority of the 53 studies reviewed being found to report proportions of between 20 and 60 percent . in addition , the vast majority of available studies of walking behavior among older people are based on self - report , making the findings subject to bias ( e.g. concluded that although evidence of a direct relationship between the built environment and elderly walking behavior was lacking , of all of the features of the built environment investigated , high density of intersections , street and traffic conditions , proximity to destinations , and green space were the factors most likely to have a direct effect on older people 's out - of - home mobility . nevertheless , a study of the consumption patterns and mode of travel used by older people ( via gps tracking ) for accessing goods and services within two different countries ( suburbs of canada and france ) showed that older people in both locations were highly reliant on travel by motor vehicle , despite substantial differences in the built environment and availability of public transport in the two study sites . this raises questions about the assumption that levels of walking outdoors among the older segment of the population will naturally follow modifications to the built environment that make them more age - friendly , without a shift away from sedentary lifestyles and a change in the propensity of the elderly to rely predominantly on motor vehicles for movement outside of the home domain . a pilot test of a behavioral intervention aimed at altering belief in the inevitability of becoming sedentary as a consequence of the aging process demonstrated that older people not only increased their walking after intervention but also experienced multiple other benefits including ease in performing daily life tasks and improvements in mental health - related quality of life , pain , energy , and quality of sleep . research undertaken in berlin with community - based and institutionalized adults aged 70 years and over , for example , indicates that older people spent the majority of their day alone at home ( 80% of awake time ) and less than a fifth of their day outdoors ( 18.7% ) . similarly , more recent australian research indicates that only a small proportion of time is spent away from home ( 90.2% spent at home ) by community - based adults aged 65 years and over , with the mean number of episodes spent away from home in the previous week being 6.3 ( sd = 4.5 ; range 019 ) . the current study used a mixed - methods approach to explore areas of interest within the area of active aging that are currently underresearched , including ( 1 ) the connection between older people 's self - perceptions of being active and indicators of their health and participation ( domains which are proposed by the who to be two of the three key determinants of active aging ) ; ( 2 ) older people 's perceptions of characteristics of their respective communities , which are generally conceived to facilitate aging in place ; and ( 3 ) the extent to which older people 's out - of - home physical activity differs across different built environments , based on high quality objective measures of active travel ( walking and biking ) . a convenience sampling method was used to recruit a total sample of 48 adults aged 55 years and over , comprising 4 subsamples of equal numbers ( n = 12 ) of individuals living in ( 1 ) inner city suburbs and ( 2 ) suburbs outside the inner city area of a capital city , ( 3 ) a regional city , and ( 4 ) a rural town in queensland , australia . the second recruitment method involved key community organisations and groups being approached to assist in identifying potential participants living in brisbane suburbs situated outside the inner city , toowoomba ( a regional city ) , and roma ( a rural town ) , as well as one individual to complete the brisbane inner city subsample . the population density for each of the four study locations the mean age for three of the subsamples was almost identical , but the mean age of the regional city ( rc ) subsample was between 2.5 and 2.7 years higher than the inner city ( ic ) , city suburban ( cs ) , and rural town ( rt ) groups . quantitative data were gathered from a gps device ( which captured one logged position every one minute and was used to track all out - of - home travel over seven consecutive days ) as well as responses to a brief survey contained in the front section of a travel diary . qualitative data were obtained from daily travel diary entries made by each participant ( documenting all out - of - home travel , activities undertaken outside the environment , and the mode of transport used for each trip ) . self - perceptions of being healthy and being active were tapped from responses to two single - item , five - level likert scales ( options and coding for these variables are reported in table 3 ) . travel diary entries were used in conjunction with gps data for determining the number of days when participants ventured out of home , their use of public transport , time spent travelling on foot and by bicycle outside of the home environment , and the total time spent traveling outside home over the seven - day tracking period . pearson correlation analyses were performed to identify relationships between select variables for the entire sample for exploratory purposes to determine relationships between self - perceptions of being active and being healthy and objective measures of participation ( number of days when participants travelled out of their homes ) and travel by active means outside the home environment . one - third of the whole sample identified their respective communities as being safe areas ( 33.3% ) and similar proportions reported proximity as a factor that contributed to their reasons for living in their current communities ( close to family / friends more older people living in the rc location identified being close to family / friends than those in the ic , cs , and rt groups ( 7 versus 3 ) as a reason for living in their current community , while similar numbers selected being close to shops , and so forth . only two older people living in the cs area agreed that safeness of the area was a reason for them living in their communities ( compared to 4 or 5 in the other three groups ) . the patterns of self - ratings of being active and of being healthy were each found to follow a normal distribution with a left skew ( prone to being healthy and active ) , results that are to be expected for a community - based older sample . no relationship was found to exist between self - ratings of being active and self - rated health status ( r = 0.24 ; p = 0.098 ) . self - perceptions of being active were also found to be unrelated to age ( r = 0.15 ; p = 0.317 ) , but a statistically significant , negative correlation was found between self - rated health status and age ( r = 0.32 ; p = 0.027 ) . the number of days on which participants ventured out of home was positively associated with their self - ratings of being active ( r = 0.36 ; p = 0.011 ) but was unrelated to both self - ratings of health ( r = 0.11 ; p = 0.468 ) and age ( r = 0.26 ; p = 0.072 ) , and the subsample means and medians for days travelled outside home were remarkably similar ( see table 3 ) . the actual time spent walking and time spent walking and biking combined by individuals living within the four study locations ( based on gps data ) are shown in the lower section of table 4 . no correlation was found between time spent walking and self - perceptions of being active ( r = 0.12 ; p = 0.401 ) and being healthy ( r = 0.12 ; p = 0.414 ) or age ( r = 0.06 ; p = 0.688 ) . in table 4 , it can also be seen that the number of nonwalkers in the lower density suburbs of brisbane was twice that of their counterparts living in the inner city suburbs of brisbane , with the numbers of nonwalkers in the ic group being similar to both the rc and rt groups . once time spent traveling by bicycle was taken into account , only three individuals in the cs group were found to have spent no time using active means of travel ( on foot or biking ) , but the remainder of the findings were similar to those observed for walking . time spent travelling by active means was found to be unrelated to both self - ratings of being active ( r = 0.16 ; p = 0.279 ) and healthy ( r = 0.18 ; p = 0.216 ) and to age ( r = 0.16 ; p = 0.284 ) . no significant difference between the four study locations was identified in the mean rankings of active travel ( based on actual minutes ) either ( h(3 ) = 0.520 , p = 0.915 ) . a visual representation of the lack of correlation between self - perceptions of being active and use of active means of travel is shown in figure 1 . note , for example , that self - ratings of being active among those who spent no time walking or biking are distributed across four categories , from very inactive to very active . the similarity in the overall pattern that emerged for each of the four study locations is clearly evident and the kruskal - wallis test confirmed that there was no significant difference in the mean rankings of the proportion of overall time spent travelling by active means across the four study locations ( h(3 ) = 0.457 , p = 0.928 ) . firstly , the whole sample analyses enabled determinations to be made about the distribution of self - ratings of being active among community - based adults living in australia . replication studies of bowling 's [ 7 , 10 ] research are needed in australia and elsewhere in order to further understand older people 's perceptions about the key constituents of active aging from their own perspective if we are to gauge similarities and differences across different cultures and countries . secondly , the current study was able to investigate correlates of self - ratings of being active beyond those examined by previous studies . our finding that age was unrelated to self - ratings of being active is consistent with bowling 's study . our finding that self - rated health status was unrelated to self - perceptions of being active differs however . thirdly , this study had the capacity to explore whether self - ratings of being active were connected with actual behavior . the fact that these self - ratings were positively correlated with the number of days when time was spent out of home but unrelated to levels of out - of - home physical activity raises the possibility that older people in this study have embraced the idea that active aging is primarily about one 's current participation within society . future research efforts need to be directed at exploring the degree that self - assessments of being active as people age correspond with objective measures of both participation and outdoor physical activity . active aging objectives are unlikely to be met if a gap exists between the meanings ascribed to active aging by older people and their actual behavior with respect to the latter . if this level of outdoor physical activity is representative of these participants ' normal pattern of walking outdoors , their long - term prospects of remaining ambulatory and capable of performing daily life activities could potentially be compromised , with this in turn eventually undermining their capacity for living independently , aging in place , and participating in society . given the accumulated evidence about barriers to walking ( see [ 16 , 27 ] ) and the difficulties associated with using public transport experienced by older people , as well as this group 's reliance on cars [ 12 , 24 ] , it may be that the developments of community - based programs that encourage walking behavior among older people with a specific focus on recreational walking in spaces where pedestrian infrastructure is in good condition and free of traffic ( e.g. this approach could potentially create opportunities for older people to overcome their fears about moving outdoors , increase their number of social interactions , maintain or improve their overall health and well - being through the gaining of regular exercise [ 18 , 31 ] , and also change expectations that an increasingly sedentary life is a natural part of the aging process . fourthly , this study was able to explore whether different built environments ( with varying public transport services and population densities ) were associated with higher or lower levels of active travel . we were able to gather a range of information about each location , including older people 's perceptions of the age- and disability - friendliness of their respective communities , their safeness and closeness to amenities and family / friends , and differences in public transport services . despite differences in public transport services in each study location and in older residents ' perceptions of the age- and disability - friendliness of their respective communities , the emergent pattern of the proportion of time traveled by active means over seven days by older people was remarkably similar this along with the finding from the whole sample analysis that only one - third engaged in active travel for more than two hours , a proportion that is consistent with findings from large population studies of older people 's walking behaviour [ 19 , 20 ] , raises the possibility that variability in the walking behavior of older people has more to do with normal variation within populations ( as a consequence of numerous factors ) than it does to features of the surrounding environment . together with the absence of any convincing evidence that there is a direct relationship between the built environment and older people 's walking behavior and the many barriers identified in the extant empirical literature which deter older people from venturing out of home on foot , our study 's findings give grounds for questioning any taken - for - granted assumption that older people will walk outdoors more frequently if the surrounding environment is made more a universal approach may need to be taken with respect to the provision of community - based walking programs for older people ( discussed earlier ) to ensure they are widely available , irrespective of the nature of the surrounding built environment of their homes , if a substantial increase in the outdoor physical activity of this segment of the population is to be achieved . fifthly , the mixed - methods approach used in this study provided the opportunity to explore connections between self - ratings of being active and objective measures of physical activity outside of the home domain via gps tracking , as well as comparison of physical activity across four different settings that differ by population density and built environment ( including public transport services ) . by instructing participants to document all modes of transport used for their out - of - home travel in combination with gps tracking , this research avoided the circumstance where the research process itself leads participants to modify their walking behavior . finally , further research based on objective measures of walking ( as well as larger samples recruited from urban and rural settings ) is needed if evidence - based determinations are to be made about the extent to which particular built environments foster greater levels of walking among older people ( as proposed by giles - corti et al . ) it is imperative that older people understand the nature of all the constituents of the active aging concept as outlined by the who if the objectives of its policy framework are to be met . currently , evidence related to the meanings older people ascribe to this concept as well as the particular constituents of active aging that inform this group 's self - ratings of being active is limited . more research is needed to determine if older populations are prone to connect active aging with getting out and about and doing things in the present more than they do behaviors such as walking outdoors as a means to maintain their health into the future . active aging is extremely broad in scope compared to its conceptual predecessors ( successful , it may therefore be necessary for messages communicated to the public under the banner of active aging to clearly articulate and emphasize the need for physical activity as a means to maintain health and prolong older people 's participation within society . furthermore , the flurry of research interest in the relationship between the built environment and older people 's walking behavior that appears to have been sparked by the who 's global age - friendly cities publication has not been matched by similar interest in other areas pertaining to active aging .

pharmacological treatment with psychotropic drugs ( i.e. antipsychotics , anxiolytics , hypnotics , and antidepressants ) is usually standard treatment and provision of psychotherapy is scarce in this age group ( alvidrez and arean 2002 ) . in addition to treatment of mental health problems , such as depression , anxiety , and insomnia , psychotopic drugs are also prescribed to older patients for behavioural and psychological symptoms of dementia ( bpsd ) ( gustafsson et al . , 2013 ) . thus , psychotropic drugs are used extensively among older people ( johnell and fastbom 2012 ) and there is a risk that these drugs are used longterm and offlabel ( maust et al . , 2015a ) . with aging come altered pharmacokinetics and pharmacodynamics , which result in prolonged and increased effects of many drugs . the altered pharmacodynamics of the aging brain leads to a higher sensitivity to central nervous system acting agents ( shi et al . , 2008 ) and these drugs are estimated to cause up to 20% of all drugrelated hospitalizations in older persons ( salvi et al . , 2012 ) . psychotropic drugs are a welldocumented risk factor for fall injuries ( bloch et al . , 2011 ) , which can cause serious adverse outcomes in older persons including increased mortality risk ( alamgir et al . , 2012 ) studies have also suggested a direct link between antipsychotic use and increased mortality in frail older persons ( huybrechts et al . , 2012 ; kales et al . , 2012 ; maust et al . , 2015b ; rochon et al . , 2008 however , few previous investigations have analyzed risk of fall injuries , hospitalizations , and mortality within the same study . the cumulative effect of use of multiple psychotropic drugs has gained little previous attention , although polypharmacy is a recognized problem in old age pharmacotherapy . the burden of multiple uses of psychotropic drugs on the aging brain can increase the risk of adverse drug reactions and should therefore be avoided in elderly patients ( hartikainen et al . , 2005 ) . nevertheless , concurrent use of several psychotropics is common in the older population in sweden , although this is considered as inappropriate prescribing in national guidelines ( johnell et al . , 2007 ) . sweden has excellent possibilities for largescale epidemiological studies through the longstanding tradition of national registers with almost complete coverage . these registers can be individually recordlinked to enable detailed analyzes of rich databases with high statistical precision . the introduction of the swedish prescribed drug register in 2005 was an important addition to the national health data in sweden and represents one of the largest pharmacoepidemiological databases in the world ( wettermark et al . , 2007 ) . analyzes of these large databases can circumvent shortcomings of other studies based on small and selected samples of older individuals . thus , we aimed to investigate whether psychotropic drugs are associated with an increased risk of fall injuries , hospitalizations , and mortality in a large general population of older adults by applying a registerbased case control study based on several swedish registers through recordlinkage based on the personal identification number . we analyzed whether use of psychotropic drugs was associated with risk of three outcomes : fall injuries , hospitalizations , and mortality ( n = 1,288,875 older adults ) . first , information about hospitalizations and diagnoses were collected from the swedish patient register , which covers all inpatient and specialized outpatient care in the whole of sweden ( ludvigsson et al . , 2011 ) . diagnoses are registered according to the international classification of diseases 10 ( icd 10 ) . we selected all persons aged 65 years and older who had been hospitalized after a fall between 1 january and 31 december 2011 . similarly , we also selected all persons aged 65 years and older who had an unplanned hospitalization for any cause . however , we excluded suicides and deaths because of an event of undetermined intent ( icd 10 codes x60x84 and y10y14 ) . second , from the register of the total population , we selected five controls matched for age in 5year classes and sex by using an incidence density sampling scheme ( lubin and gail 1984 ) . hence , for each case , we selected controls from the age and gender specific atrisk populations . third , we obtained drug data from the swedish prescribed drug register , which has detailed individualbased information about all prescribed dispensed drugs in sweden including anatomical therapeutic chemical ( atc ) codes ( wettermark et al . drug data was collected for the time period of one year before the outcome ( i.e. fall injuries , hospitalizations or mortality ) . finally , information about educational level of the patients on 31 december 2010 was collected from the swedish education register , where the highest attained level of formal education is registered for individuals aged 16 years and older . fall injuries were identified through fractures of the femur ( icd 10 codes s72s74 ) following a falling accident ( icd 10 codes w00 , w01 , w03w11 , w18 , and w19 ) . number of inpatient days the year before the adverse outcome was calculated as an overall measure of comorbidity ( schneeweiss et al . , 2001 ) . was used as a continuous variable of comorbidity based on inpatient and specialized outpatient data within five years prior to the outcome . we also collected information about diagnosis of dementia ( icd 10 codes f00f03 , g30 , g31 ) within five years prior to the outcome . this information together with data on antidementia drugs ( atc code n06d ) from the drug register was used to form the dementia variable . psychotropic drugs ( wastesson et al . , 2014 ) included antipsychotics ( atc code n05 excluding n05an01 ) , anxiolytics ( n05b ) , hypnotics / sedatives ( n05c ) , and antidepressants ( n06a ) and use was defined as filling of at least three prescriptions . the individual effect of each drug class of psychotropics as well as the combined effect of use of several psychotropic drug classes were analyzed . number of other drugs ( continuous variable ) , excluding psychotropic drugs , was also used as an overall proxy for comorbidity and polypharmacy ( schneeweiss et al . , 2001 ) . educational level was categorized into primary school , secondary school , and university ( wastesson et al . , 2015 ) . both univariate and multivariate conditional logistic regression analysis was used for investigating the association between use of psychotropic drugs and the three outcomes ; fall injuries , hospitalizations , and mortality . in the univariate model , the cases and controls were matched for age , sex , and case event day , but otherwise unadjusted . the multivariate model was additionally adjusted for education , number of inpatient days , charlson comorbidity index , dementia , and number of other drugs . the results are expressed as odds ratios ( ors ) with 95% confidence intervals ( cis ) . the ors from this design , in which controls are selected from the atrisk group , can be interpreted as incidence rate ratios ( rodrigues and kirkwood 1990 ) . first , information about hospitalizations and diagnoses were collected from the swedish patient register , which covers all inpatient and specialized outpatient care in the whole of sweden ( ludvigsson et al . , 2011 ) . diagnoses are registered according to the international classification of diseases 10 ( icd 10 ) . we selected all persons aged 65 years and older who had been hospitalized after a fall between 1 january and 31 december 2011 . similarly , we also selected all persons aged 65 years and older who had an unplanned hospitalization for any cause . mortality during the same period was collected from the swedish cause of death register . however , we excluded suicides and deaths because of an event of undetermined intent ( icd 10 codes x60x84 and y10y14 ) . second , from the register of the total population , we selected five controls matched for age in 5year classes and sex by using an incidence density sampling scheme ( lubin and gail 1984 ) . hence , for each case , we selected controls from the age and gender specific atrisk populations . third , we obtained drug data from the swedish prescribed drug register , which has detailed individualbased information about all prescribed dispensed drugs in sweden including anatomical therapeutic chemical ( atc ) codes ( wettermark et al . , 2007 ) . drug data was collected for the time period of one year before the outcome ( i.e. fall injuries , hospitalizations or mortality ) . finally , information about educational level of the patients on 31 december 2010 was collected from the swedish education register , where the highest attained level of formal education is registered for individuals aged 16 years and older . fall injuries were identified through fractures of the femur ( icd 10 codes s72s74 ) following a falling accident ( icd 10 codes w00 , w01 , w03w11 , w18 , and w19 ) . number of inpatient days the year before the adverse outcome was calculated as an overall measure of comorbidity ( schneeweiss et al . , 2001 ) . was used as a continuous variable of comorbidity based on inpatient and specialized outpatient data within five years prior to the outcome . we also collected information about diagnosis of dementia ( icd 10 codes f00f03 , g30 , g31 ) within five years prior to the outcome . this information together with data on antidementia drugs ( atc code n06d ) from the drug register was used to form the dementia variable . psychotropic drugs ( wastesson et al . , 2014 ) included antipsychotics ( atc code n05 excluding n05an01 ) , anxiolytics ( n05b ) , hypnotics / sedatives ( n05c ) , and antidepressants ( n06a ) and use was defined as filling of at least three prescriptions . the individual effect of each drug class of psychotropics as well as the combined effect of use of several psychotropic drug classes were analyzed . number of other drugs ( continuous variable ) , excluding psychotropic drugs , was also used as an overall proxy for comorbidity and polypharmacy ( schneeweiss et al . , 2001 ) . educational level was categorized into primary school , secondary school , and university ( wastesson et al . , 2015 ) . both univariate and multivariate conditional logistic regression analysis was used for investigating the association between use of psychotropic drugs and the three outcomes ; fall injuries , hospitalizations , and mortality . in the univariate model , the cases and controls were matched for age , sex , and case event day , but otherwise unadjusted . the multivariate model was additionally adjusted for education , number of inpatient days , charlson comorbidity index , dementia , and number of other drugs . the results are expressed as odds ratios ( ors ) with 95% confidence intervals ( cis ) . the ors from this design , in which controls are selected from the atrisk group , can be interpreted as incidence rate ratios ( rodrigues and kirkwood 1990 ) . for the outcomes fall injuries and mortality , the mean age was similar ( 82.3 years and 83.9 years , respectively ) , whereas the subpopulation for hospitalizations was younger ( 79.1 years ) ( table 1 ) . the proportion of women was similar for hospitalizations and mortality ( 53.5% and 53.6% , respectively ) , but higher for fall injuries ( 66.6% ) . approximately 86,843 elderly persons experienced two or more of the three detrimental outcomes during the observed study period . sociodemographic characteristics and psychotropic drug use in cases ( fall injuries , hospitalizations or death ) , and controls among persons aged 65 years and older in sweden 2011 the subpopulation for hospitalizations had on average a higher educational level compared with the two other outcomes ( table 1 ) . dementia was about twice as common in the mortality group and among elderly hospitalized after a fall injury ( 13.3% and 11.5% , respectively ) than among elderly persons hospitalized for any cause . use of psychotropic drugs , also concomitantly , was most common in the fall injury and mortality groups . antidepressants were the psychotropic most strongly related to fall injuries ( adjusted or : 1.42 ; 95 ci : 1.381.45 ) ( table 2 ) . also antipsychotics , but less so hypnotics and sedatives , were associated with fall injuries . moreover , number of psychotropic drugs was associated with increased risk of fall injuries in a dose response manner ( 4 psychotropics vs 0 : adjusted or : 1.53 ; 95% ci : 1.391.68 ) . many hospital days , a high level of comorbidity , dementia , and use of many other drugs were also associated with a greater risk for fall injuries . matched unadjusted and adjusted odds ratios ( ors ) with 95% confidence intervals ( cis ) for fall injuries in persons aged 65 years and older in sweden 2011 matched for age , sex , and case event day . matched for age , sex , and case event day and adjusted for all variables in table . antipsychotics were the psychotropic most strongly related to hospitalizations ( adjusted or : 1.22 ; 95% ci : 1.191.24 ) ( table 3 ) . number of psychotropic drugs was also related to risk of hospitalization in a dose response manner ( 4 psychotropics vs 0 : adjusted or : 1.27 ; 95% ci : 1.221.33 ) . dementia and number of other drugs were also risk factors for hospitalization . matched crude and adjusted odds ratios ( ors ) with 95% confidence intervals ( cis ) for hospitalizations in persons aged 65 years and older in sweden 2011 matched for age , sex , and case event day . matched for age , sex , and case event day and adjusted for all variables in table . use of antipsychotics was strongly associated with risk of death ( adjusted or : 2.10 ; 95% ci : 2.022.17 ) ( table 4 ) . also antidepressants and anxiolytics were associated with a higher risk of death , whereas hypnotics and sedatives were associated with a lower risk . number of psychotropic drugs was also strongly related to death in a dose response manner ( 4 psychotropics vs 0 : adjusted or : 2.50 ; 95% ci : 2.332.69 ) . dementia was also a strong predictor of death ( adjusted or : 2.14 ; 95% ci : 2.082.20 ) . matched crude and adjusted odds ratios ( ors ) with 95% confidence intervals ( cis ) for mortality in persons aged 65 years and older in sweden 2011 matched for age , sex , and case event day . matched for age , sex , and case event day and adjusted for all variables in table . we also repeated the analyzes separately for the subgroup of individuals with dementia ( n = 58,984 ; data not shown in table ) . in this analysis however , antidepressants were associated with a decreased risk of hospitalizations ( adjusted or : 0.93 ; 95% ci : 0.890.96 ) . on the other hand , there was an increased risk of death associated with use of antipsychotics ( adjusted or : 1.50 ; 95% ci : 1.411.60 ) , antidepressants ( adjusted or : 1.14 ; 95% ci : 1.081.21 ) and anxiolytics ( adjusted or : 1.33 ; 95% ci : 1.251.41 ) . finally , a dose response relationship was found between number of psychotropics and risk of death ( 4 psychotropics vs 0 : adjusted or : 1.99 ; 95% ci : 1.762.25 ) . our large study of a general population of older persons shows that psychotropic drugs , particularly when combined , are associated with risk of fall injuries , hospitalizations , and mortality among older persons . this risk existed also after adjustment for comorbidity and dementia . to our knowledge , this is the largest investigation of multiple uses of psychotropics and risk of fall injuries , hospitalizations , and death within the same study . research into psychotropics as risk factors for fall injuries begun already in the late eighties ( campbell 1991 ) . since then , reviews have established use of psychotropics as a risk factor for falls ( bloch et al . , 2011 ; woolcott et al . , we can now confirm these findings in large national data and with high statistical precision . among the psychotropic drugs , antidepressants were most strongly related to fall injuries . this is in line with recent research that has pointed out the risk of falls associated with these drugs ( huang et al . , 2012 ) . given that antidepressants are used extensively by older persons ( johnell and fastbom 2009 ) , particularly in the institutional setting ( johnell and fastbom 2012 ) , the amount of exposure to these presumably fallinducing drugs in the elderly population is substantial . safer alternatives to pharmacological treatment of depression in old age might be psychotherapy in cognitively intact persons ( karlin et al . , 2015 ; krishna et al . , 2011 ) and improved care strategies for dementia patients ( american geriatrics society and american association for geriatric psychiatry , 2003 ) . in particular , our data suggest more attention to psychotropic polypharmacy as a target for preventive strategies for fall injuries in older people . concomitant use of several psychotropics was also a risk factor for hospitalizations in a dose response manner . this is , to our knowledge , the first time that this relationship has been reported in the international scientific literature . for the most serious outcome of all there is a growing body of evidence that has pointed out use of antipsychotics in advanced age as a risk factor for mortality ( huybrechts et al . , 2012 ; kales et al . , 2012 ; rochon et al . , most of these studies , however , have been conducted in dementia patients . here , we can show that antipsychotics are associated with death also in a general population of older persons , independently of dementia status . we also found that , although to a less extent , antidepressants , and anxiolytics were associated with a higher risk of death , whereas hypnotics and sedatives were associated with a lower risk . the most common type of anxiolytic benzodiazepines has previously been investigated in relation to mortality among older people . however , the findings have so far been inconsistent ( charlson et al . , we can now provide support for anxiolytics as possible risk factors for mortality in old age . however , hypnotics and sedatives were related to a lower risk of death in our study . hypnotics and sedatives may likely be used on a regular basis , whereas anxiolytics are probably used more irregularly . hypnotics and sedatives are taken before bedtime and may therefore exert influence only during sleep , whereas anxiolytics are used during day time and may therefore be more related to adverse outcomes such as day time excessive sedation , injuries , falls , and fractures ( johnell et al . , 2014 ) . smaller studies of dementia patients have also found an association between use of several psychotropics and mortality ( hartikainen et al . , 2005 ) . however , our study is probably the first to confirm this finding in a dose responsive manner in a very large and unselected population of older persons . the swedish national board of health and welfare discourages prescribing of psychotropic polypharmacy ( i.e. 3 psychotropics ) to older patients in their national guidelines ( johnell et al . , 2007 ) . this underlines that prevention and treatment of dementia must be prioritized for improving the health and wellbeing of elderly persons . the swedish prescribed drug register does not include information about the underlying indications and diagnoses for prescription of drugs . therefore , we do not know for which psychiatric symptoms the psychotropic drugs were prescribed for . the swedish prescribed drug register does not either include data on overthecounter drugs . however , the included psychotropic drugs are only available through prescriptions in sweden . in addition , the register does not include drugs used in hospitals or from drug storerooms sometimes used in nursing homes . . however , psychotropic drugs are often prescribed without a documented diagnosis to elderly patients ( akincigil et al . , 2011 ) . we also lacked data from primary care , where many older patients with psychiatric problems are treated . thus , it is difficult to obtain data about mental disorders in old patients and therefore also to adequately adjust for underlying psychiatric diagnoses . dementia status was assessed from the swedish patient register and the swedish prescribed drug register . this underestimates the number of dementia cases , as we lack information about dementia diagnoses in primary care ( not included in the patient register ) and undiagnosed dementia . we adjusted for comorbidity through the charlson comorbidity index , number of inpatient days , and number of other drugs . there are also other potential residual confounding factors , example lifestyle and physical status , which were not available in our data . finally , a general limitation related to drug register data is that actual drug use is not assessed given that adherence to treatment can be low . our large study of a general population of older persons shows that psychotropic drugs , particularly when combined , are associated with risk of fall injuries , hospitalizations , and mortality among older persons . this risk existed also after adjustment for comorbidity and dementia . to our knowledge , this is the largest investigation of multiple uses of psychotropics and risk of fall injuries , hospitalizations , and death within the same study . research into psychotropics as risk factors for fall injuries begun already in the late eighties ( campbell 1991 ) . since then , reviews have established use of psychotropics as a risk factor for falls ( bloch et al . , 2011 ; woolcott et al . , we can now confirm these findings in large national data and with high statistical precision . among the psychotropic drugs , antidepressants were most strongly related to fall injuries . this is in line with recent research that has pointed out the risk of falls associated with these drugs ( huang et al . , 2012 ) . given that antidepressants are used extensively by older persons ( johnell and fastbom 2009 ) , particularly in the institutional setting ( johnell and fastbom 2012 ) , the amount of exposure to these presumably fallinducing drugs in the elderly population is substantial . safer alternatives to pharmacological treatment of depression in old age might be psychotherapy in cognitively intact persons ( karlin et al . , 2015 ; krishna et al . , 2011 ) and improved care strategies for dementia patients ( american geriatrics society and american association for geriatric psychiatry , 2003 ) . in particular , our data suggest more attention to psychotropic polypharmacy as a target for preventive strategies for fall injuries in older people . concomitant use of several psychotropics was also a risk factor for hospitalizations in a dose response manner . this is , to our knowledge , the first time that this relationship has been reported in the international scientific literature . for the most serious outcome of all there is a growing body of evidence that has pointed out use of antipsychotics in advanced age as a risk factor for mortality ( huybrechts et al . , 2012 ; kales et al . , 2012 ; rochon et al . , most of these studies , however , have been conducted in dementia patients . here , we can show that antipsychotics are associated with death also in a general population of older persons , independently of dementia status . we also found that , although to a less extent , antidepressants , and anxiolytics were associated with a higher risk of death , whereas hypnotics and sedatives were associated with a lower risk . the most common type of anxiolytic benzodiazepines has previously been investigated in relation to mortality among older people . however , the findings have so far been inconsistent ( charlson et al . , we can now provide support for anxiolytics as possible risk factors for mortality in old age . however , hypnotics and sedatives were related to a lower risk of death in our study . hypnotics and sedatives may likely be used on a regular basis , whereas anxiolytics are probably used more irregularly . hypnotics and sedatives are taken before bedtime and may therefore exert influence only during sleep , whereas anxiolytics are used during day time and may therefore be more related to adverse outcomes such as day time excessive sedation , injuries , falls , and fractures ( johnell et al . , 2014 ) . smaller studies of dementia patients have also found an association between use of several psychotropics and mortality ( hartikainen et al . , 2005 ) . however , our study is probably the first to confirm this finding in a dose responsive manner in a very large and unselected population of older persons . the swedish national board of health and welfare discourages prescribing of psychotropic polypharmacy ( i.e. 3 psychotropics ) to older patients in their national guidelines ( johnell et al . , 2007 ) . this underlines that prevention and treatment of dementia must be prioritized for improving the health and wellbeing of elderly persons . the swedish prescribed drug register does not include information about the underlying indications and diagnoses for prescription of drugs . therefore , we do not know for which psychiatric symptoms the psychotropic drugs were prescribed for . the swedish prescribed drug register does not either include data on overthecounter drugs . however , the included psychotropic drugs are only available through prescriptions in sweden . in addition , the register does not include drugs used in hospitals or from drug storerooms sometimes used in nursing homes . our study may be affected by confounding by indication and disease severity . however , psychotropic drugs are often prescribed without a documented diagnosis to elderly patients ( akincigil et al . , 2011 ) . we also lacked data from primary care , where many older patients with psychiatric problems are treated . thus , it is difficult to obtain data about mental disorders in old patients and therefore also to adequately adjust for underlying psychiatric diagnoses . dementia status was assessed from the swedish patient register and the swedish prescribed drug register . this underestimates the number of dementia cases , as we lack information about dementia diagnoses in primary care ( not included in the patient register ) and undiagnosed dementia . we adjusted for comorbidity through the charlson comorbidity index , number of inpatient days , and number of other drugs . there are also other potential residual confounding factors , example lifestyle and physical status , which were not available in our data . finally , a general limitation related to drug register data is that actual drug use is not assessed given that adherence to treatment can be low . our large nationwide study of a general population of older persons suggests that multiple psychotropic drug use is associated with a higher risk of fall injuries , hospitalizations , and death in a dose response manner . safer nonpharmacological alternatives may be considered given the individual suffering and large societal economic burden related to these serious adverse outcomes . key points our data suggest a dose response relationship between multiple psychotropic drug use and risk of fall injuries , hospitalizations , and death among older personsour findings support a cautious prescribing of multiple psychotropic drugs to older patients . our data suggest a dose response relationship between multiple psychotropic drug use and risk of fall injuries , hospitalizations , and death among older personsour findings support a cautious prescribing of multiple psychotropic drugs to older patients . response relationship between multiple psychotropic drug use and risk of fall injuries , hospitalizations , and death among older persons our findings support a cautious prescribing of multiple psychotropic drugs to older patients .

objectiveto investigate whether psychotropics are associated with an increased risk of fall injuries , hospitalizations , and mortality in a large general population of older adults.methodswe performed a nationwide matched ( age , sex , and case event day ) case control study between 1 january and 31 december 2011 based on several swedish registers ( n = 1,288,875 persons aged 65 years ) . we used multivariate conditional logistic regression adjusted for education , number of inpatient days , charlson comorbidity index , dementia and number of other drugs.resultsantidepressants were the psychotropic most strongly related to fall injuries ( oradjusted : 1.42 ; 95% ci : 1.381.45 ) and antipsychotics to hospitalizations ( oradjusted : 1.22 ; 95% ci : 1.191.24 ) and death ( oradjusted : 2.10 ; 95% ci : 2.022.17 ) . number of psychotropics was associated with increased the risk of fall injuries , ( 4 psychotropics vs 0 : oradjusted : 1.53 ; 95% ci : 1.391.68 ) , hospitalization ( 4 psychotropics vs 0 : oradjusted : 1.27 ; 95% ci : 1.221.33 ) and death ( 4 psychotropics vs 0 : oradjusted : 2.50 ; 95% ci : 2.332.69 ) in a dose response manner . among persons with dementia ( n = 58,984 ) , a dose response relationship was found between number of psychotropics and mortality risk ( 4 psychotropics vs 0 : oradjusted : 1.99 ; 95% ci : 1.762.25).conclusionsour findings support a cautious prescribing of multiple psychotropic drugs to older patients . 2016 the authors . international journal of geriatric psychiatry published by john wiley & sons , ltd .

Introduction Material and methods Study population Measurements Statistical analysis Results Discussion Main findings Limitations Conclusion Conflict of Interest

pharmacological treatment with psychotropic drugs ( i.e. antipsychotics , anxiolytics , hypnotics , and antidepressants ) is usually standard treatment and provision of psychotherapy is scarce in this age group ( alvidrez and arean 2002 ) . in addition to treatment of mental health problems , such as depression , anxiety , and insomnia , psychotopic drugs are also prescribed to older patients for behavioural and psychological symptoms of dementia ( bpsd ) ( gustafsson et al . thus , psychotropic drugs are used extensively among older people ( johnell and fastbom 2012 ) and there is a risk that these drugs are used longterm and offlabel ( maust et al . , 2008 ) and these drugs are estimated to cause up to 20% of all drugrelated hospitalizations in older persons ( salvi et al . psychotropic drugs are a welldocumented risk factor for fall injuries ( bloch et al . , 2011 ) , which can cause serious adverse outcomes in older persons including increased mortality risk ( alamgir et al . , 2012 ) studies have also suggested a direct link between antipsychotic use and increased mortality in frail older persons ( huybrechts et al . , 2008 however , few previous investigations have analyzed risk of fall injuries , hospitalizations , and mortality within the same study . the cumulative effect of use of multiple psychotropic drugs has gained little previous attention , although polypharmacy is a recognized problem in old age pharmacotherapy . the burden of multiple uses of psychotropic drugs on the aging brain can increase the risk of adverse drug reactions and should therefore be avoided in elderly patients ( hartikainen et al . the introduction of the swedish prescribed drug register in 2005 was an important addition to the national health data in sweden and represents one of the largest pharmacoepidemiological databases in the world ( wettermark et al . analyzes of these large databases can circumvent shortcomings of other studies based on small and selected samples of older individuals . thus , we aimed to investigate whether psychotropic drugs are associated with an increased risk of fall injuries , hospitalizations , and mortality in a large general population of older adults by applying a registerbased case control study based on several swedish registers through recordlinkage based on the personal identification number . we analyzed whether use of psychotropic drugs was associated with risk of three outcomes : fall injuries , hospitalizations , and mortality ( n = 1,288,875 older adults ) . we selected all persons aged 65 years and older who had been hospitalized after a fall between 1 january and 31 december 2011 . similarly , we also selected all persons aged 65 years and older who had an unplanned hospitalization for any cause . hence , for each case , we selected controls from the age and gender specific atrisk populations . fall injuries , hospitalizations or mortality ) . finally , information about educational level of the patients on 31 december 2010 was collected from the swedish education register , where the highest attained level of formal education is registered for individuals aged 16 years and older . fall injuries were identified through fractures of the femur ( icd 10 codes s72s74 ) following a falling accident ( icd 10 codes w00 , w01 , w03w11 , w18 , and w19 ) . number of inpatient days the year before the adverse outcome was calculated as an overall measure of comorbidity ( schneeweiss et al . was used as a continuous variable of comorbidity based on inpatient and specialized outpatient data within five years prior to the outcome . we also collected information about diagnosis of dementia ( icd 10 codes f00f03 , g30 , g31 ) within five years prior to the outcome . psychotropic drugs ( wastesson et al . , 2014 ) included antipsychotics ( atc code n05 excluding n05an01 ) , anxiolytics ( n05b ) , hypnotics / sedatives ( n05c ) , and antidepressants ( n06a ) and use was defined as filling of at least three prescriptions . the individual effect of each drug class of psychotropics as well as the combined effect of use of several psychotropic drug classes were analyzed . number of other drugs ( continuous variable ) , excluding psychotropic drugs , was also used as an overall proxy for comorbidity and polypharmacy ( schneeweiss et al . educational level was categorized into primary school , secondary school , and university ( wastesson et al . both univariate and multivariate conditional logistic regression analysis was used for investigating the association between use of psychotropic drugs and the three outcomes ; fall injuries , hospitalizations , and mortality . in the univariate model , the cases and controls were matched for age , sex , and case event day , but otherwise unadjusted . the multivariate model was additionally adjusted for education , number of inpatient days , charlson comorbidity index , dementia , and number of other drugs . we selected all persons aged 65 years and older who had been hospitalized after a fall between 1 january and 31 december 2011 . similarly , we also selected all persons aged 65 years and older who had an unplanned hospitalization for any cause . fall injuries , hospitalizations or mortality ) . finally , information about educational level of the patients on 31 december 2010 was collected from the swedish education register , where the highest attained level of formal education is registered for individuals aged 16 years and older . fall injuries were identified through fractures of the femur ( icd 10 codes s72s74 ) following a falling accident ( icd 10 codes w00 , w01 , w03w11 , w18 , and w19 ) . number of inpatient days the year before the adverse outcome was calculated as an overall measure of comorbidity ( schneeweiss et al . was used as a continuous variable of comorbidity based on inpatient and specialized outpatient data within five years prior to the outcome . we also collected information about diagnosis of dementia ( icd 10 codes f00f03 , g30 , g31 ) within five years prior to the outcome . psychotropic drugs ( wastesson et al . , 2014 ) included antipsychotics ( atc code n05 excluding n05an01 ) , anxiolytics ( n05b ) , hypnotics / sedatives ( n05c ) , and antidepressants ( n06a ) and use was defined as filling of at least three prescriptions . the individual effect of each drug class of psychotropics as well as the combined effect of use of several psychotropic drug classes were analyzed . number of other drugs ( continuous variable ) , excluding psychotropic drugs , was also used as an overall proxy for comorbidity and polypharmacy ( schneeweiss et al . educational level was categorized into primary school , secondary school , and university ( wastesson et al . both univariate and multivariate conditional logistic regression analysis was used for investigating the association between use of psychotropic drugs and the three outcomes ; fall injuries , hospitalizations , and mortality . in the univariate model , the cases and controls were matched for age , sex , and case event day , but otherwise unadjusted . the multivariate model was additionally adjusted for education , number of inpatient days , charlson comorbidity index , dementia , and number of other drugs . for the outcomes fall injuries and mortality , the mean age was similar ( 82.3 years and 83.9 years , respectively ) , whereas the subpopulation for hospitalizations was younger ( 79.1 years ) ( table 1 ) . the proportion of women was similar for hospitalizations and mortality ( 53.5% and 53.6% , respectively ) , but higher for fall injuries ( 66.6% ) . sociodemographic characteristics and psychotropic drug use in cases ( fall injuries , hospitalizations or death ) , and controls among persons aged 65 years and older in sweden 2011 the subpopulation for hospitalizations had on average a higher educational level compared with the two other outcomes ( table 1 ) . use of psychotropic drugs , also concomitantly , was most common in the fall injury and mortality groups . antidepressants were the psychotropic most strongly related to fall injuries ( adjusted or : 1.42 ; 95 ci : 1.381.45 ) ( table 2 ) . also antipsychotics , but less so hypnotics and sedatives , were associated with fall injuries . moreover , number of psychotropic drugs was associated with increased risk of fall injuries in a dose response manner ( 4 psychotropics vs 0 : adjusted or : 1.53 ; 95% ci : 1.391.68 ) . many hospital days , a high level of comorbidity , dementia , and use of many other drugs were also associated with a greater risk for fall injuries . matched unadjusted and adjusted odds ratios ( ors ) with 95% confidence intervals ( cis ) for fall injuries in persons aged 65 years and older in sweden 2011 matched for age , sex , and case event day . matched for age , sex , and case event day and adjusted for all variables in table . antipsychotics were the psychotropic most strongly related to hospitalizations ( adjusted or : 1.22 ; 95% ci : 1.191.24 ) ( table 3 ) . number of psychotropic drugs was also related to risk of hospitalization in a dose response manner ( 4 psychotropics vs 0 : adjusted or : 1.27 ; 95% ci : 1.221.33 ) . dementia and number of other drugs were also risk factors for hospitalization . matched crude and adjusted odds ratios ( ors ) with 95% confidence intervals ( cis ) for hospitalizations in persons aged 65 years and older in sweden 2011 matched for age , sex , and case event day . matched for age , sex , and case event day and adjusted for all variables in table . use of antipsychotics was strongly associated with risk of death ( adjusted or : 2.10 ; 95% ci : 2.022.17 ) ( table 4 ) . also antidepressants and anxiolytics were associated with a higher risk of death , whereas hypnotics and sedatives were associated with a lower risk . number of psychotropic drugs was also strongly related to death in a dose response manner ( 4 psychotropics vs 0 : adjusted or : 2.50 ; 95% ci : 2.332.69 ) . dementia was also a strong predictor of death ( adjusted or : 2.14 ; 95% ci : 2.082.20 ) . matched crude and adjusted odds ratios ( ors ) with 95% confidence intervals ( cis ) for mortality in persons aged 65 years and older in sweden 2011 matched for age , sex , and case event day . matched for age , sex , and case event day and adjusted for all variables in table . we also repeated the analyzes separately for the subgroup of individuals with dementia ( n = 58,984 ; data not shown in table ) . in this analysis however , antidepressants were associated with a decreased risk of hospitalizations ( adjusted or : 0.93 ; 95% ci : 0.890.96 ) . on the other hand , there was an increased risk of death associated with use of antipsychotics ( adjusted or : 1.50 ; 95% ci : 1.411.60 ) , antidepressants ( adjusted or : 1.14 ; 95% ci : 1.081.21 ) and anxiolytics ( adjusted or : 1.33 ; 95% ci : 1.251.41 ) . finally , a dose response relationship was found between number of psychotropics and risk of death ( 4 psychotropics vs 0 : adjusted or : 1.99 ; 95% ci : 1.762.25 ) . our large study of a general population of older persons shows that psychotropic drugs , particularly when combined , are associated with risk of fall injuries , hospitalizations , and mortality among older persons . to our knowledge , this is the largest investigation of multiple uses of psychotropics and risk of fall injuries , hospitalizations , and death within the same study . research into psychotropics as risk factors for fall injuries begun already in the late eighties ( campbell 1991 ) . since then , reviews have established use of psychotropics as a risk factor for falls ( bloch et al . among the psychotropic drugs , antidepressants were most strongly related to fall injuries . this is in line with recent research that has pointed out the risk of falls associated with these drugs ( huang et al . given that antidepressants are used extensively by older persons ( johnell and fastbom 2009 ) , particularly in the institutional setting ( johnell and fastbom 2012 ) , the amount of exposure to these presumably fallinducing drugs in the elderly population is substantial . , 2011 ) and improved care strategies for dementia patients ( american geriatrics society and american association for geriatric psychiatry , 2003 ) . in particular , our data suggest more attention to psychotropic polypharmacy as a target for preventive strategies for fall injuries in older people . concomitant use of several psychotropics was also a risk factor for hospitalizations in a dose response manner . here , we can show that antipsychotics are associated with death also in a general population of older persons , independently of dementia status . we also found that , although to a less extent , antidepressants , and anxiolytics were associated with a higher risk of death , whereas hypnotics and sedatives were associated with a lower risk . , we can now provide support for anxiolytics as possible risk factors for mortality in old age . however , hypnotics and sedatives were related to a lower risk of death in our study . hypnotics and sedatives are taken before bedtime and may therefore exert influence only during sleep , whereas anxiolytics are used during day time and may therefore be more related to adverse outcomes such as day time excessive sedation , injuries , falls , and fractures ( johnell et al . smaller studies of dementia patients have also found an association between use of several psychotropics and mortality ( hartikainen et al . however , our study is probably the first to confirm this finding in a dose responsive manner in a very large and unselected population of older persons . the swedish national board of health and welfare discourages prescribing of psychotropic polypharmacy ( i.e. 3 psychotropics ) to older patients in their national guidelines ( johnell et al . therefore , we do not know for which psychiatric symptoms the psychotropic drugs were prescribed for . however , the included psychotropic drugs are only available through prescriptions in sweden . however , psychotropic drugs are often prescribed without a documented diagnosis to elderly patients ( akincigil et al . we also lacked data from primary care , where many older patients with psychiatric problems are treated . this underestimates the number of dementia cases , as we lack information about dementia diagnoses in primary care ( not included in the patient register ) and undiagnosed dementia . we adjusted for comorbidity through the charlson comorbidity index , number of inpatient days , and number of other drugs . finally , a general limitation related to drug register data is that actual drug use is not assessed given that adherence to treatment can be low . our large study of a general population of older persons shows that psychotropic drugs , particularly when combined , are associated with risk of fall injuries , hospitalizations , and mortality among older persons . to our knowledge , this is the largest investigation of multiple uses of psychotropics and risk of fall injuries , hospitalizations , and death within the same study . research into psychotropics as risk factors for fall injuries begun already in the late eighties ( campbell 1991 ) . since then , reviews have established use of psychotropics as a risk factor for falls ( bloch et al . among the psychotropic drugs , antidepressants were most strongly related to fall injuries . this is in line with recent research that has pointed out the risk of falls associated with these drugs ( huang et al . given that antidepressants are used extensively by older persons ( johnell and fastbom 2009 ) , particularly in the institutional setting ( johnell and fastbom 2012 ) , the amount of exposure to these presumably fallinducing drugs in the elderly population is substantial . , 2011 ) and improved care strategies for dementia patients ( american geriatrics society and american association for geriatric psychiatry , 2003 ) . in particular , our data suggest more attention to psychotropic polypharmacy as a target for preventive strategies for fall injuries in older people . concomitant use of several psychotropics was also a risk factor for hospitalizations in a dose response manner . here , we can show that antipsychotics are associated with death also in a general population of older persons , independently of dementia status . we also found that , although to a less extent , antidepressants , and anxiolytics were associated with a higher risk of death , whereas hypnotics and sedatives were associated with a lower risk . , we can now provide support for anxiolytics as possible risk factors for mortality in old age . however , hypnotics and sedatives were related to a lower risk of death in our study . hypnotics and sedatives are taken before bedtime and may therefore exert influence only during sleep , whereas anxiolytics are used during day time and may therefore be more related to adverse outcomes such as day time excessive sedation , injuries , falls , and fractures ( johnell et al . smaller studies of dementia patients have also found an association between use of several psychotropics and mortality ( hartikainen et al . however , our study is probably the first to confirm this finding in a dose responsive manner in a very large and unselected population of older persons . the swedish national board of health and welfare discourages prescribing of psychotropic polypharmacy ( i.e. 3 psychotropics ) to older patients in their national guidelines ( johnell et al . therefore , we do not know for which psychiatric symptoms the psychotropic drugs were prescribed for . however , the included psychotropic drugs are only available through prescriptions in sweden . however , psychotropic drugs are often prescribed without a documented diagnosis to elderly patients ( akincigil et al . we also lacked data from primary care , where many older patients with psychiatric problems are treated . this underestimates the number of dementia cases , as we lack information about dementia diagnoses in primary care ( not included in the patient register ) and undiagnosed dementia . we adjusted for comorbidity through the charlson comorbidity index , number of inpatient days , and number of other drugs . finally , a general limitation related to drug register data is that actual drug use is not assessed given that adherence to treatment can be low . our large nationwide study of a general population of older persons suggests that multiple psychotropic drug use is associated with a higher risk of fall injuries , hospitalizations , and death in a dose response manner . safer nonpharmacological alternatives may be considered given the individual suffering and large societal economic burden related to these serious adverse outcomes . key points our data suggest a dose response relationship between multiple psychotropic drug use and risk of fall injuries , hospitalizations , and death among older personsour findings support a cautious prescribing of multiple psychotropic drugs to older patients . our data suggest a dose response relationship between multiple psychotropic drug use and risk of fall injuries , hospitalizations , and death among older personsour findings support a cautious prescribing of multiple psychotropic drugs to older patients . response relationship between multiple psychotropic drug use and risk of fall injuries , hospitalizations , and death among older persons our findings support a cautious prescribing of multiple psychotropic drugs to older patients .

but as early as 1981 , an article in the lancet reported a lack of skills among house - officers being tested on cardiopulmonary resuscitation.1 similar results were found in studies at the addenbrooke s hospital of cambridge ( uk ) in 19842 and the wellington school of medicine ( new zealand ) in 1989.3 a study from 1989 among family physicians and pediatricians in chicago ( usa ) reported that they were not trained in basic life support at all.4 possible inadequate competence of physicians relating to aspects of emergency care has later been confirmed by studies from australia ( 1999),5 the united states ( 2001),6 norway ( 2001),7 denmark ( 2002),8 south africa ( 2005)9 and the netherlands ( 2006),10 indicating that little progress has been made since . in belgium , no similar studies have been performed , but our own research in 2008 among medical students and junior graduates in their postgraduate training to become a family physician suggest at least a low confidence in their own skills and knowledge regarding emergency medical care . a number of publications express the need for better education in medical emergency skills , and better and more accessible training modules for physicians.11,12,13 also , the need for repetitive and continuous re - education has been illustrated by numerous studies,1417 suggesting that 6 months is the optimal time interval with which skills should be recapped.1820 based on these scientific results , initiatives to improve education in clinical skills and emergency care were set up in several countries . these initiatives involve , for example , reviewing of university curricula in medicine,21 supporting rural family physicians in acquiring and retaining urgency skills,22,23 formulating clearer final qualifications for medicine graduates,24,25 evaluating clinical skills using objective structured clinical examinations11 and incorporating clinical skills assessments in medical license exams.25 even though the study groups and assessed skills were rather heterogeneous among the different publications , we can safely say that practical knowledge of emergency medical care among physicians seems to be insufficient worldwide . however , almost no data is available concerning physicians in belgium.11 it is essential for belgian family physicians to be able to manage emergency conditions . basic emergency care is done by family physicians within their own practice or during on - call hours . especially outside of the cities , people usually do nt seek care in emergency departments without first seeing their own family physician . belgium is said to have one of the highest health care standards in the world , which implies that its physicians are probably confident and skilled . the aim of this study was to ascertain how confident dutch - speaking family physicians in belgium feel about their ability to give adequate emergency medical care and to examine their assessment of knowledge of relevant medical conditions . all local family physician organizations of dutch - speaking physicians in belgium were asked to invite their members to take part in our study in september 2009 . a convenience sample consisting of 974 family practitioners was contacted through email and asked to complete a web - based questionnaire . physicians in their postgraduate training to become a family physician were also included , since they work independently as qualified physicians and are an integral part of the medical corps in belgium . we opted for this subgroup of belgian physicians because they consist of a homogenous group . the two main language groups in belgium ( french and dutch ) have a different organization of medical studies . curricula are different , and contrary to their french - speaking counterparts , flemish physicians have been trained in an interuniversity setting for more than 25 years . because the vast majority of dutch - speaking physicians work in flanders and brussels , we performed our study only in these two parts of the country . we opted for an online questionnaire , hosted on server space of the vrije universiteit brussel . cookies as well as ip address analysis were used to avoid single users filling in the questionnaire multiple times . for each skill , the participant was asked for a self - assessment , using a likert scale with the following possibilities : 0 , has no experience or knowledge whatsoever ; 1 , has theoretical knowledge ; 2 , has had a demonstration of the skill or has observed it ; 3 , is able to perform the skill independently ; and 4 , has proficiency in the respective skill . the second part consisted of 31 questions ascertaining the participant s knowledge of conditions relevant to urgent medical care . again , a likert scale was used as follows : 0 , has never heard of the disease or condition ; 1 , has heard of the disease / condition but is unable to manage it ; 2 , can manage the disease / condition ; 3 , can diagnose the disease / condition ; and 4 , can independently treat the disease / condition . our questionnaire was based on a list used by hekkert in a similar study in the netherlands.21 a test study has been performed on medical students , using the same methodology and questionnaire.11 using the information gained from that study , changes were made to scales , descriptions , questions , and questionnaire setup . however , we limited these changes to the essential ones , in order to maintain our possibility to compare . it is unclear in what way the original instrument was validated by the composers , but since hekkert used the validated criteria set from the dutch raamplan 200124 and based on consistency within and comparison with other studies using the same questionnaire , we estimated the questionnaire to be valid and reliable . the dutch raamplan is also used in belgium , as a basis for the final qualifications in medical studies . the participants were divided into subgroups : based on sex , age ( five groups ) , geographical location ( rural , urban , metropolitan ) , size of the family practice ( four groups ) , and on the organization of the practice ( five groups ) . they were also asked if and when they had taken any additional training in emergency care . the context of this training was not further defined , meaning that every initiative being regarded as additional training by the participant was included . for each participant , a mean score for skills and although a decimal score on a likert scale has no meaning as such , we assumed that means would provide more information than the raw score itself . questionnaires in which a participant indicated the same score on each question , or where the pattern of answers showed a consequent algorithm , were assumed to be intentionally misleading and were excluded . the raamplan states that at the end of the medical studies , a physician should be able to perform skills in an appropriate manner , not always by acting independently but also by referring out , if needed.24 in our opinion , having had a demonstration of a skill ( likert score 2 ) does not comply with this criterion . therefore , we set the minimum criterion to meet our standards at likert score 3 , meaning the participant was able to perform the skill independently . even though it could be theoretically assumed that a licensed physician meets this criterion in all mentioned skills , this assumption is highly unrealistic . hence , we assumed that a participant met our standards in a reasonable manner when scoring 3 on at least half of the questioned skills . for assessing knowledge of relevant conditions , we used the appendix to the raamplan , which differentiates between the conditions . we assumed that a participant fulfilled the criteria in a reasonable way if he or she met the set criterion on at least half of the questioned pathologies . for statistical interpretation , we used spss statistics for windows , version 19.0 ( ibm corporation , armonk , ny , usa ) . due to the small subgroups and the presence of some extreme results , we could not assume a normal distribution of our data , as was indicated by a shapiro however , we decided to include extremes in our statistics , in line with growing statistical belief.26 in most cases , we used a kruskal wallis test to compare subgroups . we used a mann whitney u - test to compare men and women , and to compare holders of an ecg or spirometry certificate to those without . in order to compare the number of physicians in the subgroups meeting our set standards , we used a chi - square test however , we kept the nonparametric results , with their stronger stratification and bigger distribution independence , since this did not differentiate any additional significance . all local family physician organizations of dutch - speaking physicians in belgium were asked to invite their members to take part in our study in september 2009 . a convenience sample consisting of 974 family practitioners was contacted through email and asked to complete a web - based questionnaire . physicians in their postgraduate training to become a family physician were also included , since they work independently as qualified physicians and are an integral part of the medical corps in belgium . we opted for this subgroup of belgian physicians because they consist of a homogenous group . the two main language groups in belgium ( french and dutch ) have a different organization of medical studies . curricula are different , and contrary to their french - speaking counterparts , flemish physicians have been trained in an interuniversity setting for more than 25 years . because the vast majority of dutch - speaking physicians work in flanders and brussels , we performed our study only in these two parts of the country . we opted for an online questionnaire , hosted on server space of the vrije universiteit brussel . cookies as well as ip address analysis were used to avoid single users filling in the questionnaire multiple times . for each skill , the participant was asked for a self - assessment , using a likert scale with the following possibilities : 0 , has no experience or knowledge whatsoever ; 1 , has theoretical knowledge ; 2 , has had a demonstration of the skill or has observed it ; 3 , is able to perform the skill independently ; and 4 , has proficiency in the respective skill . the second part consisted of 31 questions ascertaining the participant s knowledge of conditions relevant to urgent medical care . again , a likert scale was used as follows : 0 , has never heard of the disease or condition ; 1 , has heard of the disease / condition but is unable to manage it ; 2 , can manage the disease / condition ; 3 , can diagnose the disease / condition ; and 4 , can independently treat the disease / condition . our questionnaire was based on a list used by hekkert in a similar study in the netherlands.21 a test study has been performed on medical students , using the same methodology and questionnaire.11 using the information gained from that study , changes were made to scales , descriptions , questions , and questionnaire setup . however , we limited these changes to the essential ones , in order to maintain our possibility to compare . it is unclear in what way the original instrument was validated by the composers , but since hekkert used the validated criteria set from the dutch raamplan 200124 and based on consistency within and comparison with other studies using the same questionnaire , we estimated the questionnaire to be valid and reliable . the dutch raamplan is also used in belgium , as a basis for the final qualifications in medical studies . the participants were divided into subgroups : based on sex , age ( five groups ) , geographical location ( rural , urban , metropolitan ) , size of the family practice ( four groups ) , and on the organization of the practice ( five groups ) . they were also asked if and when they had taken any additional training in emergency care . the context of this training was not further defined , meaning that every initiative being regarded as additional training by the participant was included . for each participant , a mean score for skills and although a decimal score on a likert scale has no meaning as such , we assumed that means would provide more information than the raw score itself . questionnaires in which a participant indicated the same score on each question , or where the pattern of answers showed a consequent algorithm , were assumed to be intentionally misleading and were excluded . the raamplan states that at the end of the medical studies , a physician should be able to perform skills in an appropriate manner , not always by acting independently but also by referring out , if needed.24 in our opinion , having had a demonstration of a skill ( likert score 2 ) does not comply with this criterion . therefore , we set the minimum criterion to meet our standards at likert score 3 , meaning the participant was able to perform the skill independently . even though it could be theoretically assumed that a licensed physician meets this criterion in all mentioned skills , this assumption is highly unrealistic . hence , we assumed that a participant met our standards in a reasonable manner when scoring 3 on at least half of the questioned skills . for assessing knowledge of relevant conditions , we used the appendix to the raamplan , which differentiates between the conditions . we assumed that a participant fulfilled the criteria in a reasonable way if he or she met the set criterion on at least half of the questioned pathologies . for statistical interpretation , we used spss statistics for windows , version 19.0 ( ibm corporation , armonk , ny , usa ) . due to the small subgroups and the presence of some extreme results , we could not assume a normal distribution of our data , as was indicated by a shapiro however , we decided to include extremes in our statistics , in line with growing statistical belief.26 in most cases , we used a kruskal wallis test to compare subgroups . we used a mann whitney u - test to compare men and women , and to compare holders of an ecg or spirometry certificate to those without . in order to compare the number of physicians in the subgroups meeting our set standards , we used a chi - square test however , we kept the nonparametric results , with their stronger stratification and bigger distribution independence , since this did not differentiate any additional significance . the recruitment rate to our email invitation was approximately 22% ( n = 210 ) . after exclusion of incomplete questionnaires ( n = 51 ) or those with obviously intentional misleading answers ( n = 1 ) , our study group consisted of 158 family physicians , meaning a completion rate of 75% . this sample size was adequate , and by comparing its composition to the demographic composition of belgian family physicians , it seemed to be highly representative . additional training in emergency medical care had been completed by 39% of participants , an ecg certificate was held by 92% , and 61% held a spirometry certificate . holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.94 and 2.72 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 3.07 and 2.72 respectively ) . any influence of time ( delay ) pertaining to additional training no other significant differences between subgroups were found . the mean likert score on skills was 2.50 . based on the questionnaire results , the least - known skills were : tilting to recovery position ( mean , 1.30 ) , use of an external defibrillator ( 1.79 ) , and resuscitation of a child ( 1.93 ) . the best - known skills were : removal of a foreign body ( 3.53 ) , applying bandages ( 3.09 ) , and external heart massage ( 3.08 ) . our minimum criteria were met by 64% of participants . in 14 of the 22 skill items , the skills criteria met by the highest number of participants were : removal of a foreign body ( 92% ) , external heart massage ( 87% ) , and applying bandages ( 80% ) . the skills criteria met by the lowest number of participants were : tilting to recovery position ( 19% ) , use of the external defibrillator ( 28% ) , and safe transport of an accident victim ( 29% ) ( table 1 ) . holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.61 and 2.34 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 2.81 and 2.31 respectively ) , with more meeting our criteria ( 77% versus 56% ; p = 0.006 ) . the least - known diseases or conditions were : a fall from great height ( mean , 2.09 ) , skull base fracture ( 2.28 ) , and electrocution ( 2.28 ) . the best - known diseases or conditions were : superficial wounds ( 3.91 ) , insect bites ( 3.86 ) , and hyperventilation ( 3.85 ) . our minimum criteria were met by 55% of participants . on 24 of the 31 diseases / conditions , at least half of the physicians met our criteria . the skills criteria met by the highest number of participants were : superficial wounds ( 99% ) , cerebrovascular abnormalities ( 99% ) , and epilepsy ( 97% ) . the skills criteria met by the lowest number of participants were : electrocution ( 11% ) , poisoning ( 11% ) , and a fall from great height ( 13% ) ( table 3 ) . more holders of a spirometry certificate met our criteria than their colleagues without this certificate ( 63% versus 43% ; p = 0.013 ) . also , family physicians with additional training in emergency care had clearly higher scores than those without ( p = 0.003 ; means , 3.33 and 2.14 respectively ) , with more meeting our criteria ( 70% versus 45% ; p = 0.002 ) . the mean score of physicians working in rural areas was higher than that of those working in urban or metropolitan areas ( p = 0.033 ; means , 3.25 and 3.15 respectively ) . more physicians from rural areas met our criteria compared to those from urban or metropolitan areas ( 61% versus 45% ; p = 0.047 ) ( table 2 ) . no significant differences between men and women , age groups , or practice size could be found . whether or not the physician was the holder of an ecg certificate did not have a significant influence on the results . the recruitment rate to our email invitation was approximately 22% ( n = 210 ) . after exclusion of incomplete questionnaires ( n = 51 ) or those with obviously intentional misleading answers ( n = 1 ) , our study group consisted of 158 family physicians , meaning a completion rate of 75% . this sample size was adequate , and by comparing its composition to the demographic composition of belgian family physicians , it seemed to be highly representative . additional training in emergency medical care had been completed by 39% of participants , an ecg certificate was held by 92% , and 61% held a spirometry certificate . holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.94 and 2.72 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 3.07 and 2.72 respectively ) . any influence of time ( delay ) pertaining to additional training could not be proven from our results ( p = 0.091 ) . no other significant differences between subgroups were found . the mean likert score on skills was 2.50 . based on the questionnaire results , the least - known skills were : tilting to recovery position ( mean , 1.30 ) , use of an external defibrillator ( 1.79 ) , and resuscitation of a child ( 1.93 ) . the best - known skills were : removal of a foreign body ( 3.53 ) , applying bandages ( 3.09 ) , and external heart massage ( 3.08 ) . our minimum criteria were met by 64% of participants . in 14 of the 22 skill items , at least half of the physicians met our criteria . the skills criteria met by the highest number of participants were : removal of a foreign body ( 92% ) , external heart massage ( 87% ) , and applying bandages ( 80% ) . the skills criteria met by the lowest number of participants were : tilting to recovery position ( 19% ) , use of the external defibrillator ( 28% ) , and safe transport of an accident victim ( 29% ) ( table 1 ) . holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.61 and 2.34 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 2.81 and 2.31 respectively ) , with more meeting our criteria ( 77% versus 56% ; p = 0.006 ) . the least - known diseases or conditions were : a fall from great height ( mean , 2.09 ) , skull base fracture ( 2.28 ) , and electrocution ( 2.28 ) . the best - known diseases or conditions were : superficial wounds ( 3.91 ) , insect bites ( 3.86 ) , and hyperventilation ( 3.85 ) . our minimum criteria were met by 55% of participants . on 24 of the 31 diseases / conditions , at least half of the physicians met our criteria . the skills criteria met by the highest number of participants were : superficial wounds ( 99% ) , cerebrovascular abnormalities ( 99% ) , and epilepsy ( 97% ) . the skills criteria met by the lowest number of participants were : electrocution ( 11% ) , poisoning ( 11% ) , and a fall from great height ( 13% ) ( table 3 ) . more holders of a spirometry certificate met our criteria than their colleagues without this certificate ( 63% versus 43% ; p = 0.013 ) . also , family physicians with additional training in emergency care had clearly higher scores than those without ( p = 0.003 ; means , 3.33 and 2.14 respectively ) , with more meeting our criteria ( 70% versus 45% ; p = 0.002 ) . the mean score of physicians working in rural areas was higher than that of those working in urban or metropolitan areas ( p = 0.033 ; means , 3.25 and 3.15 respectively ) . more physicians from rural areas met our criteria compared to those from urban or metropolitan areas ( 61% versus 45% ; p = 0.047 ) ( table 2 ) . no significant differences between men and women , age groups , or practice size could be found . whether or not the physician was the holder of an ecg certificate based on the results of our study , we can confidently state that dutch - speaking family physicians in belgium do not feel able to adequately administer most aspects of emergency care . as described in the introduction , this is in line with studies in many other countries.110 our results were also highly comparable with those obtained in our similar study from 2008 by junior doctors in their postgraduate training to become family physicians.11 factors of influence on skills and knowledge relating to urgent medical care seem limited . having taken an additional training in emergency medicine is the most influencing positive factor on both skills and knowledge . this sounds logical , but could be a result of the profile of the participant rather than of the training itself . holding a spirometry certificate proves to be of positive influence on the physician s self - assessed skills and knowledge . holding an ecg certificate , however , had no influence on the physician s skills and knowledge in emergency medical care , probably because the certified training is incorporated in the basic curriculum for most medicine students and is therefore independent of the student s profile . in most cases , obtaining an ecg certification is independent of the physician s personal interests and preparedness to take additional training , whereas obtaining a spirometry certificate is highly influenced by these factors . it is plausible that a doctor seeking additional training has an overall more proactive professional approach . therefore , the found link between the certified emergency , ecg or spirometry training , and the assessed confidence could be a result of the physician s profile rather than of the certified training itself . this seems likely , because spirometry is of little practical use in emergency medical care . according to our results , family physicians in rural areas are more confident about relevant knowledge than their urban or metropolitan counterparts . this could be explained by the proximity of hospital emergency services for urban patients , lowering the threshold and thus surpassing the family physician . even though the participants indicated a lack of confidence in skills and knowledge relating to emergency care , this needs to be seen in perspective . most physicians felt quite confident in topics they had experience with or that were common in daily practice . the items on which they indicated low confidence were generally less common and mostly treated by specialist care anyway . this may limit the practical implications of our results . also , we need to stress that it was not our aim to test individual skills , but to assess the overall confidence of the physician . however , we did include a mention of individual skills , since this provides a good idea of the specific areas in which the physician lacks confidence . specific confidence or skill in the different aspects of individual emergencies needs to be the subject of further research . . these may be the organization of education in emergency medicine in our universities , the lack of additional training and recapping , a noticeable inconsistency for some guidelines , or the frequency with which these guidelines change.2729 finally , it is important to underline that this study only assessed physician s confidence in a very specific area . our study does show , however , that dutch - speaking belgian family physicians do nt feel very confident in the area of emergency care , regardless of the quality of the care they provide . throughout our study , we have guarded the validity and reliability of the instruments used , thus ensuring the quality and relevance of our results . we used an existing questionnaire11,21 based on final qualifications described in the raamplan.24 the relevancy of our results obviously depends on the relevancy of the criteria set in the raamplan . but these qualifications were established after a long and careful process , likely ensuring their relevancy . we tried to avoid as many confounders as possible and limit expectation and confirmation bias by carefully designing and respecting the study protocol . we are aware of the fact that our method held a risk of selection bias , but by randomly choosing the potential participants , we attempted to limit this as well . self - assessment is not the most adequate instrument to evaluate skills.9,30 an objective structured clinical examination would have been more appropriate,18 but was not feasible within the context of this study . also , the tested items are only surrogate markers . it is plausible that with lesser confidence , training , or knowledge a good outcome is still possible with a proper attitude and awareness of the physician s own limitations . it is regrettable that only a small sample of family physicians were prepared to take part in our study . it is possible that due to the small study group , we were not able to prove the influence of all relating factors . however , in comparison to the official rapport of the belgian medical corps,31 we are confident that our study group is representative of the entire population of family physicians ( table 4 ) . many epidemiological studies have been performed with a representative sample of only 1%2%.32 even with the limitations of our study in mind , we feel secure in stating that the scientific value of our study is assured . throughout our study , we have guarded the validity and reliability of the instruments used , thus ensuring the quality and relevance of our results . we used an existing questionnaire11,21 based on final qualifications described in the raamplan.24 the relevancy of our results obviously depends on the relevancy of the criteria set in the raamplan . but these qualifications were established after a long and careful process , likely ensuring their relevancy . we tried to avoid as many confounders as possible and limit expectation and confirmation bias by carefully designing and respecting the study protocol . we are aware of the fact that our method held a risk of selection bias , but by randomly choosing the potential participants , we attempted to limit this as well . self - assessment is not the most adequate instrument to evaluate skills.9,30 an objective structured clinical examination would have been more appropriate,18 but was not feasible within the context of this study . also , the tested items are only surrogate markers . the real clinical relevance of emergency care is determined by patient outcome . it is plausible that with lesser confidence , training , or knowledge a good outcome is still possible with a proper attitude and awareness of the physician s own limitations . it is regrettable that only a small sample of family physicians were prepared to take part in our study . it is possible that due to the small study group , we were not able to prove the influence of all relating factors . however , in comparison to the official rapport of the belgian medical corps,31 we are confident that our study group is representative of the entire population of family physicians ( table 4 ) . many epidemiological studies have been performed with a representative sample of only 1%2%.32 even with the limitations of our study in mind , we feel secure in stating that the scientific value of our study is assured . almost half of the family physicians in the dutch - speaking area of belgium feel insufficiently competent to offer most aspects of emergency medical care . it is not clear if this lack of confidence has any consequences for the quality and outcome of patient care . it does prove , however , that family physicians know the limits of their own medical skills .

backgroundpractical knowledge of emergency medical care among physicians seems to be insufficient worldwide . research specifically aimed at family physicians is rather scarce . additionally , in belgium there are no data on this subject.purposesour aim was to ascertain how confident belgian family physicians feel about their ability to give adequate emergency care and to examine their assessment of their knowledge of relevant medical conditions.methodswe used a web - based questionnaire for which a convenience sample of 974 dutch - speaking family practitioners was invited through email . the survey assessed how these physicians perceived their own emergency skills and their knowledge of relevant medical conditions.resultsthe survey had a recruitment rate of 22% ( n = 210 ) , with a 75% completion rate . the minimum criteria formulated pertaining to skills and knowledge were met by 64% and 55% of the participants , respectively . the mean cumulative scores on skills and knowledge were 2.5 and 3.2 , respectively ( on a scale from 0 to 4 ) . physicians with additional training in emergency care ( 3.07 versus 2.72 ) , or with a spirometry certificate ( 2.94 versus 2.72 ) scored better than those without . practitioners from rural areas felt more confident than those from urbanized regions ( 3.25 versus 3.15 ) . physicians felt more competent in aspects of emergency care where they had experience.conclusionalmost half of the dutch - speaking family physicians in belgium felt insufficiently competent to offer emergency medical care .

Introduction Methods Participants Questionnaire Interpretation Statistics Results Participants Self-assessment of skills and knowledge Discussion Quality and limitations of our study Conclusion

but as early as 1981 , an article in the lancet reported a lack of skills among house - officers being tested on cardiopulmonary resuscitation.1 similar results were found in studies at the addenbrooke s hospital of cambridge ( uk ) in 19842 and the wellington school of medicine ( new zealand ) in 1989.3 a study from 1989 among family physicians and pediatricians in chicago ( usa ) reported that they were not trained in basic life support at all.4 possible inadequate competence of physicians relating to aspects of emergency care has later been confirmed by studies from australia ( 1999),5 the united states ( 2001),6 norway ( 2001),7 denmark ( 2002),8 south africa ( 2005)9 and the netherlands ( 2006),10 indicating that little progress has been made since . in belgium , no similar studies have been performed , but our own research in 2008 among medical students and junior graduates in their postgraduate training to become a family physician suggest at least a low confidence in their own skills and knowledge regarding emergency medical care . a number of publications express the need for better education in medical emergency skills , and better and more accessible training modules for physicians.11,12,13 also , the need for repetitive and continuous re - education has been illustrated by numerous studies,1417 suggesting that 6 months is the optimal time interval with which skills should be recapped.1820 based on these scientific results , initiatives to improve education in clinical skills and emergency care were set up in several countries . these initiatives involve , for example , reviewing of university curricula in medicine,21 supporting rural family physicians in acquiring and retaining urgency skills,22,23 formulating clearer final qualifications for medicine graduates,24,25 evaluating clinical skills using objective structured clinical examinations11 and incorporating clinical skills assessments in medical license exams.25 even though the study groups and assessed skills were rather heterogeneous among the different publications , we can safely say that practical knowledge of emergency medical care among physicians seems to be insufficient worldwide . however , almost no data is available concerning physicians in belgium.11 it is essential for belgian family physicians to be able to manage emergency conditions . basic emergency care is done by family physicians within their own practice or during on - call hours . especially outside of the cities , people usually do nt seek care in emergency departments without first seeing their own family physician . the aim of this study was to ascertain how confident dutch - speaking family physicians in belgium feel about their ability to give adequate emergency medical care and to examine their assessment of knowledge of relevant medical conditions . all local family physician organizations of dutch - speaking physicians in belgium were asked to invite their members to take part in our study in september 2009 . a convenience sample consisting of 974 family practitioners was contacted through email and asked to complete a web - based questionnaire . physicians in their postgraduate training to become a family physician were also included , since they work independently as qualified physicians and are an integral part of the medical corps in belgium . because the vast majority of dutch - speaking physicians work in flanders and brussels , we performed our study only in these two parts of the country . for each skill , the participant was asked for a self - assessment , using a likert scale with the following possibilities : 0 , has no experience or knowledge whatsoever ; 1 , has theoretical knowledge ; 2 , has had a demonstration of the skill or has observed it ; 3 , is able to perform the skill independently ; and 4 , has proficiency in the respective skill . again , a likert scale was used as follows : 0 , has never heard of the disease or condition ; 1 , has heard of the disease / condition but is unable to manage it ; 2 , can manage the disease / condition ; 3 , can diagnose the disease / condition ; and 4 , can independently treat the disease / condition . it is unclear in what way the original instrument was validated by the composers , but since hekkert used the validated criteria set from the dutch raamplan 200124 and based on consistency within and comparison with other studies using the same questionnaire , we estimated the questionnaire to be valid and reliable . the participants were divided into subgroups : based on sex , age ( five groups ) , geographical location ( rural , urban , metropolitan ) , size of the family practice ( four groups ) , and on the organization of the practice ( five groups ) . they were also asked if and when they had taken any additional training in emergency care . for each participant , a mean score for skills and although a decimal score on a likert scale has no meaning as such , we assumed that means would provide more information than the raw score itself . questionnaires in which a participant indicated the same score on each question , or where the pattern of answers showed a consequent algorithm , were assumed to be intentionally misleading and were excluded . the raamplan states that at the end of the medical studies , a physician should be able to perform skills in an appropriate manner , not always by acting independently but also by referring out , if needed.24 in our opinion , having had a demonstration of a skill ( likert score 2 ) does not comply with this criterion . due to the small subgroups and the presence of some extreme results , we could not assume a normal distribution of our data , as was indicated by a shapiro however , we decided to include extremes in our statistics , in line with growing statistical belief.26 in most cases , we used a kruskal wallis test to compare subgroups . we used a mann whitney u - test to compare men and women , and to compare holders of an ecg or spirometry certificate to those without . in order to compare the number of physicians in the subgroups meeting our set standards , we used a chi - square test however , we kept the nonparametric results , with their stronger stratification and bigger distribution independence , since this did not differentiate any additional significance . all local family physician organizations of dutch - speaking physicians in belgium were asked to invite their members to take part in our study in september 2009 . a convenience sample consisting of 974 family practitioners was contacted through email and asked to complete a web - based questionnaire . physicians in their postgraduate training to become a family physician were also included , since they work independently as qualified physicians and are an integral part of the medical corps in belgium . because the vast majority of dutch - speaking physicians work in flanders and brussels , we performed our study only in these two parts of the country . for each skill , the participant was asked for a self - assessment , using a likert scale with the following possibilities : 0 , has no experience or knowledge whatsoever ; 1 , has theoretical knowledge ; 2 , has had a demonstration of the skill or has observed it ; 3 , is able to perform the skill independently ; and 4 , has proficiency in the respective skill . the second part consisted of 31 questions ascertaining the participant s knowledge of conditions relevant to urgent medical care . it is unclear in what way the original instrument was validated by the composers , but since hekkert used the validated criteria set from the dutch raamplan 200124 and based on consistency within and comparison with other studies using the same questionnaire , we estimated the questionnaire to be valid and reliable . the participants were divided into subgroups : based on sex , age ( five groups ) , geographical location ( rural , urban , metropolitan ) , size of the family practice ( four groups ) , and on the organization of the practice ( five groups ) . they were also asked if and when they had taken any additional training in emergency care . for each participant , a mean score for skills and although a decimal score on a likert scale has no meaning as such , we assumed that means would provide more information than the raw score itself . questionnaires in which a participant indicated the same score on each question , or where the pattern of answers showed a consequent algorithm , were assumed to be intentionally misleading and were excluded . the raamplan states that at the end of the medical studies , a physician should be able to perform skills in an appropriate manner , not always by acting independently but also by referring out , if needed.24 in our opinion , having had a demonstration of a skill ( likert score 2 ) does not comply with this criterion . hence , we assumed that a participant met our standards in a reasonable manner when scoring 3 on at least half of the questioned skills . we assumed that a participant fulfilled the criteria in a reasonable way if he or she met the set criterion on at least half of the questioned pathologies . due to the small subgroups and the presence of some extreme results , we could not assume a normal distribution of our data , as was indicated by a shapiro however , we decided to include extremes in our statistics , in line with growing statistical belief.26 in most cases , we used a kruskal wallis test to compare subgroups . we used a mann whitney u - test to compare men and women , and to compare holders of an ecg or spirometry certificate to those without . in order to compare the number of physicians in the subgroups meeting our set standards , we used a chi - square test however , we kept the nonparametric results , with their stronger stratification and bigger distribution independence , since this did not differentiate any additional significance . the recruitment rate to our email invitation was approximately 22% ( n = 210 ) . after exclusion of incomplete questionnaires ( n = 51 ) or those with obviously intentional misleading answers ( n = 1 ) , our study group consisted of 158 family physicians , meaning a completion rate of 75% . this sample size was adequate , and by comparing its composition to the demographic composition of belgian family physicians , it seemed to be highly representative . additional training in emergency medical care had been completed by 39% of participants , an ecg certificate was held by 92% , and 61% held a spirometry certificate . holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.94 and 2.72 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 3.07 and 2.72 respectively ) . any influence of time ( delay ) pertaining to additional training no other significant differences between subgroups were found . the mean likert score on skills was 2.50 . the best - known skills were : removal of a foreign body ( 3.53 ) , applying bandages ( 3.09 ) , and external heart massage ( 3.08 ) . our minimum criteria were met by 64% of participants . in 14 of the 22 skill items , the skills criteria met by the highest number of participants were : removal of a foreign body ( 92% ) , external heart massage ( 87% ) , and applying bandages ( 80% ) . the skills criteria met by the lowest number of participants were : tilting to recovery position ( 19% ) , use of the external defibrillator ( 28% ) , and safe transport of an accident victim ( 29% ) ( table 1 ) . holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.61 and 2.34 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 2.81 and 2.31 respectively ) , with more meeting our criteria ( 77% versus 56% ; p = 0.006 ) . the best - known diseases or conditions were : superficial wounds ( 3.91 ) , insect bites ( 3.86 ) , and hyperventilation ( 3.85 ) . our minimum criteria were met by 55% of participants . the skills criteria met by the highest number of participants were : superficial wounds ( 99% ) , cerebrovascular abnormalities ( 99% ) , and epilepsy ( 97% ) . more holders of a spirometry certificate met our criteria than their colleagues without this certificate ( 63% versus 43% ; p = 0.013 ) . also , family physicians with additional training in emergency care had clearly higher scores than those without ( p = 0.003 ; means , 3.33 and 2.14 respectively ) , with more meeting our criteria ( 70% versus 45% ; p = 0.002 ) . the mean score of physicians working in rural areas was higher than that of those working in urban or metropolitan areas ( p = 0.033 ; means , 3.25 and 3.15 respectively ) . more physicians from rural areas met our criteria compared to those from urban or metropolitan areas ( 61% versus 45% ; p = 0.047 ) ( table 2 ) . the recruitment rate to our email invitation was approximately 22% ( n = 210 ) . after exclusion of incomplete questionnaires ( n = 51 ) or those with obviously intentional misleading answers ( n = 1 ) , our study group consisted of 158 family physicians , meaning a completion rate of 75% . this sample size was adequate , and by comparing its composition to the demographic composition of belgian family physicians , it seemed to be highly representative . additional training in emergency medical care had been completed by 39% of participants , an ecg certificate was held by 92% , and 61% held a spirometry certificate . holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.94 and 2.72 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 3.07 and 2.72 respectively ) . any influence of time ( delay ) pertaining to additional training could not be proven from our results ( p = 0.091 ) . the mean likert score on skills was 2.50 . the best - known skills were : removal of a foreign body ( 3.53 ) , applying bandages ( 3.09 ) , and external heart massage ( 3.08 ) . our minimum criteria were met by 64% of participants . the skills criteria met by the highest number of participants were : removal of a foreign body ( 92% ) , external heart massage ( 87% ) , and applying bandages ( 80% ) . the skills criteria met by the lowest number of participants were : tilting to recovery position ( 19% ) , use of the external defibrillator ( 28% ) , and safe transport of an accident victim ( 29% ) ( table 1 ) . holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.61 and 2.34 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 2.81 and 2.31 respectively ) , with more meeting our criteria ( 77% versus 56% ; p = 0.006 ) . the least - known diseases or conditions were : a fall from great height ( mean , 2.09 ) , skull base fracture ( 2.28 ) , and electrocution ( 2.28 ) . the best - known diseases or conditions were : superficial wounds ( 3.91 ) , insect bites ( 3.86 ) , and hyperventilation ( 3.85 ) . our minimum criteria were met by 55% of participants . on 24 of the 31 diseases / conditions , at least half of the physicians met our criteria . the skills criteria met by the highest number of participants were : superficial wounds ( 99% ) , cerebrovascular abnormalities ( 99% ) , and epilepsy ( 97% ) . more holders of a spirometry certificate met our criteria than their colleagues without this certificate ( 63% versus 43% ; p = 0.013 ) . also , family physicians with additional training in emergency care had clearly higher scores than those without ( p = 0.003 ; means , 3.33 and 2.14 respectively ) , with more meeting our criteria ( 70% versus 45% ; p = 0.002 ) . the mean score of physicians working in rural areas was higher than that of those working in urban or metropolitan areas ( p = 0.033 ; means , 3.25 and 3.15 respectively ) . more physicians from rural areas met our criteria compared to those from urban or metropolitan areas ( 61% versus 45% ; p = 0.047 ) ( table 2 ) . whether or not the physician was the holder of an ecg certificate based on the results of our study , we can confidently state that dutch - speaking family physicians in belgium do not feel able to adequately administer most aspects of emergency care . as described in the introduction , this is in line with studies in many other countries.110 our results were also highly comparable with those obtained in our similar study from 2008 by junior doctors in their postgraduate training to become family physicians.11 factors of influence on skills and knowledge relating to urgent medical care seem limited . having taken an additional training in emergency medicine is the most influencing positive factor on both skills and knowledge . holding a spirometry certificate proves to be of positive influence on the physician s self - assessed skills and knowledge . holding an ecg certificate , however , had no influence on the physician s skills and knowledge in emergency medical care , probably because the certified training is incorporated in the basic curriculum for most medicine students and is therefore independent of the student s profile . in most cases , obtaining an ecg certification is independent of the physician s personal interests and preparedness to take additional training , whereas obtaining a spirometry certificate is highly influenced by these factors . this seems likely , because spirometry is of little practical use in emergency medical care . according to our results , family physicians in rural areas are more confident about relevant knowledge than their urban or metropolitan counterparts . even though the participants indicated a lack of confidence in skills and knowledge relating to emergency care , this needs to be seen in perspective . most physicians felt quite confident in topics they had experience with or that were common in daily practice . specific confidence or skill in the different aspects of individual emergencies needs to be the subject of further research . these may be the organization of education in emergency medicine in our universities , the lack of additional training and recapping , a noticeable inconsistency for some guidelines , or the frequency with which these guidelines change.2729 finally , it is important to underline that this study only assessed physician s confidence in a very specific area . our study does show , however , that dutch - speaking belgian family physicians do nt feel very confident in the area of emergency care , regardless of the quality of the care they provide . it is plausible that with lesser confidence , training , or knowledge a good outcome is still possible with a proper attitude and awareness of the physician s own limitations . however , in comparison to the official rapport of the belgian medical corps,31 we are confident that our study group is representative of the entire population of family physicians ( table 4 ) . it is plausible that with lesser confidence , training , or knowledge a good outcome is still possible with a proper attitude and awareness of the physician s own limitations . it is regrettable that only a small sample of family physicians were prepared to take part in our study . however , in comparison to the official rapport of the belgian medical corps,31 we are confident that our study group is representative of the entire population of family physicians ( table 4 ) . many epidemiological studies have been performed with a representative sample of only 1%2%.32 even with the limitations of our study in mind , we feel secure in stating that the scientific value of our study is assured . almost half of the family physicians in the dutch - speaking area of belgium feel insufficiently competent to offer most aspects of emergency medical care . it does prove , however , that family physicians know the limits of their own medical skills .

in the international classification of functioning , disability , and health model ( icf ) , pre - existing health conditions in an individual can give rise to functional impairments , which in turn cause activity limitation and restriction in social participation ( grimby & smedby , 2001 ; world health organization , 2011 ) . disability in turn results from the interaction between these factors in the individual and the surrounding environment . in general , no attempt is made by the icf to distinguish between cognitive and physical causes of disability , which often interact . the world mental health survey initiative has demonstrated that mental disorders were more likely to be associated with severe disability than physical ones ( scott et al . , 2009 ) . the key improvement from the earlier disability model , the international classification of impairments , disabilities , and handicaps ( icidh ) , was a shift from a purely medical to a bio - psycho - social framework reflecting the interaction between an individual s health status and ambient factors , both personal and environmental . in addition , there was an emphasis on viewing disability as an expression of the aforementioned interaction , rather than as a characteristic of the individual ( world health organization , 2011 ) . previously , a person with paralysis after a stroke was deemed to be disabled due to loss of limb function . when viewed through the lens of the icf model , the stroke is a chronic disease that gives rise to functional impairment ( limb paralysis ) , which limits activity performance ( driving ) , which in turn restricts social participation ( going to play poker ) . the stroke itself may also limit activity performance and social participation directly by making it harder to retain a driving license or getting fewer invitations to play poker . personal factors such as motivation ( loves to play poker ) , and environmental factors ( poker buddy who can drive ) enable the affected person to still participate in his favorite social activity in spite of significant functional impairment or activity limitation . the interaction of all these elements thus gives rise to a certain level of disability in that person . although the above example was simplified for clarity , it can be observed that disability originates from the underlying chronic disease as it directly gives rise to functional impairment , and affects activity performance and social participation both directly and indirectly . in older people especially , multiple chronic diseases may co - exist contributing to disability , although not everyone with a high chronic disease burden is necessarily disabled . the next observation is that for a certain degree of functional impairment , the level of restriction in activity and participation is variable depending on the personal and environmental factors surrounding the individual . the last observation is that functional impairment directly impacts on activity and participation , and although the correlation between them may be lessened by the aforementioned personal and environmental factors , it is still likely to be fairly strong . earlier studies on the icf were restricted to examining disability in a particular disease - specific context or outcome , rather than looking at the general validity of the model as a whole ( dale et al . , 2012 ; faria - fortini , michaelsen , cassiano , & teixeira - salmela , 2011 ; jonsson , ekholm , & schult , 2008 ; pollard , johnston , & dieppe , 2011 ) . these disease - specific studies may also have limited generalizability to the elderly population , where there are often significant and concurrent co - morbidities contributing to disability . the use of a single large data set in examining the icf model has the advantage of analyzing the connections between icf constructs simultaneously , thus allowing a comparison of strengths between these inter - relationships . it would also be useful to determine how cognitive factors contribute to disability in the icf model . thus , our study aimed to examine in a cohort of community - dwelling older adults some of the observations or hypotheses stemming from the icf model , which are that ( a ) chronic disease burden is strongly related to disability as a whole ; ( b ) for a given level of functional impairment , individuals vary significantly in performance of activities ; and ( c ) functional impairment is strongly related to activity performance , which in turn correlates with social participation ( world health organization , 2011 ) . we also studied where relevant , the impact of dementia status on disability as well as the relationships between the icf constructs . this study was based on the determinants of wellness among older malaysians : a health promotion perspective survey conducted by trained interviewers from universiti putra malaysia and the malaysian ministry of health in 2008 to 2009 . the questions were set in english and malay , and translated into the primary language of the respondents by interviewers who were native speakers where necessary . it was not feasible to pre - translate into all the local languages due to the large number of tribes and ethnic groups especially in east malaysia . the interviewers read out the individual questions and noted the responses in a set of printed study forms . the study was approved by the malaysian ministry of health ethics review board ( approval reference p08 - 219 ) in accordance with current guidelines on good clinical practice and the declaration of helsinki . specific informed consent was not required by the ethics review board as the data were analyzed anonymously . this study used a geographical cluster sampling design based on the world health organization stepwise approach to surveillance ( steps ) , with clusters represented by enumeration blocks ( ebs ) determined by the malaysian department of statistics ( department of statistics malaysia , 2010 ; world health organization , 2012 ) . the inclusion criteria were above the age of 60 years , a malaysian citizen , and willing to be interviewed . they were excluded from the study if mobility , mental functions , or hearing was sufficiently impaired to prevent them from completing the survey . each eb had a target recruitment of eight people , but less could have been recruited if suitable residents were not found in the allocated houses . the world health organization disability assessment schedule ii ( whodas - ii ) was developed to assess disability based on the icf ( world health organization , 2001b ) . it has been validated across different cultures for use in subjects with chronic diseases , older people , and is literacy - independent ( garin et al . , 2010 ; sousa et al . , 2010 ) . in our study , we used simple percentile sum scoring . severe disability was defined as a whodas - ii score above the 90th percentile for the population ( scott et al . , 2009 ) . dementia status was defined using the malaysian version of the 30-item folstein mini - mental state examination ( mmse - m ) . the sample was stratified into two groups ( with and without dementia ) based on locally validated cutoffs , with good sensitivity ( 88%-97% ) and specificity ( 75%-93% ) in classifying dementia against diagnostic and statistical manual of mental disorders ( 4th ed . the question testing attention and calculation was repeated using serial 7 s , serial 3 s , and spelling the word world backwards , and was scored according to the best performance . the respective mmse - m cutoffs for dementia classification were 21/22 , 18/19 , and 17/18 , respectively , depending on the question used ( ibrahim et al . , respondents were asked whether they had any of the chronic non - communicable disease groups , which may contribute to disability : cancer ( all types ) , respiratory ( asthma , obstructive airways disease , pulmonary tuberculosis ) , uncorrected visual or hearing impairment , renal , gastrointestinal - urinary ( dyspepsia , constipation , incontinence ) , musculoskeletal ( joint pain , gout , arthritis ) , and cardiovascular ( hypertension , diabetes mellitus , heart disease , stroke ) . they were also questioned whether the diagnoses had been medically confirmed . although pulmonary tuberculosis is not strictly a chronic non - communicable disease , it was included in the list as it can cause long - term impairment of lung function , and is commonly found among the elderly in malaysia . each of the seven chronic disease groups and dementia were marked as either present ( 1 ) or absent ( 0 ) . the timed get - up and go test ( gugt ) , short international physical activity questionnaire ( ipaq ) , and lubben social network scale ( lsns-6 ) are measures of basic functional mobility , physical activity , and social support ( bauman et al . , 2009 ; lubben , 2003 ; podsiadlo & richardson , 1991 ) . they are feasible to perform , valid , and reliable in the elderly ( forsen et al . , 2010 ; hurtig - wennlof , hagstromer , & olsson , 2010 ; nordin , lindelof , rosendahl , jensen , & lundin - olsson , 2008 ; wall , bell , campbell , & davis , 2000 ) . unless specified in the original definition , missing values were replaced by the mean of the remaining values in the scale provided internal consistency was demonstrated to be high ( cronbach s > .7 ) . up to one missing value could be replaced in this way and further missing values invalidated the score for that respondent . education level , net monthly income , smoking , alcohol consumption , ethnicity , marital status , and religion were classified according to standard census criteria ( department of statistics malaysia , 2010 ) . fall status was determined as whether the respondent had any falls resulting in injury in the previous 6 months . body mass index ( bmi ) and waist circumference were obtained using a stadiometer ( 206 cm bodymeter measuring tape , seca , hamburg , germany ) and electronic weight scale ( hd-314 digital weight scale , tanita , arlington heights , illinois , usa ) . dietary adequacy was assessed using a dietary diversity score ( dds ; kant , schatzkin , harris , ziegler , & block , 1993 ) . the overall contribution of chronic disease to severe disability was modeled using population attributable risk ( par ) . par was calculated for each chronic disease group along with a total par for their combined effect ( sousa et al . , 2009 ) . the combined par for all ( n ) disease groups is shown below ( cole & macmahon , 1971 ) . a composite chronic disease score ( cdbs ) for all ( n ) disease groups including dementia was derived for each respondent , weighted based on the par of each group as shown below . a composite chronic disease score without dementia ( cdbs - nd ) was also derived using the same method . cdbs(n)=i=1n(dipari)i=1n(pari ) , where cdbs is expressed as a percentage and di represents the presence ( 1 ) or absence ( 0 ) of the chronic disease group i. to test the first observation that chronic disease burden is strongly related to disability , a multivariate approach was used , including covariates that could potentially act as confounders . subjects were divided into two groups based on the presence or absence of severe disability . the initial list of covariates screened were age , gender , marital status , race , religion , years of formal education , net income , smoking and alcohol use , dds , fall status , bmi , and waist circumference . to reduce the number of covariates , they were tested against the grouped whodas - ii score using a pearson s chi - square test for categorical covariates , and either an unpaired t test or mann whitney test for continuous covariates ( table 1 ) . baseline characteristics of respondents , selected summary data from the malaysia 2010 census , and key outcome measures . iqr = inter - quartile range ; whodas - ii = world health organization disability assessment schedule ii ; cdbs = composite chronic disease score ; cdbs - nd = composite chronic disease score without dementia ; gugt = get - up and go test ; ipaq = international physical activity questionnaire ; lsns-6 = lubben social network scale ; met = metabolic equivalent task . the key outcome measures were shown as medians and iqrs as most of them were non - normal in distribution . significant covariates were then entered into binary logistic regression analysis , with the cdbs as the main independent variable and the grouped whodas - ii score as the binary dependent variable . the same analysis was also repeated excluding subjects with dementia on the mmse - m , utilizing cdbs - nd as the main independent variable . to test the second observation that individuals vary significantly in performance of activities for a given level of functional impairment , we plotted ipaq activity levels ( activity performance ) for each gugt functional status category ( functional impairment ) to illustrate the degree of variability in activity performance . as a comparison , lsns-6 scores ( social participation ) were divided into quartiles and similarly charted against gugt ( functional impairment ) . the dispersion for ipaq and lsns-6 scores in each gugt category was quantified using the inter - quartile range ( iqr ) . for the third observation , we tested the relationship between functional impairment and activity performance by comparing ipaq energy expenditure ( activity performance ) with gugt ( functional impairment ) using spearman s rank correlation coefficient , the kruskal the relationship between activity performance and social participation was tested by comparing the lsns-6 score ( social participation ) and ipaq activity levels ( activity performance ) in a similar manner . this analysis was repeated stratifying by dementia status on the mmse - m . as a comparison , we quantitatively tested the relationship between functional impairment and social participation in a similar manner . sample size was calculated using the steps sample size calculator from the world health organization ( 2012 ) . based on a 95% confidence level , 5% margin of error , an estimated prevalence of risk factors of 50% , a design effect correction of 3 , an expected response rate of 60% , the calculated sample size was 3,840 subjects , translating to 480 ebs of eight subjects each . all computations were performed using spss for windows version 20.0 ( ibm corp . , armonk , new york , usa ) . the overall contribution of chronic disease to severe disability was modeled using population attributable risk ( par ) . par was calculated for each chronic disease group along with a total par for their combined effect ( sousa et al . , 2009 ) . the combined par for all ( n ) disease groups is shown below ( cole & macmahon , 1971 ) . par(n)=1i=1n(1pari ) . a composite chronic disease score ( cdbs ) for all ( n ) disease groups including dementia was derived for each respondent , weighted based on the par of each group as shown below . a composite chronic disease score without dementia ( cdbs - nd ) was also derived using the same method . cdbs(n)=i=1n(dipari)i=1n(pari ) , where cdbs is expressed as a percentage and di represents the presence ( 1 ) or absence ( 0 ) of the chronic disease group i. to test the first observation that chronic disease burden is strongly related to disability , a multivariate approach was used , including covariates that could potentially act as confounders . subjects were divided into two groups based on the presence or absence of severe disability . the initial list of covariates screened were age , gender , marital status , race , religion , years of formal education , net income , smoking and alcohol use , dds , fall status , bmi , and waist circumference . to reduce the number of covariates , they were tested against the grouped whodas - ii score using a pearson s chi - square test for categorical covariates , and either an unpaired t test or mann whitney test for continuous covariates ( table 1 ) . baseline characteristics of respondents , selected summary data from the malaysia 2010 census , and key outcome measures . note . iqr = inter - quartile range ; whodas - ii = world health organization disability assessment schedule ii ; cdbs = composite chronic disease score ; cdbs - nd = composite chronic disease score without dementia ; gugt = get - up and go test ; ipaq = international physical activity questionnaire ; lsns-6 = lubben social network scale ; met = metabolic equivalent task . the key outcome measures were shown as medians and iqrs as most of them were non - normal in distribution . significant covariates were then entered into binary logistic regression analysis , with the cdbs as the main independent variable and the grouped whodas - ii score as the binary dependent variable . the same analysis was also repeated excluding subjects with dementia on the mmse - m , utilizing cdbs - nd as the main independent variable . to test the second observation that individuals vary significantly in performance of activities for a given level of functional impairment , we plotted ipaq activity levels ( activity performance ) for each gugt functional status category ( functional impairment ) to illustrate the degree of variability in activity performance . as a comparison , lsns-6 scores ( social participation ) were divided into quartiles and similarly charted against gugt ( functional impairment ) . the dispersion for ipaq and lsns-6 scores in each gugt category was quantified using the inter - quartile range ( iqr ) . for the third observation , we tested the relationship between functional impairment and activity performance by comparing ipaq energy expenditure ( activity performance ) with gugt ( functional impairment ) using spearman s rank correlation coefficient , the kruskal the relationship between activity performance and social participation was tested by comparing the lsns-6 score ( social participation ) and ipaq activity levels ( activity performance ) in a similar manner . this analysis was repeated stratifying by dementia status on the mmse - m . as a comparison , we quantitatively tested the relationship between functional impairment and social participation in a similar manner . sample size was calculated using the steps sample size calculator from the world health organization ( 2012 ) . based on a 95% confidence level , 5% margin of error , an estimated prevalence of risk factors of 50% , a design effect correction of 3 , an expected response rate of 60% , the calculated sample size was 3,840 subjects , translating to 480 ebs of eight subjects each . all computations were performed using spss for windows version 20.0 ( ibm corp . , armonk , new york , usa ) . the sample included a total of 2,563 respondents with an overall response rate of 66.7% , which was more than the expected response rate of 60% ( figure 1 ) . baseline characteristics of respondents are detailed in table 1 , along with selected equivalent summary data from the malaysia 2010 census ( department of statistics malaysia , 2010 ) . the gender and ethnic distribution approximates that from the census data , with the exception of having less malays and more indigenous people . the responses for medically confirmed diagnoses essentially followed the same pattern as self - reported conditions ( pearson s correlation coefficient r = .68 ) , and hence were not analyzed separately . median values and non - response rates for the key outcome measures are summarized in table 1 ; 21.5% were classified as having dementia using the locally validated cutoffs for mmse - m . the non - response rate for gugt testing ( 20.4% ) was disproportionately higher than for other outcome measures , and mostly attributable to lack of proper equipment or space ( 7.7% ) and refusal ( 5.5% ) . there was a significant difference in whodas - ii scores between respondents who refused gugt testing and those who agreed , with those who refused having higher scores ( mean 4.4 points , 95% ci = [ 0.6 , 8.1 ] , p = .02 ) . alpha for whodas - ii , dds , lsns-6 , cdbs , and cdbs - nd were .92 , .46 , .83 , .28 , and .28 , respectively . replacement of missing values occurred in 8% of whodas - ii scores but not for the dds , lsns-6 , cdbs , or cdbs - nd . for the first observation , the 90th percentile for whodas - ii scores used to define severe disability was 50 points . significant covariates obtained after initial testing against whodas - ii were gender , marital status , smoking , fall status , age , years of formal education , and net income ( table 2 ) . binary logistic regression using these covariates gave a good model fit with a non - significant hosmer and lemeshow statistic ( p = .26 ) , 2 log - likelihood of 1,184 , overall correct classification percentage of 92.4% , and nagelkerke r - square of .78 . the wald statistic and significance level for the cdbs were 25.5 and p < .001 . the other significant covariates from regression analysis were gender , age , education status , and social support ( table 3 ) . when the analysis was repeated excluding subjects with dementia , the wald statistic and significance level for the cdbs - nd were 28.5 and p < .001 , with a nagelkerke r - square of .78 . the combined par for chronic disease was 53.8% , whereas the par for dementia alone was 15.3% . covariates tested against whodas - ii scores using pearson s chi - square for categorical covariates and unpaired t test for continuous covariates . note . whodas - ii = world health organization disability assessment schedule ii ; ci = confidence interval . relevant statistics for the logistic regression analysis from the first observation ( cdbs only ) . note . cdbs = composite chronic disease score ; ci = confidence interval ; lsns-6 = lubben social network scale . , bar charts of ipaq activity levels for each gugt functional status category clearly showed that at low levels of functional impairment , activity performance varied considerably with a large iqr ( 2.00 ; figure 2 , right panel ) . however , at higher levels of functional impairment , activity performance was consistently low with a small iqr ( 0.00 ; figure 2 , left panel ) . in contrast , significant variability was present in social participation throughout all levels of functional impairment ( iqr = 9 - 10 ; figure 3 ) . ipaq = international physical activity questionnaire ; gugt = get - up and go test ; iqr = inter - quartile range ; ci = confidence interval . lsns = lubben social network scale ; gugt = get - up and go test ; iqr = interquartile range ; ci = confidence interval . for the third observation .001 ) in ipaq energy expenditure between gugt functional status groups , and the bar chart showed a clear rise in energy expenditure with improving functional status ( spearman s rho = 0.33 , p < 0.001 ) . when analyzed according to dementia status , the trends were generally similar although it could be seen that those without dementia were able to maintain a higher level of activity even with moderately reduced ambulatory function ( figure 4 , bottom panel ) . bar chart of mean energy expenditure against gugt functional status category stratified according to dementia status ( total , yes , no ) . gugt = get - up and go test ; ci = confidence interval ; met = metabolic equivalent task . similarly , anova showed a highly significant difference ( = 83.8 , p < .001 ) in lsns-6 scores between ipaq activity levels , whereas the bar chart demonstrated improving social participation with better activity performance ( spearman s rho = 0.18 , p < .001 ) . the presence of dementia was shown to substantially reduce social participation , and those with concurrent low physical activity and dementia had the lowest scores ( median lsns-6 = 11.0 ; figure 5 , middle panel ) . bar chart of mean lsns-6 scores against ipaq activity levels stratified according to dementia status ( total , yes , no ) . lsns = lubben social network scale ; ipaq = international physical activity questionnaire ; ci = confidence interval . the difference in lsns-6 scores between gugt groups was also significant ( = 26.3 , p < .001 ) showing improving social participation with better functional status ( spearman s rho = 0.11 , p < .001 ) . for the first observation that examined the relationship between chronic disease burden and disability , the model used for regression analysis had a good fit and high correct classification percentage . hence , the highly significant wald statistic for cdbs indicates that chronic disease burden is strongly related to disability , even after accounting for significant confounding variables and dementia status . the large nagelkerke r - square value shows that most of the variability in disability between individuals was accounted for by chronic disease and socio - demographic characteristics such as gender , age , education level , and social support . in addition , the par calculations indicate that chronic disease accounted for more than half of prevalent disability , with dementia causing a large proportion of it . these findings are all consistent with results from the multi - national 10/66 dementia research group , which reported that the significant covariates were chronic disease , age , gender , and education level . similar to our study , par for chronic disease accounted for two thirds of disability with dementia also being the largest contributor ( sousa et al . , 2009 ) . the second observation states that for a given level of functional impairment , activity performance varies significantly between individuals due to the effect of factors such as motivation , self - esteem , and a conducive environment . however , we found that this holds true only for individuals with relatively little functional impairment ( figure 2 , right panel ) . with worsening function , the role of environmental factors diminishes as the ability to perform activities becomes limited ( figure 2 , left panel ) the reduction of variability in activity performance is evidenced by a corresponding diminution of the iqr . this means that once an individual s physical function deteriorates beyond a certain point , modification of the environment can not meaningfully improve that person s disability . in contrast , functional impairment does not seem to be a major limiting factor for social participation , and it is likely that individuals retain their pre - existing social networks even after their functional status worsens ( figure 3 ) . this relates very well to antonucci s support bank theory , which postulates that social capital built up when a person is younger carries over into old age in the form of social support ( antonucci & jackson , 1990 ) . figures 4 and 5 and the highly significant anova tests all show a clear relationship between functional impairment , activity performance , and social participation , thus validating the third observation . the relative weakness of the relationship between functional impairment and social participation ( = 26.3 ) compared with the other two relationships ( = 253.3 and 83.8 ) suggests that activity performance is the intermediary construct in this model ( figure 6 ) . a path analysis could provide more clarity although this is out of the scope of the present study . diagram showing the international classification of functioning , disability , and health constructs , the instruments representing these constructs , and the relationships examined in this study . note . cdbs = composite chronic disease score ; whodas - ii = world health organization disability assessment schedule ii ; gugt = get - up and go test ; ipaq = international physical activity questionnaire ; lsns = lubben social network scale . although the cognitive aspect of disability was not studied in depth , it could clearly be seen that the presence of dementia significantly worsened both physical activity and social participation . furthermore , cognitively intact individuals with a moderate level of functional impairment retained a significant level of activity performance , implying that this group may have the potential for further improvement with appropriate rehabilitation ( figure 4 and 5 ) . in contrast , concurrent low physical activity and poor cognitive function identify individuals who are at risk of social isolation ( lubben et al . , 2006 ) . for the first observation , because chronic non - communicable disease is so strongly tied to disability , every effort should be made to identify the major conditions in each population , which contribute to it . these conditions should then be targeted with effective preventive care programs to reduce the eventual occurrence of end organ functional impairments in the community . dementia is an especially good candidate to focus on due to its prevalence in community - dwelling elderly and the high par for severe disability ( hamid , krishnaswamy , abdullah , & momtaz , 2010 ) . in addition to these primary health conditions , attention should be paid to the secondary and co - morbid conditions that arise from them , the provision of adequate health care , and removing barriers to performance and participation ( world health organization , 2011 ) . for the second observation , it can be seen that modification of the environment will be most effective in increasing activity performance in individuals with moderate functional impairment . in japan , the heart building law ( 1994 ) , barrier free transport law ( 2000 ) , and the barrier free law ( 2006 ) provide a formal legal framework to enhance accessibility for the aged and disabled . although these regulations have greatly improved facilities for the physically disabled , this study suggests that cognitive disablement is also an important issue that needs to be considered . for example , signage needs to be written in clear simple language with unambiguous universal symbols to aid understanding . for those with high levels of functional impairment especially with concurrent cognition issues , this study shows that mobility is much harder to improve even with the best barrier - free environment . resources would be better targeted in enhancing their support network , and hence social participation with interventions such as community social worker assistance and programs for organized home visits by school children and voluntary welfare organizations . for the third observation , the strong linkages between functional impairment , activity performance , and social participation , mean that success in improving any one of these will have secondary benefits for the downstream components , hence reducing overall disability . logically this means that resources would be better spent on areas that have the most potential to benefit each individual , rather than dealing with all aspects of disability simultaneously . from this study , the implication is that younger people are best targeted with prevention programs to reduce the risk of developing disability , whereas older people with milder disability should focus on removing barriers to activity performance , and those with more severe disability should be provided more support to improve their social participation ( loke , abdullah , chai , hamid , & yahaya , 2011 ) . it can be seen from figures 4 and 5 that the presence of dementia has an important effect on the relationships between the icf constructs . moreover , data from this study and the 10/66 dementia research group show that dementia contributes significantly to disability in the community - dwelling elderly ( sousa et al . , 2009 ) . seen together , there would seem to be good reason to investigate the cognitive aspects of disability in greater detail that what was achieved in this study . we opted to study disability in community - dwelling older people as very little work has been done locally in this population aside from the national health and morbidity survey , which was based on the icidh ( institute for public health , 2008 ) . mindful of the need to establish the external validity of the model , we chose instruments that were developed separately from the icf when testing the second and third observations . the relevant domains of the whodas - ii ( cognition , mobility , life activities , and participation ) were unsuitable for this role as they were co - developed with the icf . moreover , the high level of internal consistency ( cronbach s = .94-.96 within domains , .98 across domains ) virtually guarantees that the domains will cross - correlate with each other , thus defeating the intent of the study ( world health organization , 2001b ) . in addition , the instruments used focused on important aspects of an older person s life , such as mobility ( gugt ) , overall physical activity and energy expenditure ( ipaq ) , and social networking ( lsns-6 ) . there is very little overlap between these instruments , thus enabling the constructs to be clearly distinguished ( badley , 2008 ) . the instruments are also not disease specific , thus allowing generalization across a variety of conditions . the choice of instruments was made to reflect the conceptual framework behind the icf model . for example , mobility ( gugt ) was chosen to represent body functions and structures ( icf b and s codes , respectively ) as the majority of impairments ( mental , sensory , cardio - respiratory , metabolic , and neuro - musculoskeletal , corresponding to icf chapters 1 , 2 , 4 , 5 , and 7 , respectively ) will result in reduced mobility . similarly , physical activity ( ipaq ) and social networking ( lsns-6 ) were chosen to represent activities and participation ( icf d codes ) as these are consequent manifestations of an individual s mobility in relation to the icf model ( world health organization , 2001a ) . although the whodas - ii has been superseded by the more recent whodas 2.0 , the 12-item instrument used is virtually identical in wording with the updated version ( world health organization , 2015 ) . this means that the findings and conclusions made in this article equally apply to the newer instrument and the icf in general . conservative values were chosen for sample size calculation as a significant proportion of the survey was conducted in sparsely populated rural areas . a design effect correction of 3 was used rather than the recommended value of 1.5 as a study demonstrated that in such circumstances this value can range from 1 to 5 ( rowe , lama , onikpo , & deming , 2002 ) . the expected response rate was also reduced as certain allocated ebs were impossible to access due to geographical isolation . considering this , the overall non - response rate for the study was within expectation . the individual item response rates were very good aside from gugt , which was likely because some older people can be fairly averse to physical testing . whereas the complete icf model depicts the interplay between disease states , human functioning , and environmental factors , this study is primarily scoped toward disease states and human functioning . a related study by the authors examined the contribution of environmental factors ( icf e codes ) in greater detail ( hamid et al . , 2010 ) . there is a systematic bias against severe disability due to the sampling method , which excluded people who were unable to complete the survey because of this . however , there is no good way of working around this issue as the alternative would be to rely on secondary information from caregivers , which would be vulnerable to confirmation bias . although there may have been under - sampling of the chinese population due to language barriers , the proportion of ethnic chinese in the study is similar to that from the census data , suggesting that the magnitude of under - sampling was small ( table 1 ) . moreover , this should only give rise to minimal bias as whodas - ii scores in this ethnic group are fairly similar to the other major races ( malays and indians ) . although there was possibly some minor element of bias from the gugt non - response , this should not have affected the conclusions drawn as the results were all highly significant with large effect sizes . most of the instruments used in this study were validated in english , and there were no validation studies either in malay or the many languages and dialects used locally . once again there is no realistic way around this issue , and this study should be interpreted with this in mind . although the validation article for mmse - m recommended that serial 7 s or serial 3 s be tried as a first choice , followed by the spell backwards method , we found that most of the elderly in rural communities could not cope with this due to low levels of education . the mmse - m was validated in city hospitals with subjects who had much better educational exposure , so the project team made a decision to use the best result of the three methods as a compromise ( ibrahim et al . , 2009 ) . although this may potentially alter the diagnosis of dementia to a small degree , this risk was judged to be acceptable as cognition was not the main focus for the study . lastly , certain aspects of the study were either abbreviated or simplified to keep the questionnaire time to less than an hour . although extending the time or using a multi - stage interview might have allowed collection of additional data , there was a strong likelihood that those with high levels of functional impairment would drop out or refuse in a developing country setting ( prince , 2000 ) . the cognitive aspect of disability was also not examined in depth , as other parallel studies by the authors will address this issue separately in greater detail ( hamid et al . , 2010 ; loke et al . , it would have been desirable to examine the impact of each disease group individually , but the cdbs was used in this study as a compromise between survey time and greater detail . in any case , the relationship between cdbs and whodas - ii was still found to be highly significant on logistic regression testing ( p < .001 ) , suggesting that this instrument was adequate for the limited purposes of this study . we opted to study disability in community - dwelling older people as very little work has been done locally in this population aside from the national health and morbidity survey , which was based on the icidh ( institute for public health , 2008 ) . mindful of the need to establish the external validity of the model , we chose instruments that were developed separately from the icf when testing the second and third observations . the relevant domains of the whodas - ii ( cognition , mobility , life activities , and participation ) were unsuitable for this role as they were co - developed with the icf . moreover , the high level of internal consistency ( cronbach s = .94-.96 within domains , .98 across domains ) virtually guarantees that the domains will cross - correlate with each other , thus defeating the intent of the study ( world health organization , 2001b ) . in addition , the instruments used focused on important aspects of an older person s life , such as mobility ( gugt ) , overall physical activity and energy expenditure ( ipaq ) , and social networking ( lsns-6 ) . there is very little overlap between these instruments , thus enabling the constructs to be clearly distinguished ( badley , 2008 ) . the instruments are also not disease specific , thus allowing generalization across a variety of conditions . the choice of instruments was made to reflect the conceptual framework behind the icf model . for example , mobility ( gugt ) was chosen to represent body functions and structures ( icf b and s codes , respectively ) as the majority of impairments ( mental , sensory , cardio - respiratory , metabolic , and neuro - musculoskeletal , corresponding to icf chapters 1 , 2 , 4 , 5 , and 7 , respectively ) will result in reduced mobility . similarly , physical activity ( ipaq ) and social networking ( lsns-6 ) were chosen to represent activities and participation ( icf d codes ) as these are consequent manifestations of an individual s mobility in relation to the icf model ( world health organization , 2001a ) . although the whodas - ii has been superseded by the more recent whodas 2.0 , the 12-item instrument used is virtually identical in wording with the updated version ( world health organization , 2015 ) . this means that the findings and conclusions made in this article equally apply to the newer instrument and the icf in general . conservative values were chosen for sample size calculation as a significant proportion of the survey was conducted in sparsely populated rural areas . a design effect correction of 3 was used rather than the recommended value of 1.5 as a study demonstrated that in such circumstances this value can range from 1 to 5 ( rowe , lama , onikpo , & deming , 2002 ) . the expected response rate was also reduced as certain allocated ebs were impossible to access due to geographical isolation . considering this , the individual item response rates were very good aside from gugt , which was likely because some older people can be fairly averse to physical testing . whereas the complete icf model depicts the interplay between disease states , human functioning , and environmental factors , this study is primarily scoped toward disease states and human functioning . a related study by the authors examined the contribution of environmental factors ( icf e codes ) in greater detail ( hamid et al . , 2010 ) . there is a systematic bias against severe disability due to the sampling method , which excluded people who were unable to complete the survey because of this . however , there is no good way of working around this issue as the alternative would be to rely on secondary information from caregivers , which would be vulnerable to confirmation bias . although there may have been under - sampling of the chinese population due to language barriers , the proportion of ethnic chinese in the study is similar to that from the census data , suggesting that the magnitude of under - sampling was small ( table 1 ) . moreover , this should only give rise to minimal bias as whodas - ii scores in this ethnic group are fairly similar to the other major races ( malays and indians ) . although there was possibly some minor element of bias from the gugt non - response , this should not have affected the conclusions drawn as the results were all highly significant with large effect sizes . most of the instruments used in this study were validated in english , and there were no validation studies either in malay or the many languages and dialects used locally . once again there is no realistic way around this issue , and this study should be interpreted with this in mind . although the validation article for mmse - m recommended that serial 7 s or serial 3 s be tried as a first choice , followed by the spell backwards method , we found that most of the elderly in rural communities could not cope with this due to low levels of education . the mmse - m was validated in city hospitals with subjects who had much better educational exposure , so the project team made a decision to use the best result of the three methods as a compromise ( ibrahim et al . , 2009 ) . although this may potentially alter the diagnosis of dementia to a small degree , this risk was judged to be acceptable as cognition was not the main focus for the study . lastly , certain aspects of the study were either abbreviated or simplified to keep the questionnaire time to less than an hour . although extending the time or using a multi - stage interview might have allowed collection of additional data , there was a strong likelihood that those with high levels of functional impairment would drop out or refuse in a developing country setting ( prince , 2000 ) the cognitive aspect of disability was also not examined in depth , as other parallel studies by the authors will address this issue separately in greater detail ( hamid et al . , 2010 ; loke et al . , it would have been desirable to examine the impact of each disease group individually , but the cdbs was used in this study as a compromise between survey time and greater detail . in any case , the relationship between cdbs and whodas - ii was still found to be highly significant on logistic regression testing ( p < .001 ) , suggesting that this instrument was adequate for the limited purposes of this study . our study builds upon the body of evidence from earlier disease - specific studies by demonstrating the validity of the earlier observations based on the icf model using empirical data from a large sample of community - dwelling older adults with a broad range of medical diagnoses . consistent with earlier studies , our results confirm that disability is strongly related to chronic disease burden , and that functional impairment correlates with activity performance and consequently with social participation . the novel finding from our study is that at low levels of functional impairment , there is considerable variability in the relationship between functional impairment and activity performance , but this diminishes at high impairment levels especially when complicated by cognitive deficits . this suggests that modification of the environment benefits those with moderate levels of functional impairment , but not those with higher grades of functional loss .

objective : this study examines the international classification of functioning , disability , and health model ( icf ) using a data set of 2,563 community - dwelling elderly with disease - independent measures of mobility , physical activity , and social networking , to represent icf constructs . method : the relationship between chronic disease and disability ( independent and dependent variables ) was examined using logistic regression . to demonstrate variability in activity performance with functional impairment , graphing was used . the relationship between functional impairment , activity performance , and social participation was examined graphically and using anova . the impact of cognitive deficits was quantified through stratifying by dementia . results : disability is strongly related to chronic disease ( wald 25.5 , p < .001 ) , functional impairment with activity performance ( f = 34.2 , p < .001 ) , and social participation ( f= 43.6 , p < .001 ) . with good function , there is considerable variability in activity performance ( inter - quartile range [ iqr ] = 2.00 ) , but diminishes with high impairment ( iqr = 0.00 ) especially with cognitive deficits . discussion : environment modification benefits those with moderate functional impairment , but not with higher grades of functional loss .

Introduction Method Analysis Results Discussion Study Considerations and Limitations Conclusion

in the international classification of functioning , disability , and health model ( icf ) , pre - existing health conditions in an individual can give rise to functional impairments , which in turn cause activity limitation and restriction in social participation ( grimby & smedby , 2001 ; world health organization , 2011 ) . the key improvement from the earlier disability model , the international classification of impairments , disabilities , and handicaps ( icidh ) , was a shift from a purely medical to a bio - psycho - social framework reflecting the interaction between an individual s health status and ambient factors , both personal and environmental . when viewed through the lens of the icf model , the stroke is a chronic disease that gives rise to functional impairment ( limb paralysis ) , which limits activity performance ( driving ) , which in turn restricts social participation ( going to play poker ) . the stroke itself may also limit activity performance and social participation directly by making it harder to retain a driving license or getting fewer invitations to play poker . personal factors such as motivation ( loves to play poker ) , and environmental factors ( poker buddy who can drive ) enable the affected person to still participate in his favorite social activity in spite of significant functional impairment or activity limitation . although the above example was simplified for clarity , it can be observed that disability originates from the underlying chronic disease as it directly gives rise to functional impairment , and affects activity performance and social participation both directly and indirectly . the next observation is that for a certain degree of functional impairment , the level of restriction in activity and participation is variable depending on the personal and environmental factors surrounding the individual . the last observation is that functional impairment directly impacts on activity and participation , and although the correlation between them may be lessened by the aforementioned personal and environmental factors , it is still likely to be fairly strong . the use of a single large data set in examining the icf model has the advantage of analyzing the connections between icf constructs simultaneously , thus allowing a comparison of strengths between these inter - relationships . thus , our study aimed to examine in a cohort of community - dwelling older adults some of the observations or hypotheses stemming from the icf model , which are that ( a ) chronic disease burden is strongly related to disability as a whole ; ( b ) for a given level of functional impairment , individuals vary significantly in performance of activities ; and ( c ) functional impairment is strongly related to activity performance , which in turn correlates with social participation ( world health organization , 2011 ) . we also studied where relevant , the impact of dementia status on disability as well as the relationships between the icf constructs . it has been validated across different cultures for use in subjects with chronic diseases , older people , and is literacy - independent ( garin et al . , respondents were asked whether they had any of the chronic non - communicable disease groups , which may contribute to disability : cancer ( all types ) , respiratory ( asthma , obstructive airways disease , pulmonary tuberculosis ) , uncorrected visual or hearing impairment , renal , gastrointestinal - urinary ( dyspepsia , constipation , incontinence ) , musculoskeletal ( joint pain , gout , arthritis ) , and cardiovascular ( hypertension , diabetes mellitus , heart disease , stroke ) . although pulmonary tuberculosis is not strictly a chronic non - communicable disease , it was included in the list as it can cause long - term impairment of lung function , and is commonly found among the elderly in malaysia . the timed get - up and go test ( gugt ) , short international physical activity questionnaire ( ipaq ) , and lubben social network scale ( lsns-6 ) are measures of basic functional mobility , physical activity , and social support ( bauman et al . cdbs(n)=i=1n(dipari)i=1n(pari ) , where cdbs is expressed as a percentage and di represents the presence ( 1 ) or absence ( 0 ) of the chronic disease group i. to test the first observation that chronic disease burden is strongly related to disability , a multivariate approach was used , including covariates that could potentially act as confounders . iqr = inter - quartile range ; whodas - ii = world health organization disability assessment schedule ii ; cdbs = composite chronic disease score ; cdbs - nd = composite chronic disease score without dementia ; gugt = get - up and go test ; ipaq = international physical activity questionnaire ; lsns-6 = lubben social network scale ; met = metabolic equivalent task . to test the second observation that individuals vary significantly in performance of activities for a given level of functional impairment , we plotted ipaq activity levels ( activity performance ) for each gugt functional status category ( functional impairment ) to illustrate the degree of variability in activity performance . the dispersion for ipaq and lsns-6 scores in each gugt category was quantified using the inter - quartile range ( iqr ) . for the third observation , we tested the relationship between functional impairment and activity performance by comparing ipaq energy expenditure ( activity performance ) with gugt ( functional impairment ) using spearman s rank correlation coefficient , the kruskal the relationship between activity performance and social participation was tested by comparing the lsns-6 score ( social participation ) and ipaq activity levels ( activity performance ) in a similar manner . this analysis was repeated stratifying by dementia status on the mmse - m . as a comparison , we quantitatively tested the relationship between functional impairment and social participation in a similar manner . a composite chronic disease score without dementia ( cdbs - nd ) was also derived using the same method . cdbs(n)=i=1n(dipari)i=1n(pari ) , where cdbs is expressed as a percentage and di represents the presence ( 1 ) or absence ( 0 ) of the chronic disease group i. to test the first observation that chronic disease burden is strongly related to disability , a multivariate approach was used , including covariates that could potentially act as confounders . to reduce the number of covariates , they were tested against the grouped whodas - ii score using a pearson s chi - square test for categorical covariates , and either an unpaired t test or mann whitney test for continuous covariates ( table 1 ) . iqr = inter - quartile range ; whodas - ii = world health organization disability assessment schedule ii ; cdbs = composite chronic disease score ; cdbs - nd = composite chronic disease score without dementia ; gugt = get - up and go test ; ipaq = international physical activity questionnaire ; lsns-6 = lubben social network scale ; met = metabolic equivalent task . to test the second observation that individuals vary significantly in performance of activities for a given level of functional impairment , we plotted ipaq activity levels ( activity performance ) for each gugt functional status category ( functional impairment ) to illustrate the degree of variability in activity performance . as a comparison , lsns-6 scores ( social participation ) were divided into quartiles and similarly charted against gugt ( functional impairment ) . the dispersion for ipaq and lsns-6 scores in each gugt category was quantified using the inter - quartile range ( iqr ) . for the third observation , we tested the relationship between functional impairment and activity performance by comparing ipaq energy expenditure ( activity performance ) with gugt ( functional impairment ) using spearman s rank correlation coefficient , the kruskal the relationship between activity performance and social participation was tested by comparing the lsns-6 score ( social participation ) and ipaq activity levels ( activity performance ) in a similar manner . this analysis was repeated stratifying by dementia status on the mmse - m . as a comparison , we quantitatively tested the relationship between functional impairment and social participation in a similar manner . there was a significant difference in whodas - ii scores between respondents who refused gugt testing and those who agreed , with those who refused having higher scores ( mean 4.4 points , 95% ci = [ 0.6 , 8.1 ] , p = .02 ) . binary logistic regression using these covariates gave a good model fit with a non - significant hosmer and lemeshow statistic ( p = .26 ) , 2 log - likelihood of 1,184 , overall correct classification percentage of 92.4% , and nagelkerke r - square of .78 . the wald statistic and significance level for the cdbs were 25.5 and p < .001 . the other significant covariates from regression analysis were gender , age , education status , and social support ( table 3 ) . when the analysis was repeated excluding subjects with dementia , the wald statistic and significance level for the cdbs - nd were 28.5 and p < .001 , with a nagelkerke r - square of .78 . , bar charts of ipaq activity levels for each gugt functional status category clearly showed that at low levels of functional impairment , activity performance varied considerably with a large iqr ( 2.00 ; figure 2 , right panel ) . however , at higher levels of functional impairment , activity performance was consistently low with a small iqr ( 0.00 ; figure 2 , left panel ) . in contrast , significant variability was present in social participation throughout all levels of functional impairment ( iqr = 9 - 10 ; figure 3 ) . ipaq = international physical activity questionnaire ; gugt = get - up and go test ; iqr = inter - quartile range ; ci = confidence interval . for the third observation .001 ) in ipaq energy expenditure between gugt functional status groups , and the bar chart showed a clear rise in energy expenditure with improving functional status ( spearman s rho = 0.33 , p < 0.001 ) . similarly , anova showed a highly significant difference ( = 83.8 , p < .001 ) in lsns-6 scores between ipaq activity levels , whereas the bar chart demonstrated improving social participation with better activity performance ( spearman s rho = 0.18 , p < .001 ) . the presence of dementia was shown to substantially reduce social participation , and those with concurrent low physical activity and dementia had the lowest scores ( median lsns-6 = 11.0 ; figure 5 , middle panel ) . the difference in lsns-6 scores between gugt groups was also significant ( = 26.3 , p < .001 ) showing improving social participation with better functional status ( spearman s rho = 0.11 , p < .001 ) . for the first observation that examined the relationship between chronic disease burden and disability , the model used for regression analysis had a good fit and high correct classification percentage . hence , the highly significant wald statistic for cdbs indicates that chronic disease burden is strongly related to disability , even after accounting for significant confounding variables and dementia status . the large nagelkerke r - square value shows that most of the variability in disability between individuals was accounted for by chronic disease and socio - demographic characteristics such as gender , age , education level , and social support . in addition , the par calculations indicate that chronic disease accounted for more than half of prevalent disability , with dementia causing a large proportion of it . the second observation states that for a given level of functional impairment , activity performance varies significantly between individuals due to the effect of factors such as motivation , self - esteem , and a conducive environment . however , we found that this holds true only for individuals with relatively little functional impairment ( figure 2 , right panel ) . with worsening function , the role of environmental factors diminishes as the ability to perform activities becomes limited ( figure 2 , left panel ) the reduction of variability in activity performance is evidenced by a corresponding diminution of the iqr . in contrast , functional impairment does not seem to be a major limiting factor for social participation , and it is likely that individuals retain their pre - existing social networks even after their functional status worsens ( figure 3 ) . figures 4 and 5 and the highly significant anova tests all show a clear relationship between functional impairment , activity performance , and social participation , thus validating the third observation . the relative weakness of the relationship between functional impairment and social participation ( = 26.3 ) compared with the other two relationships ( = 253.3 and 83.8 ) suggests that activity performance is the intermediary construct in this model ( figure 6 ) . diagram showing the international classification of functioning , disability , and health constructs , the instruments representing these constructs , and the relationships examined in this study . cdbs = composite chronic disease score ; whodas - ii = world health organization disability assessment schedule ii ; gugt = get - up and go test ; ipaq = international physical activity questionnaire ; lsns = lubben social network scale . although the cognitive aspect of disability was not studied in depth , it could clearly be seen that the presence of dementia significantly worsened both physical activity and social participation . furthermore , cognitively intact individuals with a moderate level of functional impairment retained a significant level of activity performance , implying that this group may have the potential for further improvement with appropriate rehabilitation ( figure 4 and 5 ) . dementia is an especially good candidate to focus on due to its prevalence in community - dwelling elderly and the high par for severe disability ( hamid , krishnaswamy , abdullah , & momtaz , 2010 ) . in addition to these primary health conditions , attention should be paid to the secondary and co - morbid conditions that arise from them , the provision of adequate health care , and removing barriers to performance and participation ( world health organization , 2011 ) . for the second observation , it can be seen that modification of the environment will be most effective in increasing activity performance in individuals with moderate functional impairment . for those with high levels of functional impairment especially with concurrent cognition issues , this study shows that mobility is much harder to improve even with the best barrier - free environment . for the third observation , the strong linkages between functional impairment , activity performance , and social participation , mean that success in improving any one of these will have secondary benefits for the downstream components , hence reducing overall disability . from this study , the implication is that younger people are best targeted with prevention programs to reduce the risk of developing disability , whereas older people with milder disability should focus on removing barriers to activity performance , and those with more severe disability should be provided more support to improve their social participation ( loke , abdullah , chai , hamid , & yahaya , 2011 ) . moreover , data from this study and the 10/66 dementia research group show that dementia contributes significantly to disability in the community - dwelling elderly ( sousa et al . seen together , there would seem to be good reason to investigate the cognitive aspects of disability in greater detail that what was achieved in this study . we opted to study disability in community - dwelling older people as very little work has been done locally in this population aside from the national health and morbidity survey , which was based on the icidh ( institute for public health , 2008 ) . in addition , the instruments used focused on important aspects of an older person s life , such as mobility ( gugt ) , overall physical activity and energy expenditure ( ipaq ) , and social networking ( lsns-6 ) . for example , mobility ( gugt ) was chosen to represent body functions and structures ( icf b and s codes , respectively ) as the majority of impairments ( mental , sensory , cardio - respiratory , metabolic , and neuro - musculoskeletal , corresponding to icf chapters 1 , 2 , 4 , 5 , and 7 , respectively ) will result in reduced mobility . similarly , physical activity ( ipaq ) and social networking ( lsns-6 ) were chosen to represent activities and participation ( icf d codes ) as these are consequent manifestations of an individual s mobility in relation to the icf model ( world health organization , 2001a ) . whereas the complete icf model depicts the interplay between disease states , human functioning , and environmental factors , this study is primarily scoped toward disease states and human functioning . however , there is no good way of working around this issue as the alternative would be to rely on secondary information from caregivers , which would be vulnerable to confirmation bias . once again there is no realistic way around this issue , and this study should be interpreted with this in mind . although extending the time or using a multi - stage interview might have allowed collection of additional data , there was a strong likelihood that those with high levels of functional impairment would drop out or refuse in a developing country setting ( prince , 2000 ) . , it would have been desirable to examine the impact of each disease group individually , but the cdbs was used in this study as a compromise between survey time and greater detail . in any case , the relationship between cdbs and whodas - ii was still found to be highly significant on logistic regression testing ( p < .001 ) , suggesting that this instrument was adequate for the limited purposes of this study . in addition , the instruments used focused on important aspects of an older person s life , such as mobility ( gugt ) , overall physical activity and energy expenditure ( ipaq ) , and social networking ( lsns-6 ) . for example , mobility ( gugt ) was chosen to represent body functions and structures ( icf b and s codes , respectively ) as the majority of impairments ( mental , sensory , cardio - respiratory , metabolic , and neuro - musculoskeletal , corresponding to icf chapters 1 , 2 , 4 , 5 , and 7 , respectively ) will result in reduced mobility . similarly , physical activity ( ipaq ) and social networking ( lsns-6 ) were chosen to represent activities and participation ( icf d codes ) as these are consequent manifestations of an individual s mobility in relation to the icf model ( world health organization , 2001a ) . whereas the complete icf model depicts the interplay between disease states , human functioning , and environmental factors , this study is primarily scoped toward disease states and human functioning . most of the instruments used in this study were validated in english , and there were no validation studies either in malay or the many languages and dialects used locally . once again there is no realistic way around this issue , and this study should be interpreted with this in mind . although extending the time or using a multi - stage interview might have allowed collection of additional data , there was a strong likelihood that those with high levels of functional impairment would drop out or refuse in a developing country setting ( prince , 2000 ) the cognitive aspect of disability was also not examined in depth , as other parallel studies by the authors will address this issue separately in greater detail ( hamid et al . , it would have been desirable to examine the impact of each disease group individually , but the cdbs was used in this study as a compromise between survey time and greater detail . in any case , the relationship between cdbs and whodas - ii was still found to be highly significant on logistic regression testing ( p < .001 ) , suggesting that this instrument was adequate for the limited purposes of this study . our study builds upon the body of evidence from earlier disease - specific studies by demonstrating the validity of the earlier observations based on the icf model using empirical data from a large sample of community - dwelling older adults with a broad range of medical diagnoses . consistent with earlier studies , our results confirm that disability is strongly related to chronic disease burden , and that functional impairment correlates with activity performance and consequently with social participation . the novel finding from our study is that at low levels of functional impairment , there is considerable variability in the relationship between functional impairment and activity performance , but this diminishes at high impairment levels especially when complicated by cognitive deficits . this suggests that modification of the environment benefits those with moderate levels of functional impairment , but not those with higher grades of functional loss .

nanometer - sized crystals ( ncs ) of the lead chalcogenide family continue to intrigue researchers for several reasons . first , they show bright exciton luminescence in the near infrared ( ir ) that can be tuned over a broad energy range by the crystal dimensions . second , lead chalcogenide nanocrystals ( ncs ) can be self - assembled into thin - film quantum dot solids , in which the quantum coupling between the ncs can be tuned . thin - film systems based on ncs separated by short ligand molecules are of interest for ir photodetectors and solar cells . recently , it has been reported that interfacial ordering and epitaxial attachment resulted in nc monolayers with a square or honeycomb nanogeometry . the energy level structure , photoluminescence , and propensity to form atomically coherent two - dimensional ( 2d ) superstructures depend critically on the size and shape of the lead chalcogenide ncs and on their surface chemistry , as well . it has been established that pbse ncs that are well - capped with oleate have an excess of pb atoms . in addition , it is theoretically shown that the binding strength and configuration of oleate ligands depend on the type of facet . lead chalcogenide ncs , with pbse being of interest here , have a rock salt crystal structure . when we consider the uncoordinated crystal , the { 100 } facets , containing square ordered pb and se , have the lowest surface energy . it is thus expected that pbse ncs have a cubic shape , terminated by six { 100 } facets . the wulff reconstruction is presented in section s1 of the supporting information . however , in the case of pbse capped with oleate ligands , different nanocrystal shapes have been reported ranging from a truncated nanocube ( or rhombal cuboctahedron ) to a truncated octahedron . hence , besides the { 100 } facets , { 111 } and { 110 } facets are also observed . moreover , it is not yet clear why different pbse nc shapes are observed , and how this is related to the surface chemistry . here , we present a study of the relation between the surface chemistry and nanocrystal shape of pbse ncs ( 5.6 nm ) capped with oleate ligands . pb oleate binding is monitored by fourier transform infrared ( ftir ) absorbance and nuclear magnetic resonance ( nmr ) spectroscopy , allowing us to quantify the nature of the surface pb oleate configuration and the number of surface - attached ligands per nc . the shape of the ncs in three dimensions is obtained via high - angle annular dark field scanning transmission electron microscopy ( haadf - stem ) combined with an atom counting method that has been established previously . we have studied the relation between the shape and surface chemistry under well - defined chemical conditions . after conventional pbse synthesis , we have washed the ncs eight times to remove all remaining precursor moieties and reduce the oleate capping to a minimum . then we gradually added pb(oleate)2 to the suspension and monitored the attached oleate capping ( by nmr ) and the nc shape , as well . our results show that enhanced oleate capping results in a stabilization and extension of the { 111 } facets and a remarkable transformation of the nanocrystal shape . figure 1a shows an overview of the infrared spectrum of a pbse nanocrystal suspension in tetrachloroethylene ( tce ) that has been washed eight times with methanol . washing removes unreacted precursor molecules , excess ligand molecules , and possibly ligand molecules that are only weakly bound to the ncs ( see section s2 of the supporting information ) . in figure 1a , three regions can be distinguished . the peaks in region a are due to the carboxylic stretching vibrations and will be detailed below . the peaks in region b are due to the ch2 and ch3 stretching vibrations of the oleate . the ir absorbance in region c , starting with the peak at 0.64 ev and extending to higher energies , reflects the optical transitions in the pbse ncs . because of a calibration procedure developed by moreels et al . , we can use this peak to determine the concentration of pbse ncs in the solution . infrared spectrum of well - washed oleate - capped pbse ncs in tetrachloroethylene . ( a ) ftir spectrum of well - washed pbse ncs ( 5.6 nm ) dissolved in tetrachloroethylene showing three regions of interest : ( a ) region of c o vibrations , detailed in figure 2 , ( b ) a peak due to the ch2 asymmetric stretching vibrations of oleate molecules ( attached to the ncs or dissolved ) , and ( c ) the ir absorption peak due to the band - edge exciton in the pbse ncs . from the integration of region c , the concentration of ncs in the solution can be determined . from the integration of peak b , the total concentration of oleate in the suspension ( the sum of oleate attached to the ncs and dissolved oleate ) can be determined . this is performed by using a calibration curve ( panel b ) , i.e. , the integrated absorbance under peak b vs the concentration of pb(oleate)2 in a solution containing only pb(oleate)2 ( r = 0.999 ) . using the integrated value under peak b subsequently , we used the integrated peak value ( figure 1a ) to determine the total number of oleate moieties present in the suspension ( attached to the ncs and freely dissolved ) . combining the integrated values of peaks b and c , we can obtain the total number of oleate moieties per pbse nanocrystal present in the suspension . we remark here that peaks due to h2o and oh are completely absent in the spectra ( see figure s3 ) , excluding the presence of water , hydroxide , and oleic acid as ligands on our pbse ncs . a comparison of the ftir spectra of diphenylphosphine ( dpp ) , trioctylphosphine ( top ) , and pb(oleate)2 with the pbse nc suspension excludes dpp and indicates that top can be present in only small quantities , if it is present at all ( see figure s4 ) . these findings contrast with those reported in the literature , where for pbs ncs , the presence of oleic acid and hydroxide on different facets of the surface of ncs has been reported . can not be attributed to oxidation of the dots ( see figure s5 ) . as grisorio et al . claimed , it can be the result from the use of pb(oleate)2-hydrate in their advanced synthesis procedure for pbs nanocrystals . we also remark that we did not observe hydroxide and oleic acid signals for the ncs synthesized with the method developed by campos et al . now , we focus on the carboxyl stretch vibrations in the energy region between 1350 and 1750 cm . a detailed spectrum of the well - washed pbse ncs ( figure 2a c ) shows two broad peaks , reflecting the symmetric ( 1 ) and asymmetric carboxyl stretch ( 2 ) of the oleate moiety ( figure 2b ) . figure 2a also displays the reference spectra of dissolved pb(oleate)2 ( green ) and oleic acid ( blue ) , indicating that the pb(oleate)2 solution contains oleic acid as an impurity . a spectrum that shows only the features of a pure pb(oleate)2 solution can be obtained by subtraction ( see figure s6 ) . from comparison of the spectra in figure 2a , it becomes clear that the spectrum of the well - washed pbse ncs lacks the peak at 0.212 ev , specific for oleic acid , and has a much more pronounced 1 peak due to the symmetric carboxylic stretch vibration . a , the carboxylic regions of washed ncs ( red ) , pb(oleate)2 ( green ) , and oleic acid ( blue ) are shown . the carboxylic vibrations are sketched in panel b. symmetric and asymmetric carboxylic vibrations 1 and 2 in pbse ncs and pb(oleate)2 are each composed of two peaks . the decomposition and notation are presented in panel c ; the meaning is discussed in the text . panel d shows the carboxylic region for well - washed ncs ( red ) and titrated ncs ( blue ) . panel e presents sketches of the bridging configuration and the ( a)symmetric chelating bidentate configurations . peaks 1 and 2 each consist of a doublet ( see figure 2c ) . for peak 1 , we note the ( left ) low - energy peak as 1,b and the one at a higher energy as 1,s . notice that for 2 , the lower - energy peak is denoted as 2,s and the high - energy peak as 2,b . the peak splitting suggests that we have two types of pb oleate binding configurations . showed that the inner couple ( 1,s and 2,s ) can be attributed to an asymmetric chelating bidentate configuration ( see figure 2e ) if the splitting is between 100 and 125 cm . we find a separation between the inner peaks of 103 cm , indicating that the asymmetric pb oleate chelating bidentate configuration is one of the dominating configurations . the outer peaks ( 1,b and 2,b ) point to a bridging configuration of the oleate bound to two neighboring pb atoms . the splitting that we observe , 141 cm , is in line with such a bridging configuration . the intensity of the symmetric vibration is much larger for ncs than for dissolved pb(oleate)2 . the relative intensity of a vibrational peak increases with the increasing dipole moment of the vibrating moiety . oleate bidentate complex attached to a facet has a dipole moment that is stronger than that of the pb oleate complexes that form in solution . via comparison of the full width at half - maximum ( fwhm ) of each of the composed peaks , 1 and 2 , to the corresponding peaks of free pb(oleate)2 , it is clear that there is a strong broadening of the surface - attached pb the fwhm of the composed symmetric stretch 1 increases from 30 to 36 cm , while the asymmetric stretch 2 increases from 56 to 68 cm . it is tempting to relate this to the roughness of the facets for which we present evidence below . now , we discuss the equilibrium between freely dissolved pb(oleate)2 and pb oleate units at the surface of the pbse ncs . with the analysis described above ( peak b in figure 1 referred to the nc concentration obtained from peak c in figure 1 ) and from nmr results detailed below , we found for the well - washed nc suspension ( red curve ) an average of 258276 oleates / nc , corresponding to a ligand density of 2.22.3 oleates / nm ( assuming a spherical shape ) . to test if this is really the minimum , a small amount of halide salt dissolved in methanol is added to the suspension . the ligand density can in this way be reduced to 2.0 oleates / nm , because of ligand exchange . next , we performed a titration experiment starting from a suspension of pbse ncs , washed eight times , to which we gradually added pb(oleate)2 . in this titration experiment , we added pb(oleate)2 to the well - washed suspension and followed the evolution of the h nmr signal of the two hydrogen atoms bound to the c = c atoms of oleic acid ( figure 3a ) . it can be seen that because of the large difference in diffusion coefficients , freely dissolved pb(oleate)2 ( black curve ) can be distinguished from the oleate attached to the ncs ( red curve ) . via addition of pb(oleate)2 ( colored curves ) , the spectra combine the contributions of attached and free oleate . they can be quantified by fitting the curves on the basis of the red ( only attached oleate ) and black ( only free oleate ) spectra ( see figure s7 ) . via a reference molecule in a known amount ( an internal standard ) and the known concentration of ncs , it is also possible to calculate the number of ligands per nc and the ligand density ( figure s8 ) , as demonstrated previously . the results of the titration experiment are presented in figure 3b , showing that the number of attached oleate moieties first increases to saturate at a maximum of approximately 368 oleates / nc . this must be the maximal oleate ligand coverage possible per nanocrystal , which corresponds to a ligand density of 3.2 oleates / nm , in agreement with previous results for as - synthesized ncs . a ) close - up of the h nmr spectra for the two hydrogen atoms bound to the c = c atoms in the oleate . the black spectrum of freely dissolved pb(oleate)2 and the red spectrum of well - washed pbse ncs are normalized on their methyl integral , so spectra at similar oleate concentrations can be compared . the spectrum of well - washed ncs ( red ) shows a broad feature , indicating all oleate is bound to the surface . , spectra were obtained during gradual addition of pb(oleate)2 to the well - washed nc suspension . it can be seen that first the magnitude of the broad peak increases ( maximum at 5.3 ppm ) while with an increase in the amount of pb(oleate)2 added the sharp peak at 5.35 ppm also arises . deconvolution of both peaks ( shown in figure s7 ) allowed us to quantify the number of attached and freely dissolved pb(oleate)2 per nc during the titration experiment . this is shown in panel b , including the red dot determined via ftir . the derived ligand densities are colored orange ( 2.4 oleates / nm ) , yellow ( 2.5 oleates / nm ) , green ( 3.2 oleates / nm ) , blue ( 3.1 oleates / nm ) , and purple ( 3.2 oleates / nm ) . we emphasize that our quantification of the chemisorption process in pb(oleate)2 units does not mean that the chemisorbed entities remain preserved as pb(oleate)2 . in figure 2 and table 2 , it can be seen that for the titrated ncs the intensity of the outer peaks decreases , indicating relatively less oleate bound in bridging configurations . moreover , the peak widths are slightly reduced after the titration , indicating that the titration results in facets with less atomic disorder . it will be shown below that pb(oleate)2 chemisorption is followed by reconfiguration of the pb ligand binding and a transformation of the nanocrystal shape . we have investigated the three - dimensional ( 3d ) atomic structure of pbse ncs with the minimal and maximal values of oleate capping . because the ncs in this study are sensitive to the electron beam , high - resolution electron tomography could not be applied to determine their three - dimensional ( 3d ) shape . therefore , the 3d atomic structure is extracted from 2d haadf - stem projection images , a statistical atom counting procedure and an energy relaxation procedure , developed in previous work ( see figures s9 and s10 ) . haadf - stem images showing an overview of the particles with minimal and maximal oleate coverage are displayed in panels a and f of figure 4 , respectively . haadf - stem images acquired using a higher magnification for both samples are presented in panels b and g of figure 4 . these images have been used as input for a statistical atom counting procedure yielding the number of atoms in each projected atomic column ( figure 4c , h ) . here , counting results are obtained with 90% single - atom sensitivity , meaning that for 90% of all atom columns the number of atoms is correctly assigned , whereas the probability to miscount by 1 atom is 10% . the top row ( a e ) shows the shape analysis for low - ligand density ncs and the bottom row ( f j ) the maximal ligand densities . panels a and f are haadf - stem images of a group of pbse ncs , whereas panels b and g are magnified images for pbse ncs that are oriented in such a way that the lead atoms are oriented in columns . in panels c and h , the intensities of the columns are integrated and the numbers of lead atoms in that row are counted and color - coded . subsequently , 10 ncs of each sample are averaged ( see figure s10 for the procedure ) in panels d and i. we propose models e and j for the fitted data shown in panels d and i , respectively . notice the increased size of the { 111 } facets for the oleate - saturated ncs . starting from these numbers , we can obtain a 3d model for each individual nanocrystal using a monte carlo - based energy minimization scheme . we have performed such a procedure for 10 different ncs of both ligand densities , as presented in figure s9 . it can be observed that for the particles with a low ligand density , the crystal facets are rather small and contain several loose atoms . in line with atomic simulations of ligand - free ncs , we attribute this surface roughness to atomic surface reconstructions . the possibility that the electron beam induces some atom sublimation , however , can not be excluded , although the exposure time is kept as short as possible . the 3d shape determination shows that particles with a high ligand density have { 111 } facets that are larger than those of low - ligand density particles . to obtain a prototypical shape of the pbse ncs , an averaging method was applied that uses the 3d models of the individual ncs as a basis ( see figure s10 for a more detailed explanation of the averaging procedure ) . the result of this procedure is presented in panels d and i of figure 4 ; it can be observed that the shape of the well - washed pbse ncs ( low level of oleate capping ) is a nanocube in which truncation results in the presence of { 110 } and { 111 } facets , besides the { 100 } facets . in addition , there is a considerable degree of atomic disorder present on each of these facets that we attribute to atomic surface reconstructions of the bare crystal facets . for the ncs with maximal oleate capping , the { 111 } facets become more dominant at the expense of the { 110 } facets , although the latter do not disappear completely . thus , the added pb(oleate)2 ligands stabilize and order the { 111 } facets . the truncated octahedral shape is similar to that found for pbs crystals and for as - synthesized pbse ncs . idealized models are presented in panels e and j of figure 4 . to quantify the increase in the number of { 111 } facets for the sample with a high ligand density , the coordination number of surface pb atoms with other pb atoms can be used . this number can be related to the surface types that are present in the final configurations . the results are presented in table 2 together with the 68% confidence intervals , indicating that 22% of the surface atoms belong to a { 111 } facet ( coordination number of 9 ) for the sample with a high ligand density . for the pbse ncs with a low ligand density , the number of irregular atoms , not having a { 100 } , { 110 } , or { 111 } coordination number , decreases from 53 to 49% with an increase in ligand density . edge atoms are also included in the irregular category , because these have a lower coordination number . thus , the real facets are on average two pb atoms larger in the two orthogonal directions ( see figure 4 ) than following from table 2 . we start with the well - washed pbse ncs that have a truncated cubic shape with rather rough facets and a minimum of capping ligands . for such a nanocrystal type , the surface energies of the three facets have been calculated by schapotschnikov et al . for the case in which no capping is present : the { 100 } facets have an energy much lower than that of the { 110 } and { 111 } facets . in fact , naked { 111 } facets have such a high energy that they tend to be reconstructed in simulations : on pb - terminated { 111 } surfaces , for example , pb atoms are partially pulled inward while se atoms pop up , resulting in a reconstructed rough surface . the well - washed samples have a relatively small number of oleate ligands [ 258276 per nanocrystal ( see above ) ] ; it is reasonable to assume that they are preferentially bound to pb atoms of the { 111 } surfaces to minimize the energy of the entire nanocrystal . the amount of surface lead atoms on the { 111 } crystalline facet can be roughly estimated from the averaged model ( figure 4e ) . this results in 220 pb atoms ; this number is too small to accommodate all oleate ligands ( 258 or 276 on average ) , and thus , 20% of the oleates is bound the { 110 } or { 100 } surfaces . the ideal position of a pb oleate moiety would be in the fcc valley of three se atoms at the { 111 } facet ; the pb oleate moieties attached to the { 110 } or { 100 } facets together with surface roughness would explain the broadening of the 1 and 2 peaks in the carboxylic region ( see figure 2 ) . what happens to the well - washed pbse ncs when pb(oleate)2 is added to the pbse nc suspension ? initially , the added pb(oleate)2 can bind to any se surface atom , as an mx2 or z - type ligand . it has been reported that the ( surface ) atoms of pbse nanocrystals have a high mobility , even at room temperature . this should allow the chemisorbed pb(oleate)2 to reconfigure such that the energy of the pbse nanocrystals is minimized . we observed that the { 111 } facets become more extended at the expense of the { 110 } facets . ( i ) the chemisorbed pb(oleate)2 is split into a pb(oleate ) moiety that becomes optimally coordinated in the valley of three se surface atoms . ( ii ) the remaining oleate binds to a free pb atom at a valley position . the process of pb(oleate)2 chemisorption , splitting , and stabilization and extension of pb oleate - terminated { 111 } surfaces is schematically presented in scheme 1 . attachment of these z - type ligands on a pbse nc resulting in stabilization of a pb oleate - terminated { 111 } facet . panel a shows an unsaturated facet , typical for well - washed pbse ncs . panels b and c show the chemisorbtion of pb(oleate)2 as a z - type ligand . panel d shows the stabilization of the pb oleate - terminated { 111 } facets . this formal notation accounts for charge neutrality and mass balance . the reconfiguration results in a stabilization and extension of the { 111 } facets , in agreement with the observation . for the well - washed pbse samples , y is approximately 184 , including the edges of the { 111 } facets . after chemisorption of the added pb(oleate)2 and the reconfiguration process , the maximal excess of pb per nanocrystal amounts to y/2 + n. the maximal oleate coverage y + 2n is equal to 368 oleates / nc . this can be compared with the number of available { 111 } lead surface positions on the averaged pbse ncs presented in figure 4i , being 356 . this means that pbse nanocrystals with a maximal oleate coverage have almost all oleate ligands attached to the { 111 } facets and its edges . by the addition of pb(oleate)2 to a suspension of well - washed ncs , the ncs acquire a larger excess of lead atoms , the charge neutrality is preserved , and the ligand stabilization of the { 111 } surfaces is optimized ( see scheme 1 and figure 5 ) . this is in line with the reduced ir line widths , indicating reduced levels of atomic disorder on the facets and more ( a)symmetric carboxylic configurations ( see table 1 and figure 2d ) . moreover , the stabilization and extension of the { 111 } facets result in a shape transformation of the nanocrystal from a truncated cube to a cuboctahedron ( truncated octahedron ) solely with { 111 } and { 100 } facets . we believe that the final situation that we reach by addition of pb(oleate)2 to well - washed samples is close to that of as - synthesized pbse nanocrystals . in summary , we have used ir absorption spectroscopy and nmr to quantitatively measure the number of pb(oleate ) ligands per pbse nanocrystal . we have shown that pbse ncs can be colloidally stable with a wide range of oleate ligand densities . our washing and titration cycle suggests that the process of removing and adding oleate ligands is reversible . pb(oleate)2 chemisorption and surface reconfiguration result in a transformation of the nanocrystal shape from a truncated nanocube with rough surface facets to a truncated octahedron with larger and smooth { 111 } facets .

we present a study of the relation between the surface chemistry and nanocrystal shape of pbse nanocrystals with a variable pb - to - se stoichiometry and density of oleate ligands . the oleate ligand density and binding configuration are monitored by nuclear magnetic resonance and fourier transform infrared absorbance spectroscopy , allowing us to quantify the number of surface - attached ligands per nc and the nature of the surface pb oleate configuration . the three - dimensional shape of the pbse nanocrystals is obtained from high - angle annular dark field scanning transmission electron microscopy combined with an atom counting method . we show that the enhanced oleate capping results in a stabilization and extension of the { 111 } facets , and a crystal shape transformation from a truncated nanocube to a truncated octahedron .

Introduction Results Discussion

recently , it has been reported that interfacial ordering and epitaxial attachment resulted in nc monolayers with a square or honeycomb nanogeometry . the energy level structure , photoluminescence , and propensity to form atomically coherent two - dimensional ( 2d ) superstructures depend critically on the size and shape of the lead chalcogenide ncs and on their surface chemistry , as well . in addition , it is theoretically shown that the binding strength and configuration of oleate ligands depend on the type of facet . when we consider the uncoordinated crystal , the { 100 } facets , containing square ordered pb and se , have the lowest surface energy . the wulff reconstruction is presented in section s1 of the supporting information . however , in the case of pbse capped with oleate ligands , different nanocrystal shapes have been reported ranging from a truncated nanocube ( or rhombal cuboctahedron ) to a truncated octahedron . hence , besides the { 100 } facets , { 111 } and { 110 } facets are also observed . moreover , it is not yet clear why different pbse nc shapes are observed , and how this is related to the surface chemistry . here , we present a study of the relation between the surface chemistry and nanocrystal shape of pbse ncs ( 5.6 nm ) capped with oleate ligands . pb oleate binding is monitored by fourier transform infrared ( ftir ) absorbance and nuclear magnetic resonance ( nmr ) spectroscopy , allowing us to quantify the nature of the surface pb oleate configuration and the number of surface - attached ligands per nc . the shape of the ncs in three dimensions is obtained via high - angle annular dark field scanning transmission electron microscopy ( haadf - stem ) combined with an atom counting method that has been established previously . we have studied the relation between the shape and surface chemistry under well - defined chemical conditions . after conventional pbse synthesis , we have washed the ncs eight times to remove all remaining precursor moieties and reduce the oleate capping to a minimum . then we gradually added pb(oleate)2 to the suspension and monitored the attached oleate capping ( by nmr ) and the nc shape , as well . our results show that enhanced oleate capping results in a stabilization and extension of the { 111 } facets and a remarkable transformation of the nanocrystal shape . washing removes unreacted precursor molecules , excess ligand molecules , and possibly ligand molecules that are only weakly bound to the ncs ( see section s2 of the supporting information ) . the peaks in region b are due to the ch2 and ch3 stretching vibrations of the oleate . ( a ) ftir spectrum of well - washed pbse ncs ( 5.6 nm ) dissolved in tetrachloroethylene showing three regions of interest : ( a ) region of c o vibrations , detailed in figure 2 , ( b ) a peak due to the ch2 asymmetric stretching vibrations of oleate molecules ( attached to the ncs or dissolved ) , and ( c ) the ir absorption peak due to the band - edge exciton in the pbse ncs . using the integrated value under peak b subsequently , we used the integrated peak value ( figure 1a ) to determine the total number of oleate moieties present in the suspension ( attached to the ncs and freely dissolved ) . combining the integrated values of peaks b and c , we can obtain the total number of oleate moieties per pbse nanocrystal present in the suspension . a comparison of the ftir spectra of diphenylphosphine ( dpp ) , trioctylphosphine ( top ) , and pb(oleate)2 with the pbse nc suspension excludes dpp and indicates that top can be present in only small quantities , if it is present at all ( see figure s4 ) . these findings contrast with those reported in the literature , where for pbs ncs , the presence of oleic acid and hydroxide on different facets of the surface of ncs has been reported . a detailed spectrum of the well - washed pbse ncs ( figure 2a c ) shows two broad peaks , reflecting the symmetric ( 1 ) and asymmetric carboxyl stretch ( 2 ) of the oleate moiety ( figure 2b ) . from comparison of the spectra in figure 2a , it becomes clear that the spectrum of the well - washed pbse ncs lacks the peak at 0.212 ev , specific for oleic acid , and has a much more pronounced 1 peak due to the symmetric carboxylic stretch vibration . panel e presents sketches of the bridging configuration and the ( a)symmetric chelating bidentate configurations . notice that for 2 , the lower - energy peak is denoted as 2,s and the high - energy peak as 2,b . we find a separation between the inner peaks of 103 cm , indicating that the asymmetric pb oleate chelating bidentate configuration is one of the dominating configurations . the outer peaks ( 1,b and 2,b ) point to a bridging configuration of the oleate bound to two neighboring pb atoms . oleate bidentate complex attached to a facet has a dipole moment that is stronger than that of the pb oleate complexes that form in solution . via comparison of the full width at half - maximum ( fwhm ) of each of the composed peaks , 1 and 2 , to the corresponding peaks of free pb(oleate)2 , it is clear that there is a strong broadening of the surface - attached pb the fwhm of the composed symmetric stretch 1 increases from 30 to 36 cm , while the asymmetric stretch 2 increases from 56 to 68 cm . it is tempting to relate this to the roughness of the facets for which we present evidence below . now , we discuss the equilibrium between freely dissolved pb(oleate)2 and pb oleate units at the surface of the pbse ncs . with the analysis described above ( peak b in figure 1 referred to the nc concentration obtained from peak c in figure 1 ) and from nmr results detailed below , we found for the well - washed nc suspension ( red curve ) an average of 258276 oleates / nc , corresponding to a ligand density of 2.22.3 oleates / nm ( assuming a spherical shape ) . next , we performed a titration experiment starting from a suspension of pbse ncs , washed eight times , to which we gradually added pb(oleate)2 . it can be seen that because of the large difference in diffusion coefficients , freely dissolved pb(oleate)2 ( black curve ) can be distinguished from the oleate attached to the ncs ( red curve ) . they can be quantified by fitting the curves on the basis of the red ( only attached oleate ) and black ( only free oleate ) spectra ( see figure s7 ) . via a reference molecule in a known amount ( an internal standard ) and the known concentration of ncs , it is also possible to calculate the number of ligands per nc and the ligand density ( figure s8 ) , as demonstrated previously . the results of the titration experiment are presented in figure 3b , showing that the number of attached oleate moieties first increases to saturate at a maximum of approximately 368 oleates / nc . this must be the maximal oleate ligand coverage possible per nanocrystal , which corresponds to a ligand density of 3.2 oleates / nm , in agreement with previous results for as - synthesized ncs . a ) close - up of the h nmr spectra for the two hydrogen atoms bound to the c = c atoms in the oleate . it can be seen that first the magnitude of the broad peak increases ( maximum at 5.3 ppm ) while with an increase in the amount of pb(oleate)2 added the sharp peak at 5.35 ppm also arises . deconvolution of both peaks ( shown in figure s7 ) allowed us to quantify the number of attached and freely dissolved pb(oleate)2 per nc during the titration experiment . the derived ligand densities are colored orange ( 2.4 oleates / nm ) , yellow ( 2.5 oleates / nm ) , green ( 3.2 oleates / nm ) , blue ( 3.1 oleates / nm ) , and purple ( 3.2 oleates / nm ) . we emphasize that our quantification of the chemisorption process in pb(oleate)2 units does not mean that the chemisorbed entities remain preserved as pb(oleate)2 . moreover , the peak widths are slightly reduced after the titration , indicating that the titration results in facets with less atomic disorder . it will be shown below that pb(oleate)2 chemisorption is followed by reconfiguration of the pb ligand binding and a transformation of the nanocrystal shape . we have investigated the three - dimensional ( 3d ) atomic structure of pbse ncs with the minimal and maximal values of oleate capping . because the ncs in this study are sensitive to the electron beam , high - resolution electron tomography could not be applied to determine their three - dimensional ( 3d ) shape . therefore , the 3d atomic structure is extracted from 2d haadf - stem projection images , a statistical atom counting procedure and an energy relaxation procedure , developed in previous work ( see figures s9 and s10 ) . these images have been used as input for a statistical atom counting procedure yielding the number of atoms in each projected atomic column ( figure 4c , h ) . here , counting results are obtained with 90% single - atom sensitivity , meaning that for 90% of all atom columns the number of atoms is correctly assigned , whereas the probability to miscount by 1 atom is 10% . the top row ( a e ) shows the shape analysis for low - ligand density ncs and the bottom row ( f j ) the maximal ligand densities . panels a and f are haadf - stem images of a group of pbse ncs , whereas panels b and g are magnified images for pbse ncs that are oriented in such a way that the lead atoms are oriented in columns . in panels c and h , the intensities of the columns are integrated and the numbers of lead atoms in that row are counted and color - coded . notice the increased size of the { 111 } facets for the oleate - saturated ncs . it can be observed that for the particles with a low ligand density , the crystal facets are rather small and contain several loose atoms . the 3d shape determination shows that particles with a high ligand density have { 111 } facets that are larger than those of low - ligand density particles . to obtain a prototypical shape of the pbse ncs , an averaging method was applied that uses the 3d models of the individual ncs as a basis ( see figure s10 for a more detailed explanation of the averaging procedure ) . the result of this procedure is presented in panels d and i of figure 4 ; it can be observed that the shape of the well - washed pbse ncs ( low level of oleate capping ) is a nanocube in which truncation results in the presence of { 110 } and { 111 } facets , besides the { 100 } facets . for the ncs with maximal oleate capping , the { 111 } facets become more dominant at the expense of the { 110 } facets , although the latter do not disappear completely . thus , the added pb(oleate)2 ligands stabilize and order the { 111 } facets . to quantify the increase in the number of { 111 } facets for the sample with a high ligand density , the coordination number of surface pb atoms with other pb atoms can be used . the results are presented in table 2 together with the 68% confidence intervals , indicating that 22% of the surface atoms belong to a { 111 } facet ( coordination number of 9 ) for the sample with a high ligand density . for the pbse ncs with a low ligand density , the number of irregular atoms , not having a { 100 } , { 110 } , or { 111 } coordination number , decreases from 53 to 49% with an increase in ligand density . we start with the well - washed pbse ncs that have a truncated cubic shape with rather rough facets and a minimum of capping ligands . for such a nanocrystal type , the surface energies of the three facets have been calculated by schapotschnikov et al . for the case in which no capping is present : the { 100 } facets have an energy much lower than that of the { 110 } and { 111 } facets . in fact , naked { 111 } facets have such a high energy that they tend to be reconstructed in simulations : on pb - terminated { 111 } surfaces , for example , pb atoms are partially pulled inward while se atoms pop up , resulting in a reconstructed rough surface . the well - washed samples have a relatively small number of oleate ligands [ 258276 per nanocrystal ( see above ) ] ; it is reasonable to assume that they are preferentially bound to pb atoms of the { 111 } surfaces to minimize the energy of the entire nanocrystal . the amount of surface lead atoms on the { 111 } crystalline facet can be roughly estimated from the averaged model ( figure 4e ) . this results in 220 pb atoms ; this number is too small to accommodate all oleate ligands ( 258 or 276 on average ) , and thus , 20% of the oleates is bound the { 110 } or { 100 } surfaces . the ideal position of a pb oleate moiety would be in the fcc valley of three se atoms at the { 111 } facet ; the pb oleate moieties attached to the { 110 } or { 100 } facets together with surface roughness would explain the broadening of the 1 and 2 peaks in the carboxylic region ( see figure 2 ) . what happens to the well - washed pbse ncs when pb(oleate)2 is added to the pbse nc suspension ? it has been reported that the ( surface ) atoms of pbse nanocrystals have a high mobility , even at room temperature . this should allow the chemisorbed pb(oleate)2 to reconfigure such that the energy of the pbse nanocrystals is minimized . we observed that the { 111 } facets become more extended at the expense of the { 110 } facets . the process of pb(oleate)2 chemisorption , splitting , and stabilization and extension of pb oleate - terminated { 111 } surfaces is schematically presented in scheme 1 . attachment of these z - type ligands on a pbse nc resulting in stabilization of a pb oleate - terminated { 111 } facet . panel d shows the stabilization of the pb oleate - terminated { 111 } facets . the reconfiguration results in a stabilization and extension of the { 111 } facets , in agreement with the observation . for the well - washed pbse samples , y is approximately 184 , including the edges of the { 111 } facets . after chemisorption of the added pb(oleate)2 and the reconfiguration process , the maximal excess of pb per nanocrystal amounts to y/2 + n. the maximal oleate coverage y + 2n is equal to 368 oleates / nc . this can be compared with the number of available { 111 } lead surface positions on the averaged pbse ncs presented in figure 4i , being 356 . this means that pbse nanocrystals with a maximal oleate coverage have almost all oleate ligands attached to the { 111 } facets and its edges . by the addition of pb(oleate)2 to a suspension of well - washed ncs , the ncs acquire a larger excess of lead atoms , the charge neutrality is preserved , and the ligand stabilization of the { 111 } surfaces is optimized ( see scheme 1 and figure 5 ) . moreover , the stabilization and extension of the { 111 } facets result in a shape transformation of the nanocrystal from a truncated cube to a cuboctahedron ( truncated octahedron ) solely with { 111 } and { 100 } facets . we believe that the final situation that we reach by addition of pb(oleate)2 to well - washed samples is close to that of as - synthesized pbse nanocrystals . in summary , we have used ir absorption spectroscopy and nmr to quantitatively measure the number of pb(oleate ) ligands per pbse nanocrystal . we have shown that pbse ncs can be colloidally stable with a wide range of oleate ligand densities . our washing and titration cycle suggests that the process of removing and adding oleate ligands is reversible . pb(oleate)2 chemisorption and surface reconfiguration result in a transformation of the nanocrystal shape from a truncated nanocube with rough surface facets to a truncated octahedron with larger and smooth { 111 } facets .

a pcr amplicon from the human insulin basal promoter region was cloned into a promoterless luciferase vector , pgl3-basic ( promega ) as pins - basal . based on this construct , six individual class i vntr alleles , including their 5-flanking regions of about 1.1 kb , were first pcr amplified from individual human genomic dna samples and subcloned into the pins - basal construct to create the luciferase reporter construct series referred to as td , hm , pq , pw , ke , and rc ( genomic dna samples were obtained from human biological data interchange , philadelphia , pa ) . the insert sequence of these constructs corresponds to the same genomic dna on human chromosome 11p15.5 , which includes the 5-flanking region , vntr , and the basal insulin promoter . the primer sequences used for cloning are listed as follow : inspf , gactcgagacagcagcgcaaagagccc ; inspr , cttaagcttgcagcctgtcctggagggc ; vntr-5 , tacacgctgctgggatcctggatct ; vntr-3 , cttggaacagacctgcttga . the luciferase reporter vector for the class iii vntr was constructed from a hi-3 clone ( 18 ) . an artificial class iii construct was assembled by linking four class i vntrs 5-upstream of the human insulin basal promoter sequentially . the 1.1 kb sequence 5-upstream of the vntr was deleted during the cloning process . all of the pcr - generated amplicons were fully sequenced before use in any functional assay . for the heterologous promoter study , the insulin basal promoter was replaced by a sv40 promoter or sv40 promoter and enhancer for use in the reporter assays . a complete set of reporter construct without the 1.1 kb region were also constructed and described in fig . 3a . hit cell line ( atcc ) was grown in low - glucose dulbecco 's modified eagle 's medium supplemented with 10% fbs . the human thymic epithelial cell ( htec ) line derived from cortical epithelium ( 19 ) was cultured in rpmi 1,640 medium supplemented with 10% fbs . the primary thymic epithelial cells from e1618 rat fetal thymi were isolated and cultured as previously described ( 20 ) . luciferase assays were performed in a 24-well plate format using lipofectamine 2000 according to the manufacturer 's instructions ( invitrogen ) . for all of the constructs assayed , equal moles of promoter equivalent were used ( 50 ng ) . luciferase activities were normalized with a cotransfected null - renilla plasmid ( 2 ng ) ( promega ) . since endogenous aire protein is not detected in the htec cell line ( 21 ) or transfection - competent rat primary thymic epithelial cells , an exogenous source of aire or control -galactosidase ( -gal ) was introduced into the cells using a mammalian expression vector ( 50 ng ) . several passages of freshly cultured primary cells were maintained until the cells became competent for transfection . luciferase activities were measured using the dual - glo luciferase assay kit ( promega ) . activities were based on three to six experiments , performed in duplicate or triplicate wells for each sample , and either one representative result or an average of all the experiments was presented . in parallel with the luciferase reporter gene assay , separate wells of samples were collected to verify the expression of aire protein with a goat anti - aire antibody that recognizes the epitope 159 - 172 amino acids in aire protein ( abcam ) . in addition , a second rabbit antibody recognizes the epitope 246 - 545 amino acids in aire protein ( santa cruz ) was used to detect the immunoprecipitated aire proteins . equal sample loading on all the western blots was confirmed by an anti--actin antibody ( sigma ) that recognizes a protein with a molecular weight of 43 kda . human tec cells were infected with an ad - aire viral vector for 2 days . total cell lysates were prepared in a pull - down buffer ( 10 mmol / l tris buffer , ph 7.5 containing 50 mmol / l nacl , 1 mmol / l edta , and 1 mmol / l dtt ) with three freeze - thaw cycles . aire protein expression was detected by western blot analysis using an anti - aire antibody ( abcam ) . the insulin basal promoter probe ( 0.4 kb ) , ins - vntr - class i basal ( 2.0 kb ) , and iii - basal ( 3.5 kb ) probes were prepared from the reporter vectors after restriction digestion with kpni and hindiii . dna probes were radiolabeled at the 5-terminal phosphate with t4 polynucleotide kinase and -p - atp ( perkin elmer life sciences ) . aire expressing htec cell lysate ( 100 g ) was mixed with 50,000 cpm of each individual probe in pull - down buffer for 6 h at room temperature with constant rotation . normal goat igg or goat anti - aire antibody was added to the mixture for an overnight incubation . the following morning , 50 l of 50% slurry of salmon sperm dna - treated protein g - beads was added to the mixture for a 1-h incubation before being subjected to three washes with pull - down buffer . the washed beads were digested with proteinase k at 55c for 1 h and subjected to 1% agarose gel separation . the agarose gel was dried and exposed to x - ray film for probe detection . a pcr amplicon from the human insulin basal promoter region was cloned into a promoterless luciferase vector , pgl3-basic ( promega ) as pins - basal . based on this construct , six individual class i vntr alleles , including their 5-flanking regions of about 1.1 kb , were first pcr amplified from individual human genomic dna samples and subcloned into the pins - basal construct to create the luciferase reporter construct series referred to as td , hm , pq , pw , ke , and rc ( genomic dna samples were obtained from human biological data interchange , philadelphia , pa ) . the insert sequence of these constructs corresponds to the same genomic dna on human chromosome 11p15.5 , which includes the 5-flanking region , vntr , and the basal insulin promoter . the primer sequences used for cloning are listed as follow : inspf , gactcgagacagcagcgcaaagagccc ; inspr , cttaagcttgcagcctgtcctggagggc ; vntr-5 , tacacgctgctgggatcctggatct ; vntr-3 , cttggaacagacctgcttga . the luciferase reporter vector for the class iii vntr was constructed from a hi-3 clone ( 18 ) . an artificial class iii construct was assembled by linking four class i vntrs 5-upstream of the human insulin basal promoter sequentially . the 1.1 kb sequence 5-upstream of the vntr was deleted during the cloning process . all of the pcr - generated amplicons were fully sequenced before use in any functional assay . for the heterologous promoter study , the insulin basal promoter was replaced by a sv40 promoter or sv40 promoter and enhancer for use in the reporter assays . a complete set of reporter construct without the 1.1 kb region were also constructed and described in fig . 3a . hit cell line ( atcc ) was grown in low - glucose dulbecco 's modified eagle 's medium supplemented with 10% fbs . the human thymic epithelial cell ( htec ) line derived from cortical epithelium ( 19 ) was cultured in rpmi 1,640 medium supplemented with 10% fbs . the primary thymic epithelial cells from e1618 rat fetal thymi were isolated and cultured as previously described ( 20 ) . luciferase assays were performed in a 24-well plate format using lipofectamine 2000 according to the manufacturer 's instructions ( invitrogen ) . for all of the constructs assayed , equal moles of promoter equivalent were used ( 50 ng ) . luciferase activities were normalized with a cotransfected null - renilla plasmid ( 2 ng ) ( promega ) . since endogenous aire protein is not detected in the htec cell line ( 21 ) or transfection - competent rat primary thymic epithelial cells , an exogenous source of aire or control -galactosidase ( -gal ) was introduced into the cells using a mammalian expression vector ( 50 ng ) . several passages of freshly cultured primary cells were maintained until the cells became competent for transfection . luciferase activities were measured using the dual - glo luciferase assay kit ( promega ) . activities were based on three to six experiments , performed in duplicate or triplicate wells for each sample , and either one representative result or an average of all the experiments was presented . in parallel with the luciferase reporter gene assay , separate wells of samples were collected to verify the expression of aire protein with a goat anti - aire antibody that recognizes the epitope 159 - 172 amino acids in aire protein ( abcam ) . in addition , a second rabbit antibody recognizes the epitope 246 - 545 amino acids in aire protein ( santa cruz ) was used to detect the immunoprecipitated aire proteins . equal sample loading on all the western blots was confirmed by an anti--actin antibody ( sigma ) that recognizes a protein with a molecular weight of 43 kda . human tec cells were infected with an ad - aire viral vector for 2 days . total cell lysates were prepared in a pull - down buffer ( 10 mmol / l tris buffer , ph 7.5 containing 50 mmol / l nacl , 1 mmol / l edta , and 1 mmol / l dtt ) with three freeze - thaw cycles . aire protein expression was detected by western blot analysis using an anti - aire antibody ( abcam ) . the insulin basal promoter probe ( 0.4 kb ) , ins - vntr - class i basal ( 2.0 kb ) , and iii - basal ( 3.5 kb ) probes were prepared from the reporter vectors after restriction digestion with kpni and hindiii . dna probes were radiolabeled at the 5-terminal phosphate with t4 polynucleotide kinase and -p - atp ( perkin elmer life sciences ) . aire expressing htec cell lysate ( 100 g ) was mixed with 50,000 cpm of each individual probe in pull - down buffer for 6 h at room temperature with constant rotation . normal goat igg or goat anti - aire antibody was added to the mixture for an overnight incubation . the following morning , 50 l of 50% slurry of salmon sperm dna - treated protein g - beads was added to the mixture for a 1-h incubation before being subjected to three washes with pull - down buffer . the washed beads were digested with proteinase k at 55c for 1 h and subjected to 1% agarose gel separation . the agarose gel was dried and exposed to x - ray film for probe detection . iddm2 or ins is one of the multiple loci that encode susceptibility to type 1 diabetes , a prototype of human autoimmunity ( 10,2224 ) . ins - vntr situated 5-upstream of the ins promoter , is regarded as a causal variant at the locus after a close correlation was found between vntr haplotypes and insulin thymic expression ( 7,8 ) . however , comprehensive resequencing of the locus revealed two polymorphisms that are potentially functional and genetically indistinguishable from the vntr ( 2527 ) . therefore , direct evidence is required to support that the vntr allele controls insulin transcript expression in the human thymus . to establish a functional role of the vntr in the differential thymic insulin gene expression , the psti ( uncut by psti ) polymorphism is in strong linkage disequilibrium with the diabetes - predisposing class i allele and psti ( cut by psti ) with diabetes - protective class iii vntr haplotypes ( 10 ) . the insulin gene locus at 11p15.5 spanning the vntr region was pcr amplified and digested with psti to determine the haplotype . class i homozygous individuals will display a single 290 bp band ; class iii homozygous individuals will display two bands 200 and 90 bp ; and class i / iii heterozygous individuals display three bands 290 , 200 , and 90 bp in size . of the six dna samples analyzed , samples td and ke are heterozygous class i / iii and the other four are homozygous class i ( fig . to determine the relative repeat number of each individual dna sample , a ncoi / bglii double restriction endonuclease digest was performed . each sample contains a 450 , 230 , and 100 bp band as shown in the schematic diagram in fig . finally , sequence analysis revealed the exact number of repeat units contained in each of the unique human dna samples and are shown according to the convention established by owerbach and gabbary ( 24 ) ( fig . . six novel class i vntr alleles and the restriction profile of the class i and iii alleles of the constructs . a : six human genomic dna samples were genotyped using the + 1,127 psti polymorphism . psti ( uncut by psti ) and psti ( cut by psti ) variants are in strong linkage disequilibrium with diabetic class i and nondiabetic class iii vntr haplotypes , respectively ( 10 ) . by designing a pair of primers ( 5-taaatgcagaagcgtggcattgtggaac-3 and 5-ctgcatgctgggcctggccgg-3 ) , the pcr products amplified from genomic dna were digested with psti to generate a single band of 290 bp from class i homozygotes ; two bands of 200 bp and 90 bp from class iii homozygotes ; and three bands of 290 bp , 200 bp , and 90 bp from i / iii heterozygotes . td and ke are heterozygous ( i / iii ) and the other four are homozygous class i. psti + and are control dna of class i and class iii homozygotes , respectively . c : schematic explanation for the profile shown on the gel b or quantitated in table d. the band sizes are indicated by base pairs and marked by n ( ncoi ) and b ( bglii ) ; 230 + vntr in c corresponds to vntr in b or restriction profile in d. d : estimation of the sizes of individual vntr alleles . e : sequence of six class i vntr alleles in the format of repeat unit array and according to the convention set by previous reference ( 24 ) . the number of repeat units is shown to the right of each sequence . to directly dissect any transcriptional activity contributed by the vntr alleles , we made a set of reporter constructs bearing the vntr sequences and excluding the two other putative causal variants , 23 hph i and + 1,140 a / c in the ins locus ( figs . 1 and 3a ) . because of the selective tissue expression of the insulin gene , we first tested the class i or class iii vntr - basal insulin luciferase reporter constructs in a hamster pancreatic -cell line , hit . consistent with previous reports , the class i vntr construct activity was approximately twofold higher than the class iii vntr constructs in the hit cells ( fig . the luciferase data confirmed the previous observation that the type 1 diabetes susceptible shorter class i vntr ( most commonly 3040 repeats ) was associated with a 1.5- to 3-fold higher insulin expression than the class iii vntr in the pancreas ( 5,10 ) , although inconsistent results have been reported ( 4 ) and discussed ( 10 ) . pancreas - based insulin expression pattern associated with the iddm2-vntr locus . in comparison with the activity in thymus , the pancreatic insulin expression pattern , represented by the luciferase reporter activity , was confirmed in a hit cell line ( a and b ) . an average of 1.58-fold increase was observed in reporter activities driven by class i over class iii vntr haplotypes . the class iii activity was expressed as an average of three experiments from one construct . the constructs assayed are the same set of reporter constructs as those used in the htec line or rat primary cells in fig . we artificially overexpressed aire in hit cells to examine the responses of class i and iii vntrs . in the presence of aire , both classes of vntr display similar activities , with the class i vntr being slightly higher than the class iii vntr . recently , correlation studies suggested an involvement of the autoimmune regulator ( aire ) in the promiscuous expression of insulin in human thymus ( 28,29 ) , whereas thymus - specific deletion of insulin induces autoimmune diabetes ( 30 ) . nevertheless , how the iddm2 locus transduces a differential expression signal in thymus in the context of aire remains to be elucidated . in contrast to the pattern in pancreas , insulin expression in thymus was reversed , with an average threefold increase of class iii over class i alleles , as represented by the luciferase activity driven by six different class i and one class iii haplotypes in a nontransformed human thymic epithelial cell line ( htec ) ( 19 ) ( fig . this observation suggests that the diabetes susceptible class i vntr might predispose individuals to type 1 diabetes by lowering insulin expression in the thymus and rendering less tolerance induction . evidently , without the other two polymorphisms , the vntr alone accounts for quantitative expression of insulin , which is consistent with the report that 23 hph i , one of the two snps , did not play a significant role in thymus ( 31 ) . additionally , we confirmed the independent effect of the vntr using shorter constructs lacking the 1.1 kb 5-flanking regions of the polymorphism ( fig . 3f and g ) . in addition to the htec line , primary epithelial cells dissected from fetal rat thymus ( e16 - 18 ) were transfected with the same set of constructs , and the results confirmed the expression profile observed in the human cell line ( fig . remarkably , the observed expression pattern persisted only in the presence of aire ( fig . 3h ) . when a control -gal expression plasmid was administered , the differential expression signal transduced by the ins - vntr / iddm2 locus was totally lost and the insulin expression driven by either allele decreased to a basal level ( fig . this line of evidence warrants further investigation of aire regulation in the context of the vntr for insulin quantitative expression in thymic epithelial cells . since there is only one natural class iii dna construct available for the comparison study with the six class i vntr constructs , we further assembled an artificial class iii construct ( 162 tandem repeats ) based upon the assumption that the differential transcriptional activities from class iii versus class i vntr are due to the number of tandem repeats . as shown in supplementary fig . 2 in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0255/dc1 , artificial class iii displays higher activity than the natural class iii construct ( 140 tandem repeats ) . additionally , this construct also displays a higher activity in the no aire setting , probably caused by the deletion of the 5-upstream 1.1 kb sequence of the vntr ( similar to fig . iddm2-vntr locus transduces a quantitative insulin expression signal in the presence of aire . a : diagrammatic representation of the th - ins - igf2 interval on chromosome 11p15.5 . ins or iddm2 locus is bordered by th ( tyrosine hydroxylase ) and igf2 ( insulin - like growth factor 2 ) loci . the three polymorphisms are designated by their positions with respect to the first base of the ins start codon that is represented by a solid triangle and marked as + 1 . the allelic variations at the vntr begin at 603 , positioned immediately before the basal promoter . the average length of a class i vntr is 0.5 kb among the alleles we cloned and the class iii used is 2.0 kb . a gray arrow depicts the basal insulin promoter region ( 365 bp ) and open boxes denote the three insulin exons . the black bold lines represent the regions cloned into the luciferase reporter constructs including the vntr , its 5-flanking 1.1 kb region , the basal insulin promoter , and excluding the other two polymorphisms . the longer version bearing the 5-flanking 1.1 kb in addition to the vntr region and basal promoter construct , as well as a shorter construct without the 1.1 kb 5-flanking region , were analyzed . b : a luciferase reporter gene is driven by class i or class iii vntr with insulin basal promoter ( 50 ng ) and transfected in a htec line . the relative activities both without ( -gal control ) and with exogenous aire ( 50 ng ) are shown . the class iii activity was expressed as an average of three experiments from one construct . c : detailed activities of each individual vntr allele in the presence of aire were tabulated . d and e : the same set of constructs was assayed using rat primary thymic epithelial cells following the same strategy as in b and c. f and g : to rule out the possibility that the upstream 5-flanking genomic region ( 1.1 kb ) might contribute to the differential insulin expression in thymus , another set of constructs was made by removing this region ( a ) and assaying in the htec line . the average difference in thymic expression is consistently a threefold increase of class iii over i vntr haplotypes . h : western blot analysis to verify aire expression in the transfected cell line . as a loading control , next , we determined how the class i or class iii vntr allele responded to an increasing dosage of aire expression plasmid . each vntr haplotype transduced a sensitive and differential signal in the htec line . as little as 10 ng of the aire expression plasmid generated a differential response between class i ( pq ) and class iii vntrs ( fig . the difference between the two classes of haplotypes was consistently about threefold at each dose tested , except at the zero dose ( fig . the level of aire protein expression was verified with an aire antibody by western blot ( fig . 4b ) . additionally , we chose the natural class iii and the artificial class iii paired with two other class i ( hm and rc ) constructs to perform the dosage response curves ( supplementary fig . class i vntrs response to aire is much lower than both natural and artificial class iii vntrs . a class i vntr ( pq ) and a class iii vntr reporter construct ( 50 ng ) were cotransfected with aire expression vector or control vector -gal dna into a htec line . the amount of aire expression vector added to each sample of a 24-well plate format is indicated . b : western blot analysis was performed to confirm that the increasing expression of aire protein corresponded to the amounts of added expression vector . equal loading of each sample was confirmed by incubation with an anti--actin antibody . in the presence of aire , the insulin basal promoter alone ( 365 bp ) displays relatively low activity in the human tec and rat primary tec ( fig . the class i vntr is two to threefold higher relative to the insulin basal promoter activity , whereas the class iii vntr increases seven to ninefold over the insulin basal promoter activity , suggesting that the aire transcriptional regulator could activate the ins - vntr sequence in the context of the insulin basal promoter . to validate the interaction between the aire protein and the vntr sequence , we performed a dna - protein pull - down experiment using adenovirus - aire transduced htec cell lysate and radiolabeled dna probes ( 50,000 cpm ) derived from the insulin basal promoter , class i - vntr - ins - basal promoter , and class iii - vntr - ins - basal promoter sequences ( fig . 5c ) . both class i and iii dna probes were pulled - down by the anti - aire antibody . a very weak signal , if any , was detected with the insulin basal promoter probe . this result suggests that aire protein can interact directly or indirectly through the recruitment of a transcriptional complex to bind the vntr sequences . a and b : insulin basal promoter , vntr - class i - ins - basal and vntr - class iii - ins - basal vectors demonstrated differential promoter activities in both htec and rat primary tec in response to aire . c : dna probes derived from insulin basal promoter ( 0.4 kb ) , vntr - class i - ins - basal ( 2.0 kb ) , and vntr - class iii - ins - basal ( 3.5 kb ) were radiolabeled with t4 polynucleotide kinase and -p - atp as shown in the input lanes . an ad - aire transduced htec total lysate ( 100 g ) was mixed with 50,000 cpm of each radiolabeled dna probe for 6 h at room temperature and subsequently added 1 g of goat anti - aire antibody for an overnight incubation . the immune complex was precipitated by salmon sperm dna - treated protein g - agarose beads , washed , and eluted from beads for separation . transfection of the construct lacking the insulin basal promoter with the aire expression vector in human tec was measured for luciferase reporter activity . e : addition of a sv40 enhancer to the construct greatly enhanced sv40 promoter activity , but the class i or iii vntr had no effect on the promoter / enhancer combination . we designed experiments to evaluate whether the vntr functions in an insulin promoter - specific fashion or if it could function effectively with a heterologous promoter and/or in combination with other enhancer elements . a representative class i or iii vntr was subcloned 5-upstream of the sv40 promoter driven luciferase vector or the sv40 promoter - enhancer driven luciferase vector . cotransfection of the luciferase constructs with an aire expression vector into the human tec showed no effect to the luciferase activity with either class i or iii vntr in conjunction with the sv40 promoter . the characteristic activation pattern of class i and iii vntrs seen when linked directly to the insulin basal promoter disappeared when the insulin basal promoter was replaced by the sv40 promoter and/or sv40 promoter / enhancer ( fig . 5d and e ) . this result indicates that the vntr sequence has to function in conjunction with the insulin basal promoter to respond to aire . in this paper , we have revealed a novel role of the aire protein with the vntr region in the establishment of differential tissue regulation of the insulin mrna in the thymus . iddm2 or ins is one of the multiple loci that encode susceptibility to type 1 diabetes , a prototype of human autoimmunity ( 10,2224 ) . ins - vntr situated 5-upstream of the ins promoter , is regarded as a causal variant at the locus after a close correlation was found between vntr haplotypes and insulin thymic expression ( 7,8 ) . however , comprehensive resequencing of the locus revealed two polymorphisms that are potentially functional and genetically indistinguishable from the vntr ( 2527 ) . therefore , direct evidence is required to support that the vntr allele controls insulin transcript expression in the human thymus . to establish a functional role of the vntr in the differential thymic insulin gene expression , the psti ( uncut by psti ) polymorphism is in strong linkage disequilibrium with the diabetes - predisposing class i allele and psti ( cut by psti ) with diabetes - protective class iii vntr haplotypes ( 10 ) . the insulin gene locus at 11p15.5 spanning the vntr region was pcr amplified and digested with psti to determine the haplotype . class i homozygous individuals will display a single 290 bp band ; class iii homozygous individuals will display two bands 200 and 90 bp ; and class i / iii heterozygous individuals display three bands 290 , 200 , and 90 bp in size . of the six dna samples analyzed , samples td and ke are heterozygous class i / iii and the other four are homozygous class i ( fig . to determine the relative repeat number of each individual dna sample , a ncoi / bglii double restriction endonuclease digest was performed . each sample contains a 450 , 230 , and 100 bp band as shown in the schematic diagram in fig . finally , sequence analysis revealed the exact number of repeat units contained in each of the unique human dna samples and are shown according to the convention established by owerbach and gabbary ( 24 ) ( fig . . six novel class i vntr alleles and the restriction profile of the class i and iii alleles of the constructs . a : six human genomic dna samples were genotyped using the + 1,127 psti polymorphism . psti ( uncut by psti ) and psti ( cut by psti ) variants are in strong linkage disequilibrium with diabetic class i and nondiabetic class iii vntr haplotypes , respectively ( 10 ) . by designing a pair of primers ( 5-taaatgcagaagcgtggcattgtggaac-3 and 5-ctgcatgctgggcctggccgg-3 ) , the pcr products amplified from genomic dna were digested with psti to generate a single band of 290 bp from class i homozygotes ; two bands of 200 bp and 90 bp from class iii homozygotes ; and three bands of 290 bp , 200 bp , and 90 bp from i / iii heterozygotes . td and ke are heterozygous ( i / iii ) and the other four are homozygous class i. psti + and are control dna of class i and class iii homozygotes , respectively . c : schematic explanation for the profile shown on the gel b or quantitated in table d. the band sizes are indicated by base pairs and marked by n ( ncoi ) and b ( bglii ) ; 230 + vntr in c corresponds to vntr in b or restriction profile in d. d : estimation of the sizes of individual vntr alleles . e : sequence of six class i vntr alleles in the format of repeat unit array and according to the convention set by previous reference ( 24 ) . the number of repeat units is shown to the right of each sequence . to directly dissect any transcriptional activity contributed by the vntr alleles , we made a set of reporter constructs bearing the vntr sequences and excluding the two other putative causal variants , 23 hph i and + 1,140 a / c in the ins locus ( figs . 1 and 3a ) . because of the selective tissue expression of the insulin gene , we first tested the class i or class iii vntr - basal insulin luciferase reporter constructs in a hamster pancreatic -cell line , hit . consistent with previous reports , the class i vntr construct activity was approximately twofold higher than the class iii vntr constructs in the hit cells ( fig . 2a , left panel ) . the luciferase data confirmed the previous observation that the type 1 diabetes susceptible shorter class i vntr ( most commonly 3040 repeats ) was associated with a 1.5- to 3-fold higher insulin expression than the class iii vntr in the pancreas ( 5,10 ) , although inconsistent results have been reported ( 4 ) and discussed ( 10 ) . pancreas - based insulin expression pattern associated with the iddm2-vntr locus . in comparison with the activity in thymus , the pancreatic insulin expression pattern , represented by the luciferase reporter activity , was confirmed in a hit cell line ( a and b ) . an average of 1.58-fold increase was observed in reporter activities driven by class i over class iii vntr haplotypes . the class i activity was expressed as an average of six constructs . the class iii activity was expressed as an average of three experiments from one construct . the constructs assayed are the same set of reporter constructs as those used in the htec line or rat primary cells in fig . we artificially overexpressed aire in hit cells to examine the responses of class i and iii vntrs . in the presence of aire , both classes of vntr display similar activities , with the class i vntr being slightly higher than the class iii vntr . recently , correlation studies suggested an involvement of the autoimmune regulator ( aire ) in the promiscuous expression of insulin in human thymus ( 28,29 ) , whereas thymus - specific deletion of insulin induces autoimmune diabetes ( 30 ) . nevertheless , how the iddm2 locus transduces a differential expression signal in thymus in the context of aire remains to be elucidated . in contrast to the pattern in pancreas , insulin expression in thymus was reversed , with an average threefold increase of class iii over class i alleles , as represented by the luciferase activity driven by six different class i and one class iii haplotypes in a nontransformed human thymic epithelial cell line ( htec ) ( 19 ) ( fig . this observation suggests that the diabetes susceptible class i vntr might predispose individuals to type 1 diabetes by lowering insulin expression in the thymus and rendering less tolerance induction . evidently , without the other two polymorphisms , the vntr alone accounts for quantitative expression of insulin , which is consistent with the report that 23 hph i , one of the two snps , did not play a significant role in thymus ( 31 ) . additionally , we confirmed the independent effect of the vntr using shorter constructs lacking the 1.1 kb 5-flanking regions of the polymorphism ( fig . 3f and g ) . in addition to the htec line , primary epithelial cells dissected from fetal rat thymus ( e16 - 18 ) were transfected with the same set of constructs , and the results confirmed the expression profile observed in the human cell line ( fig . remarkably , the observed expression pattern persisted only in the presence of aire ( fig . when a control -gal expression plasmid was administered , the differential expression signal transduced by the ins - vntr / iddm2 locus was totally lost and the insulin expression driven by either allele decreased to a basal level ( fig . this line of evidence warrants further investigation of aire regulation in the context of the vntr for insulin quantitative expression in thymic epithelial cells . since there is only one natural class iii dna construct available for the comparison study with the six class i vntr constructs , we further assembled an artificial class iii construct ( 162 tandem repeats ) based upon the assumption that the differential transcriptional activities from class iii versus class i vntr are due to the number of tandem repeats . as shown in supplementary fig . 2 in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0255/dc1 , artificial class iii displays higher activity than the natural class iii construct ( 140 tandem repeats ) . additionally , this construct also displays a higher activity in the no aire setting , probably caused by the deletion of the 5-upstream 1.1 kb sequence of the vntr ( similar to fig . a : diagrammatic representation of the th - ins - igf2 interval on chromosome 11p15.5 . ins or iddm2 locus is bordered by th ( tyrosine hydroxylase ) and igf2 ( insulin - like growth factor 2 ) loci . the three polymorphisms are designated by their positions with respect to the first base of the ins start codon that is represented by a solid triangle and marked as + 1 . the allelic variations at the vntr begin at 603 , positioned immediately before the basal promoter . the average length of a class i vntr is 0.5 kb among the alleles we cloned and the class iii used is 2.0 kb . a gray arrow depicts the basal insulin promoter region ( 365 bp ) and open boxes denote the three insulin exons . the black bold lines represent the regions cloned into the luciferase reporter constructs including the vntr , its 5-flanking 1.1 kb region , the basal insulin promoter , and excluding the other two polymorphisms . the longer version bearing the 5-flanking 1.1 kb in addition to the vntr region and basal promoter construct , as well as a shorter construct without the 1.1 kb 5-flanking region , were analyzed . b : a luciferase reporter gene is driven by class i or class iii vntr with insulin basal promoter ( 50 ng ) and transfected in a htec line . the relative activities both without ( -gal control ) and with exogenous aire ( 50 ng ) are shown . the class iii activity was expressed as an average of three experiments from one construct . c : detailed activities of each individual vntr allele in the presence of aire were tabulated . d and e : the same set of constructs was assayed using rat primary thymic epithelial cells following the same strategy as in b and c. f and g : to rule out the possibility that the upstream 5-flanking genomic region ( 1.1 kb ) might contribute to the differential insulin expression in thymus , another set of constructs was made by removing this region ( a ) and assaying in the htec line . the average difference in thymic expression is consistently a threefold increase of class iii over i vntr haplotypes . h : western blot analysis to verify aire expression in the transfected cell line . as a loading control , next , we determined how the class i or class iii vntr allele responded to an increasing dosage of aire expression plasmid . as little as 10 ng of the aire expression plasmid generated a differential response between class i ( pq ) and class iii vntrs ( fig . the difference between the two classes of haplotypes was consistently about threefold at each dose tested , except at the zero dose ( fig . the level of aire protein expression was verified with an aire antibody by western blot ( fig . 4b ) . additionally , we chose the natural class iii and the artificial class iii paired with two other class i ( hm and rc ) constructs to perform the dosage response curves ( supplementary fig . class i vntrs response to aire is much lower than both natural and artificial class iii vntrs . a class i vntr ( pq ) and a class iii vntr reporter construct ( 50 ng ) were cotransfected with aire expression vector or control vector -gal dna into a htec line . the amount of aire expression vector added to each sample of a 24-well plate format is indicated . b : western blot analysis was performed to confirm that the increasing expression of aire protein corresponded to the amounts of added expression vector . in the presence of aire , the insulin basal promoter alone ( 365 bp ) displays relatively low activity in the human tec and rat primary tec ( fig . the class i vntr is two to threefold higher relative to the insulin basal promoter activity , whereas the class iii vntr increases seven to ninefold over the insulin basal promoter activity , suggesting that the aire transcriptional regulator could activate the ins - vntr sequence in the context of the insulin basal promoter . to validate the interaction between the aire protein and the vntr sequence , we performed a dna - protein pull - down experiment using adenovirus - aire transduced htec cell lysate and radiolabeled dna probes ( 50,000 cpm ) derived from the insulin basal promoter , class i - vntr - ins - basal promoter , and class iii - vntr - ins - basal promoter sequences ( fig . 5c ) . both class i and iii dna probes were pulled - down by the anti - aire antibody . a very weak signal , if any , was detected with the insulin basal promoter probe . this result suggests that aire protein can interact directly or indirectly through the recruitment of a transcriptional complex to bind the vntr sequences . a and b : insulin basal promoter , vntr - class i - ins - basal and vntr - class iii - ins - basal vectors demonstrated differential promoter activities in both htec and rat primary tec in response to aire . c : dna probes derived from insulin basal promoter ( 0.4 kb ) , vntr - class i - ins - basal ( 2.0 kb ) , and vntr - class iii - ins - basal ( 3.5 kb ) were radiolabeled with t4 polynucleotide kinase and -p - atp as shown in the input lanes . an ad - aire transduced htec total lysate ( 100 g ) was mixed with 50,000 cpm of each radiolabeled dna probe for 6 h at room temperature and subsequently added 1 g of goat anti - aire antibody for an overnight incubation . the immune complex was precipitated by salmon sperm dna - treated protein g - agarose beads , washed , and eluted from beads for separation . transfection of the construct lacking the insulin basal promoter with the aire expression vector in human tec was measured for luciferase reporter activity . e : addition of a sv40 enhancer to the construct greatly enhanced sv40 promoter activity , but the class i or iii vntr had no effect on the promoter / enhancer combination . we designed experiments to evaluate whether the vntr functions in an insulin promoter - specific fashion or if it could function effectively with a heterologous promoter and/or in combination with other enhancer elements . a representative class i or iii vntr was subcloned 5-upstream of the sv40 promoter driven luciferase vector or the sv40 promoter - enhancer driven luciferase vector . cotransfection of the luciferase constructs with an aire expression vector into the human tec showed no effect to the luciferase activity with either class i or iii vntr in conjunction with the sv40 promoter . the characteristic activation pattern of class i and iii vntrs seen when linked directly to the insulin basal promoter disappeared when the insulin basal promoter was replaced by the sv40 promoter and/or sv40 promoter / enhancer ( fig . 5d and e ) . this result indicates that the vntr sequence has to function in conjunction with the insulin basal promoter to respond to aire . in this paper , we have revealed a novel role of the aire protein with the vntr region in the establishment of differential tissue regulation of the insulin mrna in the thymus . type 1 insulin - dependent diabetes results from the autoimmune destruction of the insulin producing cells of the pancreas . type 1 diabetes is a polygenic disease and multiple susceptibility loci have been mapped ( 32 ) . a strong correlation between the variable numbers of tandem repeat ( vntr ) or iddm2 locus and the levels of insulin mrna expression in the thymus have been clearly established ( 4,5,7,8 ) . the short class i allele is predisposing to type 1 diabetes and is associated with lower thymus insulin expression , whereas the class iii allele is protective and associated with three to fourfold higher thymic insulin expression . the autoimmune regulator , aire , has been shown to direct the expression of hundreds of tissue - specific antigens in the thymus and is crucial for establishment of immune tolerance ( 16 ) . to definitively establish a role of the vntr in insulin gene regulation in thymus , our cloning strategy spanning the vntr region of the ins locus first excluded the two additional polymorphisms ( 23 hph i and + 1,140 a / c ) that are present within the iddm2 locus and may contribute to the differential insulin levels in the thymus . without these two polymorphisms , reporter constructs that lacked the 1.1 kb 5-upstream genomic region ( to the vntr ) showed a similar promoter activity as the construct that included this region . these results further confirmed that the thymic insulin gene regulation by aire is mediated by the vntr . the transfection studies with the ins - vntr class i and iii constructs unequivocally show that aire controls differential insulin gene expression through the ins - vntr region . to study the role of the aire protein in regulating the insulin promoter via the vntr region , we further examined the physical interaction between aire and the vntr . the aire protein has been reported to bind to target dna as a homodimer or a tetramer , but not a monomer . an oligonucleotide library screen showed that aire protein bound to two nucleotide sequences , ttatta and two tandem repeats of attggta ( 33 ) . it is difficult to envision that the aire protein recognizes a specific binding sequence present in hundreds or thousands of target gene promoter sequences . particularly , the aire binding site is not found in the ins - vntr region . additionally , our data show that the insulin basal promoter region in conjunction with the vntr is required for regulation by aire protein . intriguingly , linkage of the ins - vntr class i or class iii vntr region with a heterologous promoter sequence resulted in the complete loss of aire transcriptional regulation . therefore , it is likely that aire must interact with a protein or complex that is formed on the basal insulin promoter to modulate insulin gene expression in the thymus . we speculate that aire 's binding to the target genes other studies have shown that aire recruits creb - binding protein , positive transcription elongation factor b , and interacts with target genes lacking methylated histone h3k4 to regulate tissue - restricted antigen expression in the thymus ( 3436 ) . recent studies reported that aire protein could partner with four major functional classes : nuclear transport , chromatin binding / structure , transcription , and premrna processing for targeting and inducing peripheral tissue self - antigen ( 37 ) . a recent review article described the molecular mechanisms of central tolerance , focusing on the transcriptional regulation by aire ( 39 ) . although this paper discussed the general mechanisms of how the aire protein could interact with multiple target genes , it suggests a sharing of a common transcriptional complex to exert its activation activities . the insulin basal promoter has been identified as a target for aire regulation , but these constructs always lacked the vntr region . in response to aire , our data show that in the presence of the vntr sequence ( as in the human insulin gene locus ) insulin basal promoter activity is significantly enhanced in htecs . labeling of the basal insulin promoter , class i and class iii basal insulin promoter regions and incubation with an aire - expressing htec cell lysates unambiguously showed preferential binding of the aire protein with the vntr containing fragments ( fig . the tandem repeat regions of the insulin vntr have been shown to form g - quadruplex structures ( 40 ) . the enhanced regulation of the class iii constructs is most likely due to the larger number of tandem repeats forming a more stable intramolecular secondary structure that is required for aire protein binding and formation of a transcriptional complex . we have constructed an artificial class iii vntr from four different copies of class i vntr sequences derived from patient samples . as expected , the artificial class iii vntr ( 162 tandem repeats ) demonstrated a higher transcriptional activity as compared with the natural class iii vntr ( 140 tandem repeats ) and exhibited approximately threefold higher activity than class i vntr ( supplementary fig . 2 ) . together , we show that the aire protein is capable of selectively pulling - down the vntr insulin basal promoter sequence . definitively , these data support the physical interaction between aire and the ins - vntr , but we can not rule out the involvement of other cellular components that may be involved in mediating this interaction . in summary , we reveal a dual control mechanism governing the insulin quantitative expression in thymus that may account for part of the genetic predisposition observed in type 1 diabetic patients . aire - mediated differential regulation of insulin gene expression via the vntr locus in the thymus may influence tolerance and susceptibility to type 1 diabetes . a similar mechanism was also reported for myasthenia gravis , another autoimmune disease ( 21 ) . these findings may together suggest a commonly adopted regulation strategy that , in addition to genetic variations endowed by each of the individual genes , encode hundreds and/or even more than a thousand tissue - restricted antigens in the human thymus ( 41 ) .

objectivepolymorphic ins - vntr plays an important role in regulating insulin transcript expression in the human thymus that leads to either insulin autoimmunity or tolerance . the molecular mechanisms underlying the ins - vntr haplotype - dependent insulin expression are still unclear . in this study , we determined the mechanistic components underlying the differential insulin gene expression in human thymic epithelial cells , which should have profound effects on the insulin autoimmune tolerance induction.research design and methodsa repetitive dna region designated as a variable number of tandem repeats ( vntr ) is located upstream of the human insulin gene and correlates with the incidence of type 1 diabetes . we generated six class i and two class iii vntr constructs linked to the human insulin basal promoter or sv40 heterologous promoter / enhancer and demonstrated that aire protein modulates the insulin promoter activities differentially through binding to the vntr region.resultshere we show that in the presence of the autoimmune regulator ( aire ) , the class iii vntr haplotype is responsible for an average of three - fold higher insulin expression than class i vntr in thymic epithelial cells . in a protein - dna pull - down experiment , aire protein is capable of binding to vntr class i and iii probes . further , the transcriptional activation of the ins - vntr by aire requires the insulin basal promoter . the vntr sequence loses its activation activity when linked to a heterologous promoter and/or enhancer.conclusionsthese findings demonstrate a type 1 diabetes predisposition encoded by the ins - vntr locus and a critical function played by aire , which constitute a dual control mechanisms regulating quantitative expression of insulin in human thymic epithelial cells .

RESEARCH DESIGN AND METHODS Constructs. Luciferase reporter assay. Western blot analysis. INS-VNTR pull-down experiment. RESULTS Molecular cloning of six novel class I VNTR alleles and the restriction profiles of the class I and III alleles. None VNTR responded sensitively and differentially to AIRE in a human thymic epithelial cell line. AIRE regulates the insulin promoter activity mediated through the INS-VNTR element. DISCUSSION Supplementary Material

an artificial class iii construct was assembled by linking four class i vntrs 5-upstream of the human insulin basal promoter sequentially . for the heterologous promoter study , the insulin basal promoter was replaced by a sv40 promoter or sv40 promoter and enhancer for use in the reporter assays . since endogenous aire protein is not detected in the htec cell line ( 21 ) or transfection - competent rat primary thymic epithelial cells , an exogenous source of aire or control -galactosidase ( -gal ) was introduced into the cells using a mammalian expression vector ( 50 ng ) . the insulin basal promoter probe ( 0.4 kb ) , ins - vntr - class i basal ( 2.0 kb ) , and iii - basal ( 3.5 kb ) probes were prepared from the reporter vectors after restriction digestion with kpni and hindiii . an artificial class iii construct was assembled by linking four class i vntrs 5-upstream of the human insulin basal promoter sequentially . for the heterologous promoter study , the insulin basal promoter was replaced by a sv40 promoter or sv40 promoter and enhancer for use in the reporter assays . since endogenous aire protein is not detected in the htec cell line ( 21 ) or transfection - competent rat primary thymic epithelial cells , an exogenous source of aire or control -galactosidase ( -gal ) was introduced into the cells using a mammalian expression vector ( 50 ng ) . the insulin basal promoter probe ( 0.4 kb ) , ins - vntr - class i basal ( 2.0 kb ) , and iii - basal ( 3.5 kb ) probes were prepared from the reporter vectors after restriction digestion with kpni and hindiii . therefore , direct evidence is required to support that the vntr allele controls insulin transcript expression in the human thymus . to establish a functional role of the vntr in the differential thymic insulin gene expression , the psti ( uncut by psti ) polymorphism is in strong linkage disequilibrium with the diabetes - predisposing class i allele and psti ( cut by psti ) with diabetes - protective class iii vntr haplotypes ( 10 ) . six novel class i vntr alleles and the restriction profile of the class i and iii alleles of the constructs . to directly dissect any transcriptional activity contributed by the vntr alleles , we made a set of reporter constructs bearing the vntr sequences and excluding the two other putative causal variants , 23 hph i and + 1,140 a / c in the ins locus ( figs . because of the selective tissue expression of the insulin gene , we first tested the class i or class iii vntr - basal insulin luciferase reporter constructs in a hamster pancreatic -cell line , hit . consistent with previous reports , the class i vntr construct activity was approximately twofold higher than the class iii vntr constructs in the hit cells ( fig . the luciferase data confirmed the previous observation that the type 1 diabetes susceptible shorter class i vntr ( most commonly 3040 repeats ) was associated with a 1.5- to 3-fold higher insulin expression than the class iii vntr in the pancreas ( 5,10 ) , although inconsistent results have been reported ( 4 ) and discussed ( 10 ) . an average of 1.58-fold increase was observed in reporter activities driven by class i over class iii vntr haplotypes . in the presence of aire , both classes of vntr display similar activities , with the class i vntr being slightly higher than the class iii vntr . recently , correlation studies suggested an involvement of the autoimmune regulator ( aire ) in the promiscuous expression of insulin in human thymus ( 28,29 ) , whereas thymus - specific deletion of insulin induces autoimmune diabetes ( 30 ) . in contrast to the pattern in pancreas , insulin expression in thymus was reversed , with an average threefold increase of class iii over class i alleles , as represented by the luciferase activity driven by six different class i and one class iii haplotypes in a nontransformed human thymic epithelial cell line ( htec ) ( 19 ) ( fig . this observation suggests that the diabetes susceptible class i vntr might predispose individuals to type 1 diabetes by lowering insulin expression in the thymus and rendering less tolerance induction . evidently , without the other two polymorphisms , the vntr alone accounts for quantitative expression of insulin , which is consistent with the report that 23 hph i , one of the two snps , did not play a significant role in thymus ( 31 ) . when a control -gal expression plasmid was administered , the differential expression signal transduced by the ins - vntr / iddm2 locus was totally lost and the insulin expression driven by either allele decreased to a basal level ( fig . this line of evidence warrants further investigation of aire regulation in the context of the vntr for insulin quantitative expression in thymic epithelial cells . since there is only one natural class iii dna construct available for the comparison study with the six class i vntr constructs , we further assembled an artificial class iii construct ( 162 tandem repeats ) based upon the assumption that the differential transcriptional activities from class iii versus class i vntr are due to the number of tandem repeats . b : a luciferase reporter gene is driven by class i or class iii vntr with insulin basal promoter ( 50 ng ) and transfected in a htec line . the class iii activity was expressed as an average of three experiments from one construct . d and e : the same set of constructs was assayed using rat primary thymic epithelial cells following the same strategy as in b and c. f and g : to rule out the possibility that the upstream 5-flanking genomic region ( 1.1 kb ) might contribute to the differential insulin expression in thymus , another set of constructs was made by removing this region ( a ) and assaying in the htec line . as a loading control , next , we determined how the class i or class iii vntr allele responded to an increasing dosage of aire expression plasmid . a class i vntr ( pq ) and a class iii vntr reporter construct ( 50 ng ) were cotransfected with aire expression vector or control vector -gal dna into a htec line . in the presence of aire , the insulin basal promoter alone ( 365 bp ) displays relatively low activity in the human tec and rat primary tec ( fig . the class i vntr is two to threefold higher relative to the insulin basal promoter activity , whereas the class iii vntr increases seven to ninefold over the insulin basal promoter activity , suggesting that the aire transcriptional regulator could activate the ins - vntr sequence in the context of the insulin basal promoter . to validate the interaction between the aire protein and the vntr sequence , we performed a dna - protein pull - down experiment using adenovirus - aire transduced htec cell lysate and radiolabeled dna probes ( 50,000 cpm ) derived from the insulin basal promoter , class i - vntr - ins - basal promoter , and class iii - vntr - ins - basal promoter sequences ( fig . a and b : insulin basal promoter , vntr - class i - ins - basal and vntr - class iii - ins - basal vectors demonstrated differential promoter activities in both htec and rat primary tec in response to aire . c : dna probes derived from insulin basal promoter ( 0.4 kb ) , vntr - class i - ins - basal ( 2.0 kb ) , and vntr - class iii - ins - basal ( 3.5 kb ) were radiolabeled with t4 polynucleotide kinase and -p - atp as shown in the input lanes . transfection of the construct lacking the insulin basal promoter with the aire expression vector in human tec was measured for luciferase reporter activity . e : addition of a sv40 enhancer to the construct greatly enhanced sv40 promoter activity , but the class i or iii vntr had no effect on the promoter / enhancer combination . cotransfection of the luciferase constructs with an aire expression vector into the human tec showed no effect to the luciferase activity with either class i or iii vntr in conjunction with the sv40 promoter . the characteristic activation pattern of class i and iii vntrs seen when linked directly to the insulin basal promoter disappeared when the insulin basal promoter was replaced by the sv40 promoter and/or sv40 promoter / enhancer ( fig . this result indicates that the vntr sequence has to function in conjunction with the insulin basal promoter to respond to aire . in this paper , we have revealed a novel role of the aire protein with the vntr region in the establishment of differential tissue regulation of the insulin mrna in the thymus . therefore , direct evidence is required to support that the vntr allele controls insulin transcript expression in the human thymus . to establish a functional role of the vntr in the differential thymic insulin gene expression , the psti ( uncut by psti ) polymorphism is in strong linkage disequilibrium with the diabetes - predisposing class i allele and psti ( cut by psti ) with diabetes - protective class iii vntr haplotypes ( 10 ) . six novel class i vntr alleles and the restriction profile of the class i and iii alleles of the constructs . to directly dissect any transcriptional activity contributed by the vntr alleles , we made a set of reporter constructs bearing the vntr sequences and excluding the two other putative causal variants , 23 hph i and + 1,140 a / c in the ins locus ( figs . because of the selective tissue expression of the insulin gene , we first tested the class i or class iii vntr - basal insulin luciferase reporter constructs in a hamster pancreatic -cell line , hit . consistent with previous reports , the class i vntr construct activity was approximately twofold higher than the class iii vntr constructs in the hit cells ( fig . the luciferase data confirmed the previous observation that the type 1 diabetes susceptible shorter class i vntr ( most commonly 3040 repeats ) was associated with a 1.5- to 3-fold higher insulin expression than the class iii vntr in the pancreas ( 5,10 ) , although inconsistent results have been reported ( 4 ) and discussed ( 10 ) . in the presence of aire , both classes of vntr display similar activities , with the class i vntr being slightly higher than the class iii vntr . recently , correlation studies suggested an involvement of the autoimmune regulator ( aire ) in the promiscuous expression of insulin in human thymus ( 28,29 ) , whereas thymus - specific deletion of insulin induces autoimmune diabetes ( 30 ) . in contrast to the pattern in pancreas , insulin expression in thymus was reversed , with an average threefold increase of class iii over class i alleles , as represented by the luciferase activity driven by six different class i and one class iii haplotypes in a nontransformed human thymic epithelial cell line ( htec ) ( 19 ) ( fig . this observation suggests that the diabetes susceptible class i vntr might predispose individuals to type 1 diabetes by lowering insulin expression in the thymus and rendering less tolerance induction . evidently , without the other two polymorphisms , the vntr alone accounts for quantitative expression of insulin , which is consistent with the report that 23 hph i , one of the two snps , did not play a significant role in thymus ( 31 ) . when a control -gal expression plasmid was administered , the differential expression signal transduced by the ins - vntr / iddm2 locus was totally lost and the insulin expression driven by either allele decreased to a basal level ( fig . this line of evidence warrants further investigation of aire regulation in the context of the vntr for insulin quantitative expression in thymic epithelial cells . since there is only one natural class iii dna construct available for the comparison study with the six class i vntr constructs , we further assembled an artificial class iii construct ( 162 tandem repeats ) based upon the assumption that the differential transcriptional activities from class iii versus class i vntr are due to the number of tandem repeats . b : a luciferase reporter gene is driven by class i or class iii vntr with insulin basal promoter ( 50 ng ) and transfected in a htec line . the class iii activity was expressed as an average of three experiments from one construct . d and e : the same set of constructs was assayed using rat primary thymic epithelial cells following the same strategy as in b and c. f and g : to rule out the possibility that the upstream 5-flanking genomic region ( 1.1 kb ) might contribute to the differential insulin expression in thymus , another set of constructs was made by removing this region ( a ) and assaying in the htec line . as a loading control , next , we determined how the class i or class iii vntr allele responded to an increasing dosage of aire expression plasmid . in the presence of aire , the insulin basal promoter alone ( 365 bp ) displays relatively low activity in the human tec and rat primary tec ( fig . the class i vntr is two to threefold higher relative to the insulin basal promoter activity , whereas the class iii vntr increases seven to ninefold over the insulin basal promoter activity , suggesting that the aire transcriptional regulator could activate the ins - vntr sequence in the context of the insulin basal promoter . to validate the interaction between the aire protein and the vntr sequence , we performed a dna - protein pull - down experiment using adenovirus - aire transduced htec cell lysate and radiolabeled dna probes ( 50,000 cpm ) derived from the insulin basal promoter , class i - vntr - ins - basal promoter , and class iii - vntr - ins - basal promoter sequences ( fig . a and b : insulin basal promoter , vntr - class i - ins - basal and vntr - class iii - ins - basal vectors demonstrated differential promoter activities in both htec and rat primary tec in response to aire . c : dna probes derived from insulin basal promoter ( 0.4 kb ) , vntr - class i - ins - basal ( 2.0 kb ) , and vntr - class iii - ins - basal ( 3.5 kb ) were radiolabeled with t4 polynucleotide kinase and -p - atp as shown in the input lanes . transfection of the construct lacking the insulin basal promoter with the aire expression vector in human tec was measured for luciferase reporter activity . e : addition of a sv40 enhancer to the construct greatly enhanced sv40 promoter activity , but the class i or iii vntr had no effect on the promoter / enhancer combination . cotransfection of the luciferase constructs with an aire expression vector into the human tec showed no effect to the luciferase activity with either class i or iii vntr in conjunction with the sv40 promoter . the characteristic activation pattern of class i and iii vntrs seen when linked directly to the insulin basal promoter disappeared when the insulin basal promoter was replaced by the sv40 promoter and/or sv40 promoter / enhancer ( fig . this result indicates that the vntr sequence has to function in conjunction with the insulin basal promoter to respond to aire . in this paper , we have revealed a novel role of the aire protein with the vntr region in the establishment of differential tissue regulation of the insulin mrna in the thymus . a strong correlation between the variable numbers of tandem repeat ( vntr ) or iddm2 locus and the levels of insulin mrna expression in the thymus have been clearly established ( 4,5,7,8 ) . the short class i allele is predisposing to type 1 diabetes and is associated with lower thymus insulin expression , whereas the class iii allele is protective and associated with three to fourfold higher thymic insulin expression . the autoimmune regulator , aire , has been shown to direct the expression of hundreds of tissue - specific antigens in the thymus and is crucial for establishment of immune tolerance ( 16 ) . to definitively establish a role of the vntr in insulin gene regulation in thymus , our cloning strategy spanning the vntr region of the ins locus first excluded the two additional polymorphisms ( 23 hph i and + 1,140 a / c ) that are present within the iddm2 locus and may contribute to the differential insulin levels in the thymus . the transfection studies with the ins - vntr class i and iii constructs unequivocally show that aire controls differential insulin gene expression through the ins - vntr region . to study the role of the aire protein in regulating the insulin promoter via the vntr region , we further examined the physical interaction between aire and the vntr . particularly , the aire binding site is not found in the ins - vntr region . additionally , our data show that the insulin basal promoter region in conjunction with the vntr is required for regulation by aire protein . intriguingly , linkage of the ins - vntr class i or class iii vntr region with a heterologous promoter sequence resulted in the complete loss of aire transcriptional regulation . therefore , it is likely that aire must interact with a protein or complex that is formed on the basal insulin promoter to modulate insulin gene expression in the thymus . the insulin basal promoter has been identified as a target for aire regulation , but these constructs always lacked the vntr region . in response to aire , our data show that in the presence of the vntr sequence ( as in the human insulin gene locus ) insulin basal promoter activity is significantly enhanced in htecs . labeling of the basal insulin promoter , class i and class iii basal insulin promoter regions and incubation with an aire - expressing htec cell lysates unambiguously showed preferential binding of the aire protein with the vntr containing fragments ( fig . the enhanced regulation of the class iii constructs is most likely due to the larger number of tandem repeats forming a more stable intramolecular secondary structure that is required for aire protein binding and formation of a transcriptional complex . as expected , the artificial class iii vntr ( 162 tandem repeats ) demonstrated a higher transcriptional activity as compared with the natural class iii vntr ( 140 tandem repeats ) and exhibited approximately threefold higher activity than class i vntr ( supplementary fig . together , we show that the aire protein is capable of selectively pulling - down the vntr insulin basal promoter sequence . in summary , we reveal a dual control mechanism governing the insulin quantitative expression in thymus that may account for part of the genetic predisposition observed in type 1 diabetic patients . aire - mediated differential regulation of insulin gene expression via the vntr locus in the thymus may influence tolerance and susceptibility to type 1 diabetes .

movement of eukaryotic cells , intracellular transport , signaling , cell division and cell shape are functions of the cytoskeleton [ 1 , 2 , 3 , 4 ] . the cytoskeleton is made up of three types of filaments : actin filaments , intermediate filaments and microtubules . three groups of proteins called molecular motors utilize energy from the hydrolysis of atp to move in association with the cytoskeleton : kinesins , dyneins and myosins [ 1 , 5 , 6 ] . kinesins and dyneins move along microtubules [ 5 , 7 ] and actin is utilized by myosin for motility [ 8 , 9 ] . molecular motors in non - plant systems have been extensively characterized but less is known about the presence and functions of these motors in plant cells . using antibodies to mouse dynein , two 400 kda proteins were identified in tobacco pollen during pollen germination suggesting the presence of dynein in pollen tubes . to date , no report has been published on the presence of dynein at the molecular level . using animal dynein sequences to search the arabidopsis database tair ( the arabidopsis information resource ) , no sequences similar to heavy or intermediate chains were found . however , some sequences showing similarity to light chains are present in the database . kinesins have been identified in arabidopsis and other plant systems [ 12 , 13 , 14 , 15 , 16 ] and their movement along microtubules has been analyzed [ 16 , 17 , 18 , 19 ] . kinesins are a superfamily of molecular motors containing at least nine subfamilies [ 7 , 20 ] . plant kinesins are represented in all but two of the families . using the amino - acid sequence of the motor domain of a plant kinesin , a search of the arabidopsis genome yielded 61 kinesin - like proteins . this is the largest number of kinesins in an organism per thousand genes compared to yeast , drosophila melanogaster and caenorhabditis elegans . phylogenetic analysis of known myosins in various organisms has resulted in the classification of myosins into several groups . the myosin home page ( mhp ) has a phylogenetic tree with 143 myosins classified into 17 classes . however , an analysis of the myosin superfamily in drosophila , concluded that two new mammalian myosins and a drosophila myosin make up a new class of myosins , class xviii . the classes have been named according to the order in which each class was first discovered except for myosins i and ii . myosins have three domains in common ; a motor domain that interacts with actin and binds atp , a neck domain that binds light chains or calmodulin and a tail domain . phylogenetic analysis is often based on the motor domain of the myosins . however , using the full - length sequence results in nearly the same grouping , indicating that the heads and tails have evolved together [ 23 , 24 , 25 , 26 ] . a study using the head ( motor domain ) , neck and tail domains separately for phylogenetic analysis or the head and neck / tail showed that this is generally true . the neck domain consists of one or more helical sequences termed the iq motif , which has the consensus sequence iqxxxrgxxxr . the iq motif binds the conventional myosin ii light chains and calmodulin or calmodulin - like proteins in other myosins . unlike most calmodulin - binding proteins , myosins bind calmodulin in the absence of ca . as actin is utilized by myosin for motility , the possible functions of myosin in plants are closely linked to the functions of actin . the actin cytoskeleton has been shown to be involved in many processes in plants including transportation , signaling , cell division , cytoplasmic streaming and morphogenesis [ 2 , 3 ] . much of the cytoplasmic streaming work has been done in algal cells and the direct involvement of actin and myosin has been shown [ 30 , 31 ] . genetic , biochemical and cell biological studies with trichomes during the past four years have provided interesting insights into the role of the cytoskeleton in trichome morphogenesis . these studies indicate that actin and the microtubule cytoskeleton play a pivotal role in cell expansion and branching during trichome development . localization studies and visualization of the actin cytoskeleton in live cells with an actin - binding protein tagged with green fluorescent protein ( gfp ) indicate that the organization of f - actin changes during trichome morphogenesis [ 33 , 34 ] . chemicals that promote depolymerization or stabilization of the actin cytoskeleton did not effect branching but produced distorted trichomes . the morphology of these trichomes is similar to that observed in a ' distorted ' class of mutants , suggesting that at least some of the affected genes are likely to code for proteins involved in actin organization / dynamics ( for example myosins , actin - depolymerizing factors , actin - binding proteins ) . there is also evidence that the actin cytoskeleton is involved in mitosis and during separation of daughter cells after the successful segregation of chromosomes into daughter nuclei . the actin cytoskeleton is also involved in pollen tube growth , and calcium regulation has also been shown to be involved [ 35 , 36 ] . myosins have been identified in plants both biochemically [ 37 , 38 , 39 , 40 ] and at the molecular level [ 41 , 42 , 43 ] . immunological detection of myosins using antibodies against animal myosin identified proteins of various sizes from different plants [ 44 , 45 , 46 ] . immunofluorescence studies localized myosin to the surface of organelles , the vegetative nuclei and generative cells in pollen grains and tubes , to the active streaming lanes and cortical surface in pollen tubes and , more recently , to plasmodesmata in root tissues [ 38 , 47 ] . motility assays and atpase assays [ 48 , 49 , 50 ] using myosin - like proteins isolated from plants have also demonstrated the presence of myosins in plants . since 1993 , five partial or full - length myosins from arabidopsis have been characterized at the molecular level . using pcr - based approaches , knight and kendrick - jones cloned a myosin they called atm ( arabidopsis thaliana myosin ) , kinkema and schiefelbein cloned the myosin mya1 and kinkema et al . cloned another full - length myosin , atm2 , and two partial length myosins mya2 and mya3 . kinekema et al . also identified three pcr products that coded for unique myosin motor domain sequence . phylogenetic analysis using these myosins indicated that the atm myosins were a unique class and they were named class viii . the mya myosins are somewhat related to class v myosins but as other analyses have been done , these myosins were also assigned to a new class , class xi [ 8 , 42 ] . myosins have been identified in zea mays , two of which belonged to class xi and one to class viii . pcr fragments for fern myosins have been reported [ 52 , 53 ] and sequences are available for myosins from helianthus annuus ( 0 . two fern ( anemia phyllitidis ) pcr products and the h. annuus myosins also fall either into class viii or class xi myosins [ 22 , 42 ] . two algal myosins are also members of the class xi myosins , one from chara corallina and one from chlamydomonas reinhardtii [ 22 , 54 ] . a third class of myosins ( xiii ) no animal myosins are in any of these classes and no plant myosins are in any other myosin class . however , the cellular slime mold dictyostelium discoideum has one myosin ( dd myoj ) , which is alternatively grouped with class v or class xi . how many myosins are in a plant genome ? what are the similarities and differences between plant and non - plant myosins that might help establish a function for the myosins ? until the recent completion of the sequencing of the arabidopsis genome , answers to these questions were not known . it is now possible to determine how many myosins are in the arabidopsis genome and to see if any plant myosins fall into other myosin classes . as the myosin motor domain is highly conserved , the sequence from one myosin motor can be used to search a database for all other myosins . we used the motor domain from mya1 to search the arabidopsis database for sequences with similarity to this domain . we identified 17 arabidopsis myosins , including the 5 reported myosins , in the arabidopsis genome . phylogenetic analysis using non - plant and plant myosins showed that all 17 fall into either myosin class viii or xi . an analysis of their exon / intron junctions and sequence similarities indicates that all myosins are highly conserved and some may represent gene duplication events . using the amino - acid sequence of the conserved motor domain of the plant myosin mya1 , databases were searched using blastp and tblastn at tair . other searches using the amino - acid sequence of motor domains from representatives of other classes of myosins were also done but they did not reveal any other myosin sequences . sixteen unique sequences were obtained that contain a myosin motor domain as identified by the smart ( smart modular architecture research tool ) program . the sequences obtained in this search were compared to the munich information center for protein sequences ( mips ) list of myosin domains in arabidopsis . a check of these showed that 13 of the sequences were myosins identified in our search and one was a myosin not available in the ncbi ( national center for biotechnology information ) protein database . one is a putative helicase ( at1g26370 ) with no myosin motor domain and one is a possible pseudogene ( at1g42680 ) with only 162 amino acids that have some similarity to the myosin motor domain . mips does not list three myosins identified in our search ( at xig , at xif and at xi - i ) . table 1 lists the myosins by names as given in the phylogenetic tree constructed by hodge and cope and as assigned by us . there are a total of 17 myosin genes in arabidopsis . in comparison , s. cerevisiae , schizosaccharomyces pombe , c. elegans and d. melanogaster have 5 , 5 , 20 and 13 myosins , respectively ( figure 1 ) [ 60 , 61 ] . arabidopsis has the lowest percentage ( 0.068% ) of myosin genes out of the total number of genes , as compared to s. cerevisiae and s. pombe with 0.080% and 0.093% , respectively , c. elegans with 0.11% and d. melanogaster with 0.096% ( see figure 1 ) . the number of myosins in each organism is on the left ( red column ) and the number per thousand for each organism is on the right ( blue column ) . at , arabidopsis thaliana ; dm , drosophila melanogaster ; ce , caenorhabditis elegans ; sc , saccharomyces cerevisiae ; sp , schizosaccharomyces pombe . edited by authors for full - length sequence : atdb , arabidopsis database ; mips , munich information center for protein sequences ; md , motor domain ; cc , coiled - coil region ; iq , putative calmodulin - binding motif . only 5 of the 17 arabidopsis myosins have been reported in the literature [ 41 , 42 , 43 ] . the other 12 are sequences obtained from the arabidopsis database sequenced as part of the arabidopsis genome sequencing project . these sequences are , therefore , predicted sequences that have not been verified by complete cdnas . the average sequence length of the arabidopsis myosins is 1,400 residues , with the shortest sequence prediction being 1,085 ( at viiia ) amino acids and the longest 1,730 ( at xia ) . some of the intron / exon predictions may not be correct , which could reduce or increase the size of the predicted proteins and so the sizes may change as more characterization is done for each myosin . a case in point is the cdna that was isolated by kinkema and schiefelbein for at mya1 ( at mya1 ) which codes for 1,520 amino acids , whereas the predicted protein has 1,599 because of differences in intron prediction . using the arabidopsis sequence map overview of tair , the location of each myosin was determined ( figure 2 ) . the chromosome lengths are based on the centimorgan ( cm ) scale as shown on the tair map overview . the maps reported with the announcement of the arabidopsis genome sequence show somewhat different lengths than the tair maps . all arabidopsis myosins and a selection of myosins from other organisms representing each of the myosin classes were aligned using the motor domain sequence as determined by the smart program . the alignment was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap ( 100 replicates ) method with a heuristic search of the paup 4.0b6 program ( figure 3 ) . the arabidopsis myosins all group into two classes along with other plant myosins - class viii and class xi . no animal myosins group with the plant myosins and no plant myosins group into any of the animal myosins . an algal ( chara corallina ) myosin , cc ccm , does group with the plant class xi myosins but is on a separate branch from any other class xi myosin ( figure 3 ) . the d. discoideum myosin dd myoj did not fall into a class with any of the plant myosins . in fact , three d. discoideum myosins ( dd myoi , dd myoj , and dd myom ) did not fall into any of the classes ( figure 3 ) . the phylogenetic trees of hodge and cope and the tree on the myosin home page [ 22 , 59 ] show the dd myoi branching from class vii myosins . a heuristic search without bootstrapping also showed the dd myoi myosin as a branch from class vii and domain analysis shows that dd myoi has the myth4 domain found in other class vii myosins . other phylogenetic anaylses have placed dd myoj as a branch off class xi myosins from plants [ 22 , 59 ] . however , the phylogenetic tree generated from full - length sequences of plant myosins and dd myoj ( see below ) also shows that dd myoj is separate from the plant myosins . alignment of the motor domain of representative myosins and all arabidopsis myosins was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the paup 4.0b6 program . the myosin groups , as defined by hodge and cope and yamashita et al . , are identified on the right in roman numerals . myosins from the following organisms were used : ac , acanthamoeba castellani ; acl , acetabularia cliftoni ; at , arabidopsis thaliana ; cc , chara corallina , ha , helianthus annuus ; zm , zea mays ; bt , bos taurus ; mm , mus musculus ; ce , caenorhabditis elegans ; dm , drosophila melanogaster ; rn , rattus norvegicus ; sc , saccharomyces cerevisiae ; hs , homo sapiens ; dd , dictyostelium discoideum ; lp , limulus polyphemus ; en , emericella nidulans ; pg , pyricularia grisea ; pf , plasmodium falciparum ; and tg , toxoplasma gondii . the number at the branches indicates the number of times the dichotomy was supported out of 100 bootstrap tries . myosins from another alga , acetabularia cliftonii , are classified into a separate group ( xiii ) and one myosin each from the fungi emericella nidulans and pyricularia grisea are also assigned to a separate class ( xvii ) . a second alignment was done using the full - length sequences for all arabidopsis and other known full - length plant myosins with a human heavy - chain myosin ( hs ib ) as an outgroup . the two classes of plant myosins are clearly seen ( figure 4 ) . among the class xi myosins the similarity ranges from 40 - 85% ( full length ) and 61 - 91% ( motor domain ) . the similarity between the class viii myosins ranges from 50 - 83% ( full length ) and 64 - 92% ( motor domain ) . when class viii myosins are compared to class xi myosins the similarity only ranges from 22 - 29% ( full - length ) and 35 - 42% ( motor domain ) . one subgroup consists of two pairs of arabidopsis myosins , at xib / at mya2 and at xig / at xih , which are most similar to the sunflower myosin hahamy4 and then another pair of arabidopsis myosins , at xid / at xia . the other subgroup consists of the arabidopsis myosin pair at xic / at xie and two unpaired arabidopsis myosins , at xik and at mya1 , that are most closely related to sunflower myosins hahamy2 and hahamy5 and to the maize myosin zmmyo1 . at xij , at xif and at xi - i are on separate branches that group with the other class xi myosins but not within the two subgroups . there are four class viii arabidopsis myosins that form two pairs , at atm / at viiia and at viiib / at atm2 . the first pair group with class viii myosins from z. mays and h. annuus whereas the second pair are on a separate branch . alignment of the full - length arabidopsis myosins , other full - length plant myosins available in the ncbi database and dd myoj was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the paup 4.0b4a ( ppc ) program . arabidopsis thaliana ; dd , dictyostelium discoideum ; ha , helianthus annuus ; zm , zea mays . the number at the branches indicates the number of times the dichotomy was supported out of 100 bootstrap tries . the motor domain starts at about 50 - 55 residues for the class xi myosins whereas the class viii myosins have a longer amino - terminal region before the motor domain ( 99 - 159 residues ) . the iq domains usually follow right after the motor domain but are separated slightly from the motor domain in at xid , at xi - i , and at xik . there are three or four iq domains in class viii myosins and five or six in class xi , except for at xik , which has only four . there are coiled - coil domains , that differ in length and number , in all the myosins . they often follow directly after the iq domains , but in some cases there is intervening sequence . based on the presence of the coiled - coil domains , the arabidopsis myosins are probably dimeric . the class xi myosins are much longer than the class viii myosins with the difference being in the length of the carboxy - terminal region following the conserved domains found in myosins . the numbers refer to the number in table 1 . the motor domain , iq domains , and coiled - coil domains are as indicated in the key . the first four myosins are in class viii and the following 13 are in class xi . the bar represents 100 amino acids . besides the motor , iq and coiled - coil domains , other domains have been identified in myosins from classes other than the plant classes viii and xi . these include sh3 domains ( src homology 3 domains , that bind to target proteins ) , myth4 ( a domain of unknown function found in a few classes of myosins ) , a zinc - binding domain , a pleckstrin homology domain , ferm / talin ( band 4.1/ezrin / radixin / moesin ) , gpa - rich domains and a protein kinase domain [ 8 , 22 , 26 ] . these domains are involved in protein interactions and presumably give specificity to the action of the myosin . except for the iq and coiled - coil domains , the smart program used to identify the motor domain of the myosin sequences did not identify any domains other than a few with scores less significant than the required threshold . myosins have 131 highly conserved residues spread throughout the motor domain that define a core consensus sequence . comparison of an alignment of arabidopsis myosin motor domains to these conserved sequences shows a great deal of conservation among them ( data not shown ) . one example is the atp - binding site which consists of gesgagkt ( 179 - 187 in dictyostelium myosin ii , dmyoii ) and nxnssr - fgk ( 233 - 241 , dmyoii ) . with the exception of only one residue these the conformational state of myosin changes with atp hydrolysis and a very conserved region implicated in this process has the conserved sequence ldixgfexfxxn(s the sequence in this region is ldiygfexfxxnsfeqxcine(k / r)lqqhf ( the first x is s in all but one myosin , the fourth x is f in all but one myosin ) . suggest that release of the -phosphate of atp may be through a hole in the structure centered around an absolutely conserved arginine residue ( residue 654 , dmyoii ) which is also absolutely conserved in all arabidopsis myosins . the presence of these highly conserved residues in plant myosins suggests that they are capable of motor function . in fact , in vitro motility studies with a purified myosin from chara ( myosin xi , cc ccm in figure 3 ) have confirmed that it is indeed an actin - based motor . a loop present in the motor domain called the hcm ( mutations in this loop cause hypertrophic cardiomyopathy ) is the location of a phosphorylatable serine ( s ) or threonine ( t ) in certain amoeboid myosin i molecules and myosin vi molecules . this s or t residue is 16 residues upstream from the conserved dalak sequence . the enzyme activity of the amoeboid myosins depends on phosphorylation of this site , but although phosphorylation of the myosin vi t residue has been demonstrated , the regulation of enzyme activity has not [ 8 , 63 ] . most other myosins have a constitutively negatively charged amino acid , either aspartic acid ( d ) or glutamic acid ( e ) at this site . this site has been named the teds rule site on the basis of these amino acids . the arabidopsis and other plant myosins all have aspartic acid , glutamic acid or glycine residue at this site , suggesting that they are not regulated by phosphorylation at this site . however , three residues upstream ( 19 from dalak ) , all the class xi myosins have a threonine residue . the site for each predicted or actual intron was located and is shown schematically in figure 6 . the length of each exon and the domain(s ) they code for are shown in tables 2 and 3 for class viii and class xi myosins , respectively . the exons vary in length from 12 to greater than 672 nucleotides ( the length of the beginning and last exons for each gene are not known as the predicted sizes include only the protein - coding nucleotides ) with an average of 122 nucleotides . the four class viii myosins have seven exons of the same length in the same order within the myosin motor domain ( table 2 ) . the start of the iq domains and the coiled - coil domains is more variable except for the at atm2/at viiib pair . the class xi myosins also have many exons that are of the same length and in the same order but that differ from the class viii pattern ( table 3 ) . most class xi myosins motor domains start in the third exon and end in the twentieth . most differences in exon length are in the carboxy - terminal regions ( figure 6 and table 3 ) . however , even in the carboxy - terminal region there are some exon lengths conserved between some or all of the myosins . a clustal alignment at pole bio - informatique lyonnais showed 83.88% identity , 8.19% strong similarity and 2.36% weak similarity between these two myosins . twenty - three of their introns are at the same location in the motor domain area and then following a few different size exons , there are similar sized exons again . class viii myosins at atm and at viiia have 13 exons of the same length . their full - length sequences are 79% identical with another 6.72% strongly similar and 3.52% weakly similar . is on chromosome iii whereas at viiia is on chromosome i. this again may have resulted from a gene duplication . analysis of the total arabidopsis genome revealed that a whole genome duplication occurred , followed by subsequent gene loss and extensive local gene duplications . the duplicated segments represent 58% of the arabidopsis genome . the s. cerevisiae genome has also had a complete ancient genome duplication and analysis of exon sizes in class viii myosins and the domain coded by each exon n , amino - terminal sequence ; m , motor domain ; i , iq domain ; c , coiled - coil domain ; t , tail domain . the size of the first and last exons in each gene reflects only the size of the coding region . analysis of exon sizes in class xi myosins and the domain coded by each exon n , amino - terminal sequence ; m , motor domain ; i , iq domain ; c , coiled - coil domain ; u , undefined ; t , tail domain . the size of the first and last exons in each gene reflects only the size of the coding region . if the gene pairs are the result of duplication , it is interesting to note that while exon lengths have been conserved , intron lengths have not . the average intron length is 131 nucleotides with the shortest intron at 47 nucleotides and the longest at 860 . at it contains the 860-nucleotide intron and three others that are over 500 nucleotides . in a study of 998 introns only 3.3% of the introns were longer than 500 nucleotides with sizes ranging from 59 to 1242 nucleotides . this makes at xi - i unusual in having four out of 33 introns ( 12% ) longer than 500 nucleotides . of the total 557 splice sites that were identified in the arabidopsis myosins only six ( a little more than 1% ) were over 500 nucleotides with four out of the six being in one myosin . hunt et al . found that a sv40 small - t intron only 66 nucleotides in length was spliced efficiently in tobacco cells . several of the introns in the myosins are between 66 and 70 nucleotides and so may be long enough to be spliced . only one is in a cloned myosin known to be spliced at that site ( at xij ) . there is also a predicted intron of only 47 nucleotides in length ( at xid ) which is thought to be too short for efficient splicing . brown et al . found three introns less than 66 nucleotides in length in known expressed proteins , but none of them was less than 59 nucleotides . until the expression of at xid is studied , no conclusion can be made as to the validity of this intron prediction . the significance of the range and variability of intron length is not known . in arabidopsis , in general , the range is even greater ( 47 - 6,442 ) . intron size and sequence of 5 ' and 3 ' splice sites the consensus nucleotide sequences for the 5 ' and 3 ' splice sites are a-2g-1 g+1t+2a+3a+4g+5t+6 and t-5g-4c-3a-2g-1g+1t+2 , respectively . the most conserved sequences are the 5 ' consensus g ( 100% ) t ( 99% ) at the + 1 , + 2 positions , respectively , and the 3 ' a(100% ) g(100% ) at the -2 , -1 positions , respectively . the splice sites in the reported myosins and the predicted myosins ( table 4 ) all contain the 5 ' gt and 3 ' ag sequences . the sequences in the arabidopsis myosins upstream and downstream of these two very conserved sites varied as a reflection of the less conserved nature of these nucleotides ( table 4 ) . however , these predicted sites at the 5 ' and 3 ' splice sites need to be confirmed experimentally . using the amino - acid sequence of the conserved motor domain of the plant myosin mya1 , databases were searched using blastp and tblastn at tair . other searches using the amino - acid sequence of motor domains from representatives of other classes of myosins were also done but they did not reveal any other myosin sequences . sixteen unique sequences were obtained that contain a myosin motor domain as identified by the smart ( smart modular architecture research tool ) program . the sequences obtained in this search were compared to the munich information center for protein sequences ( mips ) list of myosin domains in arabidopsis . a check of these showed that 13 of the sequences were myosins identified in our search and one was a myosin not available in the ncbi ( national center for biotechnology information ) protein database . one is a putative helicase ( at1g26370 ) with no myosin motor domain and one is a possible pseudogene ( at1g42680 ) with only 162 amino acids that have some similarity to the myosin motor domain . mips does not list three myosins identified in our search ( at xig , at xif and at xi - i ) . table 1 lists the myosins by names as given in the phylogenetic tree constructed by hodge and cope and as assigned by us . there are a total of 17 myosin genes in arabidopsis . in comparison , s. cerevisiae , schizosaccharomyces pombe , c. elegans and d. melanogaster have 5 , 5 , 20 and 13 myosins , respectively ( figure 1 ) [ 60 , 61 ] . arabidopsis has the lowest percentage ( 0.068% ) of myosin genes out of the total number of genes , as compared to s. cerevisiae and s. pombe with 0.080% and 0.093% , respectively , c. elegans with 0.11% and d. melanogaster with 0.096% ( see figure 1 ) . the number of myosins in each organism is on the left ( red column ) and the number per thousand for each organism is on the right ( blue column ) . at , arabidopsis thaliana ; dm , drosophila melanogaster ; ce , caenorhabditis elegans ; sc , saccharomyces cerevisiae ; sp , schizosaccharomyces pombe . edited by authors for full - length sequence : atdb , arabidopsis database ; mips , munich information center for protein sequences ; md , motor domain ; cc , coiled - coil region ; iq , putative calmodulin - binding motif . only 5 of the 17 arabidopsis myosins have been reported in the literature [ 41 , 42 , 43 ] . the other 12 are sequences obtained from the arabidopsis database sequenced as part of the arabidopsis genome sequencing project . these sequences are , therefore , predicted sequences that have not been verified by complete cdnas . the average sequence length of the arabidopsis myosins is 1,400 residues , with the shortest sequence prediction being 1,085 ( at viiia ) amino acids and the longest 1,730 ( at xia ) . some of the intron / exon predictions may not be correct , which could reduce or increase the size of the predicted proteins and so the sizes may change as more characterization is done for each myosin . a case in point is the cdna that was isolated by kinkema and schiefelbein for at mya1 ( at mya1 ) which codes for 1,520 amino acids , whereas the predicted protein has 1,599 because of differences in intron prediction . using the arabidopsis sequence map overview of tair , the location of each myosin was determined ( figure 2 ) . the chromosome lengths are based on the centimorgan ( cm ) scale as shown on the tair map overview . the maps reported with the announcement of the arabidopsis genome sequence show somewhat different lengths than the tair maps . all arabidopsis myosins and a selection of myosins from other organisms representing each of the myosin classes were aligned using the motor domain sequence as determined by the smart program . the alignment was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap ( 100 replicates ) method with a heuristic search of the paup 4.0b6 program ( figure 3 ) . the arabidopsis myosins all group into two classes along with other plant myosins - class viii and class xi . no animal myosins group with the plant myosins and no plant myosins group into any of the animal myosins . an algal ( chara corallina ) myosin , cc ccm , does group with the plant class xi myosins but is on a separate branch from any other class xi myosin ( figure 3 ) . the d. discoideum myosin dd myoj did not fall into a class with any of the plant myosins . in fact , three d. discoideum myosins ( dd myoi , dd myoj , and dd myom ) did not fall into any of the classes ( figure 3 ) . the phylogenetic trees of hodge and cope and the tree on the myosin home page [ 22 , 59 ] show the dd myoi branching from class vii myosins . a heuristic search without bootstrapping also showed the dd myoi myosin as a branch from class vii and domain analysis shows that dd myoi has the myth4 domain found in other class vii myosins . other phylogenetic anaylses have placed dd myoj as a branch off class xi myosins from plants [ 22 , 59 ] . however , the phylogenetic tree generated from full - length sequences of plant myosins and dd myoj ( see below ) also shows that dd myoj is separate from the plant myosins . alignment of the motor domain of representative myosins and all arabidopsis myosins was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the paup 4.0b6 program . the myosin groups , as defined by hodge and cope and yamashita et al . , are identified on the right in roman numerals . myosins from the following organisms were used : ac , acanthamoeba castellani ; acl , acetabularia cliftoni ; at , arabidopsis thaliana ; cc , chara corallina , ha , helianthus annuus ; zm , zea mays ; bt , bos taurus ; mm , mus musculus ; ce , caenorhabditis elegans ; dm , drosophila melanogaster ; rn , rattus norvegicus ; sc , saccharomyces cerevisiae ; hs , homo sapiens ; dd , dictyostelium discoideum ; lp , limulus polyphemus ; en , emericella nidulans ; pg , pyricularia grisea ; pf , plasmodium falciparum ; and tg , toxoplasma gondii . the number at the branches indicates the number of times the dichotomy was supported out of 100 bootstrap tries . myosins from another alga , acetabularia cliftonii , are classified into a separate group ( xiii ) and one myosin each from the fungi emericella nidulans and pyricularia grisea are also assigned to a separate class ( xvii ) . a second alignment was done using the full - length sequences for all arabidopsis and other known full - length plant myosins with a human heavy - chain myosin ( hs ib ) as an outgroup . the two classes of plant myosins are clearly seen ( figure 4 ) . among the class xi myosins the similarity ranges from 40 - 85% ( full length ) and 61 - 91% ( motor domain ) . the similarity between the class viii myosins ranges from 50 - 83% ( full length ) and 64 - 92% ( motor domain ) . when class viii myosins are compared to class xi myosins the similarity only ranges from 22 - 29% ( full - length ) and 35 - 42% ( motor domain ) . one subgroup consists of two pairs of arabidopsis myosins , at xib / at mya2 and at xig / at xih , which are most similar to the sunflower myosin hahamy4 and then another pair of arabidopsis myosins , at xid / at xia . the other subgroup consists of the arabidopsis myosin pair at xic / at xie and two unpaired arabidopsis myosins , at xik and at mya1 , that are most closely related to sunflower myosins hahamy2 and hahamy5 and to the maize myosin zmmyo1 . at xij , at xif and at xi - i are on separate branches that group with the other class xi myosins but not within the two subgroups . there are four class viii arabidopsis myosins that form two pairs , at atm / at viiia and at viiib / at atm2 . the first pair group with class viii myosins from z. mays and h. annuus whereas the second pair are on a separate branch . alignment of the full - length arabidopsis myosins , other full - length plant myosins available in the ncbi database and dd myoj was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the paup 4.0b4a ( ppc ) program . a human myosin ( hs 1b ) was used as an outgroup . at , arabidopsis thaliana ; the number at the branches indicates the number of times the dichotomy was supported out of 100 bootstrap tries . the motor domain starts at about 50 - 55 residues for the class xi myosins whereas the class viii myosins have a longer amino - terminal region before the motor domain ( 99 - 159 residues ) . the iq domains usually follow right after the motor domain but are separated slightly from the motor domain in at xid , at xi - i , and at xik . there are three or four iq domains in class viii myosins and five or six in class xi , except for at xik , which has only four . there are coiled - coil domains , that differ in length and number , in all the myosins . they often follow directly after the iq domains , but in some cases there is intervening sequence . based on the presence of the coiled - coil domains , the arabidopsis myosins are probably dimeric . the class xi myosins are much longer than the class viii myosins with the difference being in the length of the carboxy - terminal region following the conserved domains found in myosins . the numbers refer to the number in table 1 . the motor domain , iq domains , and coiled - coil domains are as indicated in the key . the first four myosins are in class viii and the following 13 are in class xi . the bar represents 100 amino acids . besides the motor , iq and coiled - coil domains , other domains have been identified in myosins from classes other than the plant classes viii and xi . these include sh3 domains ( src homology 3 domains , that bind to target proteins ) , myth4 ( a domain of unknown function found in a few classes of myosins ) , a zinc - binding domain , a pleckstrin homology domain , ferm / talin ( band 4.1/ezrin / radixin / moesin ) , gpa - rich domains and a protein kinase domain [ 8 , 22 , 26 ] . these domains are involved in protein interactions and presumably give specificity to the action of the myosin . except for the iq and coiled - coil domains , the smart program used to identify the motor domain of the myosin sequences did not identify any domains other than a few with scores less significant than the required threshold . myosins have 131 highly conserved residues spread throughout the motor domain that define a core consensus sequence . comparison of an alignment of arabidopsis myosin motor domains to these conserved sequences shows a great deal of conservation among them ( data not shown ) . one example is the atp - binding site which consists of gesgagkt ( 179 - 187 in dictyostelium myosin ii , dmyoii ) and nxnssr - fgk ( 233 - 241 , dmyoii ) . with the exception of only one residue these the conformational state of myosin changes with atp hydrolysis and a very conserved region implicated in this process has the conserved sequence ldixgfexfxxn(s the sequence in this region is ldiygfexfxxnsfeqxcine(k / r)lqqhf ( the first x is s in all but one myosin , the fourth x is f in all but one myosin ) . suggest that release of the -phosphate of atp may be through a hole in the structure centered around an absolutely conserved arginine residue ( residue 654 , dmyoii ) which is also absolutely conserved in all arabidopsis myosins . the presence of these highly conserved residues in plant myosins suggests that they are capable of motor function . in fact , in vitro motility studies with a purified myosin from chara ( myosin xi , cc ccm in figure 3 ) have confirmed that it is indeed an actin - based motor . a loop present in the motor domain called the hcm ( mutations in this loop cause hypertrophic cardiomyopathy ) is the location of a phosphorylatable serine ( s ) or threonine ( t ) in certain amoeboid myosin i molecules and myosin vi molecules . this s or t residue is 16 residues upstream from the conserved dalak sequence . the enzyme activity of the amoeboid myosins depends on phosphorylation of this site , but although phosphorylation of the myosin vi t residue has been demonstrated , the regulation of enzyme activity has not [ 8 , 63 ] . most other myosins have a constitutively negatively charged amino acid , either aspartic acid ( d ) or glutamic acid ( e ) at this site . this site has been named the teds rule site on the basis of these amino acids . the arabidopsis and other plant myosins all have aspartic acid , glutamic acid or glycine residue at this site , suggesting that they are not regulated by phosphorylation at this site . however , three residues upstream ( 19 from dalak ) , all the class xi myosins have a threonine residue . the site for each predicted or actual intron was located and is shown schematically in figure 6 . the length of each exon and the domain(s ) they code for are shown in tables 2 and 3 for class viii and class xi myosins , respectively . the exons vary in length from 12 to greater than 672 nucleotides ( the length of the beginning and last exons for each gene are not known as the predicted sizes include only the protein - coding nucleotides ) with an average of 122 nucleotides . the four class viii myosins have seven exons of the same length in the same order within the myosin motor domain ( table 2 ) . the start of the iq domains and the coiled - coil domains is more variable except for the at atm2/at viiib pair . the class xi myosins also have many exons that are of the same length and in the same order but that differ from the class viii pattern ( table 3 ) . most class xi myosins motor domains start in the third exon and end in the twentieth . most differences in exon length are in the carboxy - terminal regions ( figure 6 and table 3 ) . however , even in the carboxy - terminal region there are some exon lengths conserved between some or all of the myosins . a clustal alignment at pole bio - informatique lyonnais showed 83.88% identity , 8.19% strong similarity and 2.36% weak similarity between these two myosins . twenty - three of their introns are at the same location in the motor domain area and then following a few different size exons , there are similar sized exons again . class viii myosins at atm and at viiia have 13 exons of the same length . their full - length sequences are 79% identical with another 6.72% strongly similar and 3.52% weakly similar . is on chromosome iii whereas at viiia is on chromosome i. this again may have resulted from a gene duplication . analysis of the total arabidopsis genome revealed that a whole genome duplication occurred , followed by subsequent gene loss and extensive local gene duplications . the duplicated segments represent 58% of the arabidopsis genome . the s. cerevisiae genome has also had a complete ancient genome duplication and analysis of exon sizes in class viii myosins and the domain coded by each exon n , amino - terminal sequence ; m , motor domain ; i , iq domain ; c , coiled - coil domain ; t , tail domain . the size of the first and last exons in each gene reflects only the size of the coding region . analysis of exon sizes in class xi myosins and the domain coded by each exon n , amino - terminal sequence ; m , motor domain ; i , iq domain ; c , coiled - coil domain ; u , undefined ; t , tail domain . the size of the first and last exons in each gene reflects only the size of the coding region . if the gene pairs are the result of duplication , it is interesting to note that while exon lengths have been conserved , intron lengths have not . the average intron length is 131 nucleotides with the shortest intron at 47 nucleotides and the longest at 860 . at it contains the 860-nucleotide intron and three others that are over 500 nucleotides . in a study of 998 introns only 3.3% of the introns were longer than 500 nucleotides with sizes ranging from 59 to 1242 nucleotides . this makes at xi - i unusual in having four out of 33 introns ( 12% ) longer than 500 nucleotides . of the total 557 splice sites that were identified in the arabidopsis myosins only six ( a little more than 1% ) were over 500 nucleotides with four out of the six being in one myosin . hunt et al . found that a sv40 small - t intron only 66 nucleotides in length was spliced efficiently in tobacco cells . several of the introns in the myosins are between 66 and 70 nucleotides and so may be long enough to be spliced . only one is in a cloned myosin known to be spliced at that site ( at xij ) . there is also a predicted intron of only 47 nucleotides in length ( at xid ) which is thought to be too short for efficient splicing . brown et al . found three introns less than 66 nucleotides in length in known expressed proteins , but none of them was less than 59 nucleotides . until the expression of at xid is studied , no conclusion can be made as to the validity of this intron prediction . the significance of the range and variability of intron length is not known . in arabidopsis , in general , the range is even greater ( 47 - 6,442 ) . intron size and sequence of 5 ' and 3 ' splice sites the consensus nucleotide sequences for the 5 ' and 3 ' splice sites are a-2g-1 g+1t+2a+3a+4g+5t+6 and t-5g-4c-3a-2g-1g+1t+2 , respectively . the most conserved sequences are the 5 ' consensus g ( 100% ) t ( 99% ) at the + 1 , + 2 positions , respectively , and the 3 ' a(100% ) g(100% ) at the -2 , -1 positions , respectively . the splice sites in the reported myosins and the predicted myosins ( table 4 ) all contain the 5 ' gt and 3 ' ag sequences . the sequences in the arabidopsis myosins upstream and downstream of these two very conserved sites varied as a reflection of the less conserved nature of these nucleotides ( table 4 ) . however , these predicted sites at the 5 ' and 3 ' splice sites need to be confirmed experimentally . a study of myosins in lily and tobacco pollen tubes using antibodies to three animal - type myosins ia and ib , ii and v suggested the presence of three types of myosins in these plants . however , no type i , ii or v myosins have been found in any plant and only two types ( viii and xi ) have been identified . class xi are somewhat similar to class v myosins and this may explain the reaction with the type v antibody . phylogenetic analysis of arabidopsis myosins along with other plant myosins suggests that most class xi myosins ( except three ) fall into two subgroups ( figure 4 ) . the arabidopsis myosins have anywhere from three to six iq domains . the iq domain in non - plant myosins the regulation of myosin action is thought to be due to calmodulin interaction . in plants , two myosin heavy chains have been shown to associate with calmodulin [ 37 , 67 ] . a myosin - containing protein fraction from tobacco by2 cells was used in motility assays with f - actin . concentrations of ca higher than 10 m caused a significant reduction in f - actin sliding . another study with myosin isolated from lily pollen , also demonstrated a co - precipitation of myosin and calmodulin and a similar effect of caconcentration . not only did concentrations above 10 m cause inhibition of myosin activity , but the effects of concentrations higher than 10 m were not reversible upon ca removal . these studies provide evidence that plant myosins bind calmodulin in the absence of ca and are active when calmodulin is bound and inactivated when the ca concentration is increased . they also found that when the myosin fraction was pretreated with cacl2 calmodulin did not bind the myosin , suggesting that calmodulin dissociates from myosin at high concentrations of ca . the myosins in the above studies have not been cloned , and binding to specific iq domains has not been established . however , the presence of iq domains in arabidopsis and other plant myosins suggests that these are the sites of ca regulation . it would be interesting to investigate the possible phosphorylation of the threonine residue which is three residues upstream from the teds rule site in class xi myosins and to see if enzyme activity is regulated by phosphorylation of this residue . they have been shown to be involved in movement , translocation , cell division , organelle transport , g - protein - linked signal cascade and maintenance of structure within cells . cytoplasmic streaming is responsible for movement of organelles and vesicles and of generative cells and vegetative nuclei in pollen tubes . physiological studies in chara have shown that an increase in ca concentration causes cytoplasmic streaming to stop . a myosin isolated from the alga chara corallina was shown to be responsible for cytoplasmic streaming [ 30 , 69 , 70 ] . the myosin was cloned and characterized and found to be a class xi myosin related to the arabidopsis mya myosins . using immunofluorescence , myosin was localized to vesicles , organelles and generative cells and vegetative nuclei in grass pollen tubes . a myosin isolated from lily pollen has been shown to be responsible for cytoplasmic streaming in pollen tubes and two myosins were identified in tobacco cell cultures that are also thought to participate in cytoplasmic streaming [ 37 , 71 ] . antibodies to the myosins recognized a protein in vegetative cells as well as pollen tubes . liu et al . suggest that class xi myosins are likely candidates for transport of large vesicles because of the number of iq domains ( 5 - 6 ) . previous studies showed that translocational step size produced by a myosin motor is proportional to the number of iq domains and the larger the step the faster or more efficiently they are able to transport vesicles . however , the kinetic properties of the motor domain are also involved in speed and there is a wide range of movement speeds for myosin ii molecules [ 2 , 72 , 73 ] . an antibody specific to a z. mays class xi myosin was used to localize this myosin in fractions of maize proteins and maize root tip cells . the nuclear / cell wall fraction and the plastid fraction contained relatively small amounts of antigen while the mitochondrial fraction and the low density membrane fraction had most of the antigen . the root tip cells showed particulate staining in the cytoplasm , but neither the vacuole membrane nor plasma membrane were stained , although in some cells the staining was too bright to distinguish if the plasma membrane was stained or not . there are 13 class xi myosins in arabidopsis that could be involved in vesicle and organelle transport . patterns of expression were different for the cloned z. mays and arabidopsis myosins that have been analyzed [ 42 , 51 ] . plasmodesmata are interconnections between contiguous plant cells that allow direct cell - to - cell transport of ions and proteins . a recent study using an antibody to a cloned class viii arabidopsis myosin atm1 ( at atm ) localized this myosin to the plasmodesmata and the plasma membrane regions involved in the assembly of new cell walls . other studies using antibodies to animal myosins in root tissues of allium cepa , z. mays and hordeum vulagare have also indicated the presence of myosin in the plasmodesmata . however , immunolocalization studies with antibodies to animal myosins need to be interpreted with caution as there are no plant myosins that group with animal myosins . the myosin was localized mainly to the transverse walls with some punctate labeling of the longitudinal walls . during cell division the anti - class - viii myosin staining remains confined to the transverse cell walls and is strongest in the newly formed cell wall . immunogold electron microscopy showed labeling of class viii myosin associated with the plasma membrane and plasmodesmata . these studies suggest that class viii myosins may be involved in new cell wall formation and transport in the plasmodesmata . . suggest that myosin viii could act to bring islands of membrane plate material together or could trigger exocytosis of new cell wall material , or alternatively as an anchor for actin along the transverse walls . the role of myosin in the plasmodesmata was studied further by pretreating tissue with 2,3-butanedione 2-moxoxime ( bdm ) , an inhibitor of actin - myosin motility . myosin viii in the plasmodesmata could be a part of a gating complex that is thought to control the opening of the plasmodesma neck . there are four class viii myosins in arabidopsis that could be involved in these types of functions . a recent study of the effect of bdm on the distribution of myosins , f - actin , microtubules and cortical endoplasmic reticulum ( er ) suggests that myosins may link together microtubules and actin filaments involved in structural interactions . this study used antibody to myosin ii from animals and arabidopsis myosin viii for immunofluorescence studies . bdm treatment disrupted normal cellular distributions of maize myosins and the characteristic distribution of f - actin was also affected . myosin may participate in the intracellular distribution of actin filaments as was proposed for myosin xv . microtubule arrangements in cortical root cells were altered , as was the normal er network . post - mitotic cell growth was inhibited by bdm , specifically in the transition zone and the apical parts of the elongation region . the study suggested that actin fibers and microtubules interact together via myosins and that myosin - based contractility of the actin cytoskeleton is essential for the developmental progression of root cells . however , bdm has only been shown to inhibit a few myosins in vitro and is known to be a nonspecific inhibitor ; so these results must be viewed with caution . as the classification system of myosins now stands , plant myosins fall only into two classes - class viii and class xi . all animal cells examined contain at least one myosin ii gene and usually multiple myosin i genes , but this is not true for arabidopsis specifically and possibly for all plants . plant and animal cells have some common tasks such as vesicular and organelle movement , but plant cells are unique in many ways and the presence of specific plant myosins is probably a reflection of that uniqueness the large number of myosins in class xi could be the result of gene duplication or specialization of function in different tissues or different life cycle times . this work identifies the arabidopsis myosins , their domains and gene intron / exon structure . the task ahead is to analyze the protein products biochemically and try to establish the function of each myosin . using the conserved motor domain of the plant myosin at mya1 database searches were performed using blastp and tblastn at tair . the sequences were evaluated for the presence of a myosin motor domain using the smart program . all sequences with a myosin domain had blastp scores greater than 100 and e values less than 10 . the motor domains of representative myosins from other groups were also used to search the arabidopsis domain but the searches did not reveal any new myosin genes . the smart program also identified the iq and coiled - coil domains and the location of the domains . the sizes of the exons / introns were determined using the exon / intron data for each myosin sequence using the mips predictions for myosins not previously cloned . two sequences ( at xif , at xih ) were edited by comparing the upstream genome sequence translation to conserved sequences present in the other myosins but missing in the predicted sequences . sequences of myosins other than the arabidopsis myosins for phylogenetic analysis were obtained from mhp or ncbi . the motor domain sequences were used for alignment of the plant and non - plant myosins using the megalign program . the alignment was saved as a paup file and the phylogenetic analysis was done using paup 4.0b4a ( ppc ) . full - length sequences were used for analysis of the plant myosins using the same methods as above . this work was supported in part by grants from the national science foundation ( mcb-0079938 ) and nasa to a.s.n.r .

backgroundthree types of molecular motors play an important role in the organization , dynamics and transport processes associated with the cytoskeleton . the myosin family of molecular motors move cargo on actin filaments , whereas kinesin and dynein motors move cargo along microtubules . these motors have been highly characterized in non - plant systems and information is becoming available about plant motors . the actin cytoskeleton in plants has been shown to be involved in processes such as transportation , signaling , cell division , cytoplasmic streaming and morphogenesis . the role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number , types and roles of myosins in plants.resultsusing the motor domain of an arabidopsis myosin we identified 17 myosin sequences in the arabidopsis genome . phylogenetic analysis of the arabidopsis myosins with non - plant and plant myosins revealed that all the arabidopsis myosins and other plant myosins fall into two groups - class viii and class xi . these groups contain exclusively plant or algal myosins with no animal or fungal myosins . exon / intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication.conclusionsplant myosins are unlike myosins from any other organisms except algae . as a percentage of the total gene number , the number of myosins is small overall in arabidopsis compared with the other sequenced eukaryotic genomes . there are , however , a large number of class xi myosins . the function of each myosin has yet to be determined .

Background Results Identification of Phylogenetic analysis Characterization of the Discussion Conclusions Materials and methods Acknowledgements

movement of eukaryotic cells , intracellular transport , signaling , cell division and cell shape are functions of the cytoskeleton [ 1 , 2 , 3 , 4 ] . molecular motors in non - plant systems have been extensively characterized but less is known about the presence and functions of these motors in plant cells . kinesins have been identified in arabidopsis and other plant systems [ 12 , 13 , 14 , 15 , 16 ] and their movement along microtubules has been analyzed [ 16 , 17 , 18 , 19 ] . using the amino - acid sequence of the motor domain of a plant kinesin , a search of the arabidopsis genome yielded 61 kinesin - like proteins . phylogenetic analysis of known myosins in various organisms has resulted in the classification of myosins into several groups . however , an analysis of the myosin superfamily in drosophila , concluded that two new mammalian myosins and a drosophila myosin make up a new class of myosins , class xviii . phylogenetic analysis is often based on the motor domain of the myosins . as actin is utilized by myosin for motility , the possible functions of myosin in plants are closely linked to the functions of actin . the actin cytoskeleton has been shown to be involved in many processes in plants including transportation , signaling , cell division , cytoplasmic streaming and morphogenesis [ 2 , 3 ] . much of the cytoplasmic streaming work has been done in algal cells and the direct involvement of actin and myosin has been shown [ 30 , 31 ] . genetic , biochemical and cell biological studies with trichomes during the past four years have provided interesting insights into the role of the cytoskeleton in trichome morphogenesis . localization studies and visualization of the actin cytoskeleton in live cells with an actin - binding protein tagged with green fluorescent protein ( gfp ) indicate that the organization of f - actin changes during trichome morphogenesis [ 33 , 34 ] . there is also evidence that the actin cytoskeleton is involved in mitosis and during separation of daughter cells after the successful segregation of chromosomes into daughter nuclei . the actin cytoskeleton is also involved in pollen tube growth , and calcium regulation has also been shown to be involved [ 35 , 36 ] . two algal myosins are also members of the class xi myosins , one from chara corallina and one from chlamydomonas reinhardtii [ 22 , 54 ] . a third class of myosins ( xiii ) no animal myosins are in any of these classes and no plant myosins are in any other myosin class . what are the similarities and differences between plant and non - plant myosins that might help establish a function for the myosins ? it is now possible to determine how many myosins are in the arabidopsis genome and to see if any plant myosins fall into other myosin classes . we identified 17 arabidopsis myosins , including the 5 reported myosins , in the arabidopsis genome . phylogenetic analysis using non - plant and plant myosins showed that all 17 fall into either myosin class viii or xi . an analysis of their exon / intron junctions and sequence similarities indicates that all myosins are highly conserved and some may represent gene duplication events . the number of myosins in each organism is on the left ( red column ) and the number per thousand for each organism is on the right ( blue column ) . only 5 of the 17 arabidopsis myosins have been reported in the literature [ 41 , 42 , 43 ] . the average sequence length of the arabidopsis myosins is 1,400 residues , with the shortest sequence prediction being 1,085 ( at viiia ) amino acids and the longest 1,730 ( at xia ) . the maps reported with the announcement of the arabidopsis genome sequence show somewhat different lengths than the tair maps . all arabidopsis myosins and a selection of myosins from other organisms representing each of the myosin classes were aligned using the motor domain sequence as determined by the smart program . the arabidopsis myosins all group into two classes along with other plant myosins - class viii and class xi . no animal myosins group with the plant myosins and no plant myosins group into any of the animal myosins . an algal ( chara corallina ) myosin , cc ccm , does group with the plant class xi myosins but is on a separate branch from any other class xi myosin ( figure 3 ) . other phylogenetic anaylses have placed dd myoj as a branch off class xi myosins from plants [ 22 , 59 ] . alignment of the motor domain of representative myosins and all arabidopsis myosins was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the paup 4.0b6 program . among the class xi myosins the similarity ranges from 40 - 85% ( full length ) and 61 - 91% ( motor domain ) . when class viii myosins are compared to class xi myosins the similarity only ranges from 22 - 29% ( full - length ) and 35 - 42% ( motor domain ) . the other subgroup consists of the arabidopsis myosin pair at xic / at xie and two unpaired arabidopsis myosins , at xik and at mya1 , that are most closely related to sunflower myosins hahamy2 and hahamy5 and to the maize myosin zmmyo1 . alignment of the full - length arabidopsis myosins , other full - length plant myosins available in the ncbi database and dd myoj was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the paup 4.0b4a ( ppc ) program . the motor domain starts at about 50 - 55 residues for the class xi myosins whereas the class viii myosins have a longer amino - terminal region before the motor domain ( 99 - 159 residues ) . based on the presence of the coiled - coil domains , the arabidopsis myosins are probably dimeric . the class xi myosins are much longer than the class viii myosins with the difference being in the length of the carboxy - terminal region following the conserved domains found in myosins . these include sh3 domains ( src homology 3 domains , that bind to target proteins ) , myth4 ( a domain of unknown function found in a few classes of myosins ) , a zinc - binding domain , a pleckstrin homology domain , ferm / talin ( band 4.1/ezrin / radixin / moesin ) , gpa - rich domains and a protein kinase domain [ 8 , 22 , 26 ] . except for the iq and coiled - coil domains , the smart program used to identify the motor domain of the myosin sequences did not identify any domains other than a few with scores less significant than the required threshold . suggest that release of the -phosphate of atp may be through a hole in the structure centered around an absolutely conserved arginine residue ( residue 654 , dmyoii ) which is also absolutely conserved in all arabidopsis myosins . however , three residues upstream ( 19 from dalak ) , all the class xi myosins have a threonine residue . the length of each exon and the domain(s ) they code for are shown in tables 2 and 3 for class viii and class xi myosins , respectively . the four class viii myosins have seven exons of the same length in the same order within the myosin motor domain ( table 2 ) . the class xi myosins also have many exons that are of the same length and in the same order but that differ from the class viii pattern ( table 3 ) . however , even in the carboxy - terminal region there are some exon lengths conserved between some or all of the myosins . twenty - three of their introns are at the same location in the motor domain area and then following a few different size exons , there are similar sized exons again . analysis of the total arabidopsis genome revealed that a whole genome duplication occurred , followed by subsequent gene loss and extensive local gene duplications . analysis of exon sizes in class xi myosins and the domain coded by each exon n , amino - terminal sequence ; m , motor domain ; i , iq domain ; c , coiled - coil domain ; u , undefined ; t , tail domain . of the total 557 splice sites that were identified in the arabidopsis myosins only six ( a little more than 1% ) were over 500 nucleotides with four out of the six being in one myosin . several of the introns in the myosins are between 66 and 70 nucleotides and so may be long enough to be spliced . the sequences in the arabidopsis myosins upstream and downstream of these two very conserved sites varied as a reflection of the less conserved nature of these nucleotides ( table 4 ) . only 5 of the 17 arabidopsis myosins have been reported in the literature [ 41 , 42 , 43 ] . the average sequence length of the arabidopsis myosins is 1,400 residues , with the shortest sequence prediction being 1,085 ( at viiia ) amino acids and the longest 1,730 ( at xia ) . all arabidopsis myosins and a selection of myosins from other organisms representing each of the myosin classes were aligned using the motor domain sequence as determined by the smart program . the arabidopsis myosins all group into two classes along with other plant myosins - class viii and class xi . no animal myosins group with the plant myosins and no plant myosins group into any of the animal myosins . an algal ( chara corallina ) myosin , cc ccm , does group with the plant class xi myosins but is on a separate branch from any other class xi myosin ( figure 3 ) . other phylogenetic anaylses have placed dd myoj as a branch off class xi myosins from plants [ 22 , 59 ] . alignment of the motor domain of representative myosins and all arabidopsis myosins was done in megalign by the clustal method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the paup 4.0b6 program . among the class xi myosins the similarity ranges from 40 - 85% ( full length ) and 61 - 91% ( motor domain ) . when class viii myosins are compared to class xi myosins the similarity only ranges from 22 - 29% ( full - length ) and 35 - 42% ( motor domain ) . the other subgroup consists of the arabidopsis myosin pair at xic / at xie and two unpaired arabidopsis myosins , at xik and at mya1 , that are most closely related to sunflower myosins hahamy2 and hahamy5 and to the maize myosin zmmyo1 . the motor domain starts at about 50 - 55 residues for the class xi myosins whereas the class viii myosins have a longer amino - terminal region before the motor domain ( 99 - 159 residues ) . based on the presence of the coiled - coil domains , the arabidopsis myosins are probably dimeric . the class xi myosins are much longer than the class viii myosins with the difference being in the length of the carboxy - terminal region following the conserved domains found in myosins . these include sh3 domains ( src homology 3 domains , that bind to target proteins ) , myth4 ( a domain of unknown function found in a few classes of myosins ) , a zinc - binding domain , a pleckstrin homology domain , ferm / talin ( band 4.1/ezrin / radixin / moesin ) , gpa - rich domains and a protein kinase domain [ 8 , 22 , 26 ] . except for the iq and coiled - coil domains , the smart program used to identify the motor domain of the myosin sequences did not identify any domains other than a few with scores less significant than the required threshold . suggest that release of the -phosphate of atp may be through a hole in the structure centered around an absolutely conserved arginine residue ( residue 654 , dmyoii ) which is also absolutely conserved in all arabidopsis myosins . the arabidopsis and other plant myosins all have aspartic acid , glutamic acid or glycine residue at this site , suggesting that they are not regulated by phosphorylation at this site . however , three residues upstream ( 19 from dalak ) , all the class xi myosins have a threonine residue . the length of each exon and the domain(s ) they code for are shown in tables 2 and 3 for class viii and class xi myosins , respectively . the four class viii myosins have seven exons of the same length in the same order within the myosin motor domain ( table 2 ) . the class xi myosins also have many exons that are of the same length and in the same order but that differ from the class viii pattern ( table 3 ) . however , even in the carboxy - terminal region there are some exon lengths conserved between some or all of the myosins . twenty - three of their introns are at the same location in the motor domain area and then following a few different size exons , there are similar sized exons again . analysis of the total arabidopsis genome revealed that a whole genome duplication occurred , followed by subsequent gene loss and extensive local gene duplications . analysis of exon sizes in class xi myosins and the domain coded by each exon n , amino - terminal sequence ; m , motor domain ; i , iq domain ; c , coiled - coil domain ; u , undefined ; t , tail domain . of the total 557 splice sites that were identified in the arabidopsis myosins only six ( a little more than 1% ) were over 500 nucleotides with four out of the six being in one myosin . several of the introns in the myosins are between 66 and 70 nucleotides and so may be long enough to be spliced . the sequences in the arabidopsis myosins upstream and downstream of these two very conserved sites varied as a reflection of the less conserved nature of these nucleotides ( table 4 ) . phylogenetic analysis of arabidopsis myosins along with other plant myosins suggests that most class xi myosins ( except three ) fall into two subgroups ( figure 4 ) . the iq domain in non - plant myosins the regulation of myosin action is thought to be due to calmodulin interaction . however , the presence of iq domains in arabidopsis and other plant myosins suggests that these are the sites of ca regulation . they have been shown to be involved in movement , translocation , cell division , organelle transport , g - protein - linked signal cascade and maintenance of structure within cells . the myosin was cloned and characterized and found to be a class xi myosin related to the arabidopsis mya myosins . a myosin isolated from lily pollen has been shown to be responsible for cytoplasmic streaming in pollen tubes and two myosins were identified in tobacco cell cultures that are also thought to participate in cytoplasmic streaming [ 37 , 71 ] . suggest that class xi myosins are likely candidates for transport of large vesicles because of the number of iq domains ( 5 - 6 ) . however , the kinetic properties of the motor domain are also involved in speed and there is a wide range of movement speeds for myosin ii molecules [ 2 , 72 , 73 ] . there are 13 class xi myosins in arabidopsis that could be involved in vesicle and organelle transport . a recent study using an antibody to a cloned class viii arabidopsis myosin atm1 ( at atm ) localized this myosin to the plasmodesmata and the plasma membrane regions involved in the assembly of new cell walls . other studies using antibodies to animal myosins in root tissues of allium cepa , z. mays and hordeum vulagare have also indicated the presence of myosin in the plasmodesmata . however , immunolocalization studies with antibodies to animal myosins need to be interpreted with caution as there are no plant myosins that group with animal myosins . these studies suggest that class viii myosins may be involved in new cell wall formation and transport in the plasmodesmata . the role of myosin in the plasmodesmata was studied further by pretreating tissue with 2,3-butanedione 2-moxoxime ( bdm ) , an inhibitor of actin - myosin motility . there are four class viii myosins in arabidopsis that could be involved in these types of functions . a recent study of the effect of bdm on the distribution of myosins , f - actin , microtubules and cortical endoplasmic reticulum ( er ) suggests that myosins may link together microtubules and actin filaments involved in structural interactions . the study suggested that actin fibers and microtubules interact together via myosins and that myosin - based contractility of the actin cytoskeleton is essential for the developmental progression of root cells . however , bdm has only been shown to inhibit a few myosins in vitro and is known to be a nonspecific inhibitor ; so these results must be viewed with caution . as the classification system of myosins now stands , plant myosins fall only into two classes - class viii and class xi . plant and animal cells have some common tasks such as vesicular and organelle movement , but plant cells are unique in many ways and the presence of specific plant myosins is probably a reflection of that uniqueness the large number of myosins in class xi could be the result of gene duplication or specialization of function in different tissues or different life cycle times . the sizes of the exons / introns were determined using the exon / intron data for each myosin sequence using the mips predictions for myosins not previously cloned . the motor domain sequences were used for alignment of the plant and non - plant myosins using the megalign program .

isolated non - compaction of the left ventricle ( lvnc ) is a rare congenital cardiomyopathy . recognized relatively recently , it has been categorized as unclassified cardiomyopathy by the world health organization . it is caused by failure of the developing myocardium to become compact , thereby resulting in a spongy myocardium . isolated lvnc is a rare disorder of endomyocardial morphogenesis characterized by numerous prominent ventricular trabeculations and deep intratrabecular recesses ( 1 ) . during normal embryonic development , endomyocardial trabeculations emerge from the apical region of the primitive ventricles at day 32 of fetal life ( 2 ) , and involute by day 70 through a process of resorption and remodeling . it is thought to result from failure of trabecular compaction of the loose mesh of muscle fibers seen in the early stages of myocardial development . normally this process is more pronounced in the left than in the right ventricle , resulting in a smooth , flat endocardial surface . the normal left ventricle has less than three trabeculations per imaging field , mostly confined to the lateral wall ( 3 ) . in this disorder , however , there are abnormal trabeculations resulting from non - compaction scattered all over the myocardium , but most evident near the attachment of the papillary muscles of the mitral valve ( fig . right ventricular non - compaction may accompany lvnc in < 50% of patients . due to the difficulty in distinguishing normal variants in the highly trabeculated right ventricle from the pathological non - compacted ventricle , several authors dispute the existence of right ventricular non - compaction ( 4 , 5 ) . fig . isolated non - compaction of the ventricular myocardium , first described by chin et al ( 1 ) in 1990 , is characterized by persistent embryonic myocardial morphology found in the absence of other cardiac anomalies to explain the abnormal development . in such cases , the resulting deep recesses communicate with the ventricular cavity only , not the coronary circulation ( 1 ) . there is no specific histological finding in lnvc , although fibrosis has been described in numerous reports ( 6 , 7 ) ( fig . some reports have observed necrotic myocytes within the prominent trabeculations of patients with non - compaction ( 5 , 6 ) . many cases of lvnc are caused by associated anomalies that generate intraventricular pressure overload , as in the case of pulmonary atresia with intact ventricular septum ( 8) or anomalous origin of the left coronary artery from the pulmonary trunk ( 9 ) . in these hearts , the deep recesses are in continuity with the ventricular cavity and with the coronary arteries ; and therefore , are more accurately described as persistent intramyocardial sinusoids . by contrast , lvnc has no associated cardiac lesions , and persistent sinusoids are not seen . the disorder can be sporadic , but familial recurrence and associated dysmorphism has been reported ( 8 , 9 ) . the familial form has a heterogenous mode of inheritance . in the x - linked form of the disease , the locus has been found on q28 , and mutations have been reported in the g4.5 gene ( 10 , 11 ) . this gene is in close proximity to other genes responsible for myopathies , and a number of patients with hypertrabeculations have been found to suffer from neuromuscular disorders such as emery - dreifuss muscular dystrophy , barth s syndrome , becker s muscular dystrophy , metabolic myopathies , and roifmann syndrome ( 12 ) , skeletal abnormalities such as melnick - needles syndrome and nail - patella syndrome ( 13 ) . the facial dysmorphism consists of prominent forehead , strabismus , gothic palate or micrognathia . in children , lvnc can occur in barth s syndrome , a rare x - linked multi - system disorder caused by a mutation in the g4.5 gene that encodes the tafazzin family of proteins ( 14 , 15 ) . mutations in this gene in adult lvnc are rare , however , but mutations have been described in adult lvnc in the genes encoding a - dystrobrevin and cypher / zasp ( 14 , 16 ) , integral parts of the complex that link the extracellular matrix of the myocardial cell to the cytoskeleton . in the initial case series of isolated non - compaction ( 1 ) , the median age at diagnosis was 7 yrs ( range 11 months to 22 yrs ) . subsequent case reports have described this finding in adults , including the elderly ( 1 , 5 , 1719 ) . in the largest series of patients with lvnc ( 5 ) , the true prevalence is unclear since the correct diagnosis is often missed or delayed due to a lack of knowledge concerning this uncommon disease , and its similarity to other diseases of the myocardium and endocardium . men appear to be affected more than women , accounting for 5682% of cases in the largest reported series of lvnc ( 1 , 5 , 20 , 21 ) . severe systolic heart failure and increased end - diastolic pressure with restrictive cardiomyopathy are seen in > 50% of patients . bundle branch block , atrial arrhythmias and other serious ventricular arrhythmias occur in 40% of cases and can cause sudden cardiac death ( 11 ) . diastolic dysfunction in ventricular non - compaction can be related to both abnormal relaxation and restrictive filling caused by the numerous prominent trabeculae ( 22 ) . the origin of systolic dysfunction in non - compaction is unclear , but there is increasing evidence that subendocardial hypoperfusion and microcirculatory dysfunction play a role in ventricular dysfunction and arrhythmogenesis . chin et al ( 1 ) suggested that subendocardial perfusion might be abnormal in lvnc despite the absence of epicardial coronary artery disease . due to the numerous prominent trabeculae , subendocardial ischemia can be the result of isometric contraction of the endocardium and myocardium within the deep intertrabecular recesses . subendocardial perfusion defects have been described in lvnc using cardiac magnetic resonance imaging ( mri ) ( fig . positron emission tomography ( pet ) ( 24 ) and scintigraphy with thallium-201 ( 21 ) have shown transmural perfusion defects correlating with areas of non - compacted myocardium in lvnc . abnormalities in resting ecg are found in the majority of patients with lvnc , but findings are non - specific and include left ventricular ( lv ) hypertrophy , repolarization changes , inverted t waves , st segment changes , axis shifts , intraventricular conduction abnormalities and av block ( 1 , 5 , 20 , 21 ) . electrocardiographic findings of the wolff - parkinson - white syndrome have been described in up to 15% of pediatric patients ( 21 , 25 ) , but it was not observed in the two largest series of adults with isolated non - compaction ( 5 , 20 ) . arrhythmias are common in patients with ventricular non - compaction . atrial fibrillation has been reported in over 25% of adults with lvnc ( 5 , 20 ) . sudden cardiac death accounted for half of the deaths in the larger series of patients with lvnc ( 1 , 20 ) . although ventricular arrhythmias occurred in nearly 40% of patients in the initial description of lvnc by chin et al ( 1 ) , ichida et al ( 21 ) described no cases of ventricular tachycardia or sudden death in the largest series of pediatric patients with lvnc . paroxysmal supraventricular tachycardia and complete heart block have also been reported in patients with lvnc ( 20 , 21 ) . the occurrence of thromboembolic events , including cerebrovascular accident , transient ischemic attack , pulmonary embolism and mesenteric infarction , ranged from 2138% ( 5 , 20 ) . embolic complications may be related to the development of thrombi in the extensively trabeculated ventricle , depressed systolic function , or the development of atrial fibrillation ( 20 ) . interestingly , no systemic embolic events were reported in the largest pediatric series with lvnc ( 21 ) . an association between non - compaction and neuromuscular disorders has also been described ( 3 , 26 ) , as many as 82% of patients having some form of neuromuscular disorder . chin et al described an association between lvnc and facial dysmorphisms , including a prominent forehead , strabismus , high - arching palate , and micrognathia ( 1 ) . the combined echocardiographic features essential for making a diagnosis include : a two - layered ventricular myocardium consisting of an subendocardial compact layer and a thick non - compact endocardial layer with prominent trabeculations and intratrabecular recesses ; continuity between the lv cavity and the recesses with blood flowing in and out of the ventricular cavity and absence of secondary causes of increased trabeculations ( fig . 4 ) ( 13 ) . there is continuity between the lv cavity and the deep intratrabecular recesses that are filled with blood from the ventricular cavity , without evidence of communication with the epicardial coronary artery system ( 1 , 20 ) . the lv apical and inferior wall segments were involved in all patients in an adult population with lvnc studied by echocardiography ( 20 ) . the right ventricular apex was involved in 41% of patients . in the largest series of patients with lvnc ( 5 ) , in addition to the apical and mid - ventricular inferior wall segments , hypokinesis was observed occasionally in normally compacted segments as well as in the non - compacted segments of the left ventricle ( 5 ) , which may correlate with the observation of microcirculatory dysfunction in both non - compacted and normal segments in patients with lvnc . chin et al ( 1 ) described a quantitative approach for diagnosing non - compaction using a trabeculation peak to trough ratio , but it has not been used widely in clinical practice . oechslin et al ( 5 ) and jenni et al ( 4 ) described the abnormally thickened myocardium as a two - layered structure , with a normally compacted epicardial layer and a thickened endocardial layer . they proposed a quantitative evaluation for the diagnosis of lvnc by determining the ratio of maximal thickness of the non - compacted to compacted layers ( measured at end systole in a parasternal short axis view ) , with a ratio of 2.0 diagnostic of lvnc . this technique allowed the differentiation of the trabeculations of lvnc from that observed with dilated cardiomyopathy or hypertensive cardiomyopathy ( 4 ) . based on echocardiographic studies , its prevalence has been estimated at 0.05% in the general population ( 20 ) , and the finding of a ratio of 2.0 between the thickness of the non - compacted and compacted myocardial layers in systole is considered diagnostic ( 4 ) . contrast echocardiography can be helpful when standard echocardiographic image quality is limited or the diagnosis is questionable ( 27 ) . prominent trabeculations , apical hypertrophic cardiomyopathy , dilated cardiomyopathy , arrhythmogenic right ventricular dysplasia , endocardial fibroelastosis , cardiac metastasis , and lv thrombus are important differential diagnostic considerations . echocardiography can easily diagnose isolated ventricular non - compaction if the echocardiographer is familiar with this congenital disorder , and if clear - cut diagnostic criteria are used . due to the risk of familial occurrence based on the ratio of end - diastolic thickness of the non - compacted to compacted layers of the myocardium , cmr imaging is accurate when diagnosing pathological lvnc ( 28 ) . the literature supports the clinical use of cmr in diagnosing lvnc , especially for patients with poor echocardiographic windows ( 29 ) . ventriculography and computed tomography can also show the typical morphological features of non - compaction : pathognomonic combination of multiple prominent ventricular trabeculations and of multiple deep intertrabecular recesses communicating with the ventricular cavity . the current literature suggests that lvnc in adults is rare and associated with a poor prognosis ( 5 , 30 ) . in the largest series to date , 48% of patients died or underwent a heart transplant over a period of 44 months ( 5 ) . prognosis in the asymptomatic patients was clearly better than in the symptomatic patients ( 21 ) . the prognosis depends on the severity and is generally grim due to cardiovascular complications such as congestive failure ( heart failure was caused by systolic and diastolic dysfunction ) , shock , arrhythmias and fatal thromboembolic events . the high prevalence of thromboembolic events ( 24% of patients ) was consistent and was independent of lv size or function ( 1 ) . the deep recesses can aggravate the risk of thrombus formation and be an additional factor for this serious complication . the hypertrophic segments are perfused via the epicardial coronary arteries , which have no continuity with the deep recesses communicating with the lv cavity . thickened endocardium and ischemic lesions in prominent trabeculae surrounded by deep trabecular recesses were documented in histologic specimens , which could be caused by ischemia ( 20 ) . indeed , pet demonstrated restricted myocardial perfusion in areas of lvnc ( 24 ) . hypothetically , both morphology and vasomotion of the coronary vessels feeding the hypertrophic segments can be abnormal with subsequent ischemia . progressive ischemia and subsequent scar tissue can be an arrhythmogenic substrate for ventricular arrhythmias because the well - defined morphologic substrate of lvnc can not be considered inherently arrhythmogenic . in severe cases , a heart transplant may be the only option ( 1 ) . there is no specific option for the treatment of non - compaction cardiomyopathy , which is the same as for heart failure . some findings have pointed to the efficacy of carvedilol in improving lv function , hypertrophy , and both metabolic and adrenergic abnormalities in isolated lvnc ( 31 ) . cardiac transplantation has been used for those with refractory congestive heart failure . a more aggressive approach to the diagnosis and treatment of ventricular arrhythmias may be justified , and assessment for atrial and ventricular arrhythmias by ambulatory ecg monitoring should be performed annually . as more information is gathered about lvnc and the risk of sudden cardiac death , implantable defibrillator technology could have an increased role . biventricular pacemakers could play a role in the treatment of lvnc patients with heart failure , reduced lv function , and prolonged intraventricular conduction . prophylactic anticoagulation may be warranted because of the higher risk of thrombus formation within the intratrabecular recesses . isolated lvnc is a rare disorder of endomyocardial morphogenesis characterized by numerous prominent ventricular trabeculations and deep intratrabecular recesses ( 1 ) . during normal embryonic development , endomyocardial trabeculations emerge from the apical region of the primitive ventricles at day 32 of fetal life ( 2 ) , and involute by day 70 through a process of resorption and remodeling . it is thought to result from failure of trabecular compaction of the loose mesh of muscle fibers seen in the early stages of myocardial development . normally this process is more pronounced in the left than in the right ventricle , resulting in a smooth , flat endocardial surface . the normal left ventricle has less than three trabeculations per imaging field , mostly confined to the lateral wall ( 3 ) . in this disorder , however , there are abnormal trabeculations resulting from non - compaction scattered all over the myocardium , but most evident near the attachment of the papillary muscles of the mitral valve ( fig . right ventricular non - compaction may accompany lvnc in < 50% of patients . due to the difficulty in distinguishing normal variants in the highly trabeculated right ventricle from the pathological non - compacted ventricle , several authors dispute the existence of right ventricular non - compaction ( 4 , 5 ) . isolated non - compaction of the ventricular myocardium , first described by chin et al ( 1 ) in 1990 , is characterized by persistent embryonic myocardial morphology found in the absence of other cardiac anomalies to explain the abnormal development . in such cases , the resulting deep recesses communicate with the ventricular cavity only , not the coronary circulation ( 1 ) . there is no specific histological finding in lnvc , although fibrosis has been described in numerous reports ( 6 , 7 ) ( fig . some reports have observed necrotic myocytes within the prominent trabeculations of patients with non - compaction ( 5 , 6 ) . . many cases of lvnc are caused by associated anomalies that generate intraventricular pressure overload , as in the case of pulmonary atresia with intact ventricular septum ( 8) or anomalous origin of the left coronary artery from the pulmonary trunk ( 9 ) . in these hearts , the deep recesses are in continuity with the ventricular cavity and with the coronary arteries ; and therefore , are more accurately described as persistent intramyocardial sinusoids . by contrast , lvnc has no associated cardiac lesions , and persistent sinusoids are not seen . the disorder can be sporadic , but familial recurrence and associated dysmorphism has been reported ( 8 , 9 ) . the familial form has a heterogenous mode of inheritance . in the x - linked form of the disease , the locus has been found on q28 , and mutations have been reported in the g4.5 gene ( 10 , 11 ) . this gene is in close proximity to other genes responsible for myopathies , and a number of patients with hypertrabeculations have been found to suffer from neuromuscular disorders such as emery - dreifuss muscular dystrophy , barth s syndrome , becker s muscular dystrophy , metabolic myopathies , and roifmann syndrome ( 12 ) , skeletal abnormalities such as melnick - needles syndrome and nail - patella syndrome ( 13 ) . the facial dysmorphism consists of prominent forehead , strabismus , gothic palate or micrognathia . in children , lvnc can occur in barth s syndrome , a rare x - linked multi - system disorder caused by a mutation in the g4.5 gene that encodes the tafazzin family of proteins ( 14 , 15 ) . mutations in this gene in adult lvnc are rare , however , but mutations have been described in adult lvnc in the genes encoding a - dystrobrevin and cypher / zasp ( 14 , 16 ) , integral parts of the complex that link the extracellular matrix of the myocardial cell to the cytoskeleton . in the initial case series of isolated non - compaction ( 1 ) , the median age at diagnosis was 7 yrs ( range 11 months to 22 yrs ) . subsequent case reports have described this finding in adults , including the elderly ( 1 , 5 , 1719 ) . in the largest series of patients with lvnc ( 5 ) , the true prevalence is unclear since the correct diagnosis is often missed or delayed due to a lack of knowledge concerning this uncommon disease , and its similarity to other diseases of the myocardium and endocardium . men appear to be affected more than women , accounting for 5682% of cases in the largest reported series of lvnc ( 1 , 5 , 20 , 21 ) . clinical severity depends on the extent of non - compacted cardiac segments . severe systolic heart failure and increased end - diastolic pressure with restrictive cardiomyopathy bundle branch block , atrial arrhythmias and other serious ventricular arrhythmias occur in 40% of cases and can cause sudden cardiac death ( 11 ) . diastolic dysfunction in ventricular non - compaction can be related to both abnormal relaxation and restrictive filling caused by the numerous prominent trabeculae ( 22 ) . the origin of systolic dysfunction in non - compaction is unclear , but there is increasing evidence that subendocardial hypoperfusion and microcirculatory dysfunction play a role in ventricular dysfunction and arrhythmogenesis . chin et al ( 1 ) suggested that subendocardial perfusion might be abnormal in lvnc despite the absence of epicardial coronary artery disease . due to the numerous prominent trabeculae , subendocardial ischemia can be the result of isometric contraction of the endocardium and myocardium within the deep intertrabecular recesses . subendocardial perfusion defects have been described in lvnc using cardiac magnetic resonance imaging ( mri ) ( fig . 3 ) ( 23 ) . positron emission tomography ( pet ) ( 24 ) and scintigraphy with thallium-201 ( 21 ) have shown transmural perfusion defects correlating with areas of non - compacted myocardium in lvnc . abnormalities in resting ecg are found in the majority of patients with lvnc , but findings are non - specific and include left ventricular ( lv ) hypertrophy , repolarization changes , inverted t waves , st segment changes , axis shifts , intraventricular conduction abnormalities and av block ( 1 , 5 , 20 , 21 ) . electrocardiographic findings of the wolff - parkinson - white syndrome have been described in up to 15% of pediatric patients ( 21 , 25 ) , but it was not observed in the two largest series of adults with isolated non - compaction ( 5 , 20 ) . atrial fibrillation has been reported in over 25% of adults with lvnc ( 5 , 20 ) . sudden cardiac death accounted for half of the deaths in the larger series of patients with lvnc ( 1 , 20 ) . although ventricular arrhythmias occurred in nearly 40% of patients in the initial description of lvnc by chin et al ( 1 ) , ichida et al ( 21 ) described no cases of ventricular tachycardia or sudden death in the largest series of pediatric patients with lvnc . paroxysmal supraventricular tachycardia and complete heart block have also been reported in patients with lvnc ( 20 , 21 ) . the occurrence of thromboembolic events , including cerebrovascular accident , transient ischemic attack , pulmonary embolism and mesenteric infarction , ranged from 2138% ( 5 , 20 ) . embolic complications may be related to the development of thrombi in the extensively trabeculated ventricle , depressed systolic function , or the development of atrial fibrillation ( 20 ) . interestingly , no systemic embolic events were reported in the largest pediatric series with lvnc ( 21 ) . an association between non - compaction and neuromuscular disorders has also been described ( 3 , 26 ) , as many as 82% of patients having some form of neuromuscular disorder . chin et al described an association between lvnc and facial dysmorphisms , including a prominent forehead , strabismus , high - arching palate , and micrognathia ( 1 ) . the combined echocardiographic features essential for making a diagnosis include : a two - layered ventricular myocardium consisting of an subendocardial compact layer and a thick non - compact endocardial layer with prominent trabeculations and intratrabecular recesses ; continuity between the lv cavity and the recesses with blood flowing in and out of the ventricular cavity and absence of secondary causes of increased trabeculations ( fig . 4 ) ( 13 ) . there is continuity between the lv cavity and the deep intratrabecular recesses that are filled with blood from the ventricular cavity , without evidence of communication with the epicardial coronary artery system ( 1 , 20 ) . the lv apical and inferior wall segments were involved in all patients in an adult population with lvnc studied by echocardiography ( 20 ) . the right ventricular apex was involved in 41% of patients . in the largest series of patients with lvnc ( 5 ) , in addition to the apical and mid - ventricular inferior wall segments , hypokinesis was observed occasionally in normally compacted segments as well as in the non - compacted segments of the left ventricle ( 5 ) , which may correlate with the observation of microcirculatory dysfunction in both non - compacted and normal segments in patients with lvnc . chin et al ( 1 ) described a quantitative approach for diagnosing non - compaction using a trabeculation peak to trough ratio , but it has not been used widely in clinical practice . oechslin et al ( 5 ) and jenni et al ( 4 ) described the abnormally thickened myocardium as a two - layered structure , with a normally compacted epicardial layer and a thickened endocardial layer . they proposed a quantitative evaluation for the diagnosis of lvnc by determining the ratio of maximal thickness of the non - compacted to compacted layers ( measured at end systole in a parasternal short axis view ) , with a ratio of 2.0 diagnostic of lvnc . this technique allowed the differentiation of the trabeculations of lvnc from that observed with dilated cardiomyopathy or hypertensive cardiomyopathy ( 4 ) . based on echocardiographic studies , its prevalence has been estimated at 0.05% in the general population ( 20 ) , and the finding of a ratio of 2.0 between the thickness of the non - compacted and compacted myocardial layers in systole is considered diagnostic ( 4 ) . contrast echocardiography can be helpful when standard echocardiographic image quality is limited or the diagnosis is questionable ( 27 ) . prominent trabeculations , apical hypertrophic cardiomyopathy , dilated cardiomyopathy , arrhythmogenic right ventricular dysplasia , endocardial fibroelastosis , cardiac metastasis , and lv thrombus are important differential diagnostic considerations . echocardiography can easily diagnose isolated ventricular non - compaction if the echocardiographer is familiar with this congenital disorder , and if clear - cut diagnostic criteria are used . due to the risk of familial occurrence based on the ratio of end - diastolic thickness of the non - compacted to compacted layers of the myocardium , cmr imaging is accurate when diagnosing pathological lvnc ( 28 ) . the literature supports the clinical use of cmr in diagnosing lvnc , especially for patients with poor echocardiographic windows ( 29 ) . ventriculography and computed tomography can also show the typical morphological features of non - compaction : pathognomonic combination of multiple prominent ventricular trabeculations and of multiple deep intertrabecular recesses communicating with the ventricular cavity . the current literature suggests that lvnc in adults is rare and associated with a poor prognosis ( 5 , 30 ) . in the largest series to date , 48% of patients died or underwent a heart transplant over a period of 44 months ( 5 ) . prognosis in the asymptomatic patients was clearly better than in the symptomatic patients ( 21 ) . the prognosis depends on the severity and is generally grim due to cardiovascular complications such as congestive failure ( heart failure was caused by systolic and diastolic dysfunction ) , shock , arrhythmias and fatal thromboembolic events . the high prevalence of thromboembolic events ( 24% of patients ) was consistent and was independent of lv size or function ( 1 ) . the deep recesses can aggravate the risk of thrombus formation and be an additional factor for this serious complication . the hypertrophic segments are perfused via the epicardial coronary arteries , which have no continuity with the deep recesses communicating with the lv cavity . thickened endocardium and ischemic lesions in prominent trabeculae surrounded by deep trabecular recesses were documented in histologic specimens , which could be caused by ischemia ( 20 ) . indeed , pet demonstrated restricted myocardial perfusion in areas of lvnc ( 24 ) . hypothetically , both morphology and vasomotion of the coronary vessels feeding the hypertrophic segments can be abnormal with subsequent ischemia . progressive ischemia and subsequent scar tissue can be an arrhythmogenic substrate for ventricular arrhythmias because the well - defined morphologic substrate of lvnc can not be considered inherently arrhythmogenic . in severe cases , a heart transplant may be the only option ( 1 ) . there is no specific option for the treatment of non - compaction cardiomyopathy , which is the same as for heart failure . some findings have pointed to the efficacy of carvedilol in improving lv function , hypertrophy , and both metabolic and adrenergic abnormalities in isolated lvnc ( 31 ) . cardiac transplantation has been used for those with refractory congestive heart failure . a more aggressive approach to the diagnosis and treatment of ventricular arrhythmias may be justified , and assessment for atrial and ventricular arrhythmias by ambulatory ecg monitoring should be performed annually . as more information is gathered about lvnc and the risk of sudden cardiac death , implantable defibrillator technology could have an increased role . biventricular pacemakers could play a role in the treatment of lvnc patients with heart failure , reduced lv function , and prolonged intraventricular conduction . prophylactic anticoagulation may be warranted because of the higher risk of thrombus formation within the intratrabecular recesses .

non - compaction of the left ventricle ( lvnc ) is a disorder of endomyocardial morphogenesis that results in multiple trabeculations in the left ventricular ( lv ) myocardium . this rare disorder is characterized by an excessively prominent trabecular meshwork and deep intratrabecular recesses . this idiopathic cardiomyopathy is characterized by an altered structure of the myocardial wall as a result of intrauterine arrest of compaction of the myocardial fibers in the absence of any coexisting congenital lesion . it can be associated with neuromuscular disorders and can co - exist with other cardiac malformations , and it is accompanied by depressed ventricular function , systemic embolism and ventricular arrhythmia . echocardiography is the method of choice for diagnosing lvnc , but the correct diagnosis is often missed or delayed due to a lack of knowledge concerning this uncommon disease and its similarity to other diseases of the myocardium and endocardium . there is a two - layered structure of the myocardial wall consisting of a thin compacted epicardial layer and a thick non - compacted endocardial layer with prominent trabeculations and deep recesses .

INTRODUCTION Embryology and development Genetics Epidemiology Clinical features Diagnosis Prognosis Therapy

isolated non - compaction of the left ventricle ( lvnc ) is a rare congenital cardiomyopathy . it is caused by failure of the developing myocardium to become compact , thereby resulting in a spongy myocardium . isolated lvnc is a rare disorder of endomyocardial morphogenesis characterized by numerous prominent ventricular trabeculations and deep intratrabecular recesses ( 1 ) . during normal embryonic development , endomyocardial trabeculations emerge from the apical region of the primitive ventricles at day 32 of fetal life ( 2 ) , and involute by day 70 through a process of resorption and remodeling . it is thought to result from failure of trabecular compaction of the loose mesh of muscle fibers seen in the early stages of myocardial development . normally this process is more pronounced in the left than in the right ventricle , resulting in a smooth , flat endocardial surface . in this disorder , however , there are abnormal trabeculations resulting from non - compaction scattered all over the myocardium , but most evident near the attachment of the papillary muscles of the mitral valve ( fig . right ventricular non - compaction may accompany lvnc in < 50% of patients . due to the difficulty in distinguishing normal variants in the highly trabeculated right ventricle from the pathological non - compacted ventricle , several authors dispute the existence of right ventricular non - compaction ( 4 , 5 ) . isolated non - compaction of the ventricular myocardium , first described by chin et al ( 1 ) in 1990 , is characterized by persistent embryonic myocardial morphology found in the absence of other cardiac anomalies to explain the abnormal development . some reports have observed necrotic myocytes within the prominent trabeculations of patients with non - compaction ( 5 , 6 ) . many cases of lvnc are caused by associated anomalies that generate intraventricular pressure overload , as in the case of pulmonary atresia with intact ventricular septum ( 8) or anomalous origin of the left coronary artery from the pulmonary trunk ( 9 ) . the disorder can be sporadic , but familial recurrence and associated dysmorphism has been reported ( 8 , 9 ) . in the x - linked form of the disease , the locus has been found on q28 , and mutations have been reported in the g4.5 gene ( 10 , 11 ) . this gene is in close proximity to other genes responsible for myopathies , and a number of patients with hypertrabeculations have been found to suffer from neuromuscular disorders such as emery - dreifuss muscular dystrophy , barth s syndrome , becker s muscular dystrophy , metabolic myopathies , and roifmann syndrome ( 12 ) , skeletal abnormalities such as melnick - needles syndrome and nail - patella syndrome ( 13 ) . mutations in this gene in adult lvnc are rare , however , but mutations have been described in adult lvnc in the genes encoding a - dystrobrevin and cypher / zasp ( 14 , 16 ) , integral parts of the complex that link the extracellular matrix of the myocardial cell to the cytoskeleton . in the initial case series of isolated non - compaction ( 1 ) , the median age at diagnosis was 7 yrs ( range 11 months to 22 yrs ) . in the largest series of patients with lvnc ( 5 ) , the true prevalence is unclear since the correct diagnosis is often missed or delayed due to a lack of knowledge concerning this uncommon disease , and its similarity to other diseases of the myocardium and endocardium . diastolic dysfunction in ventricular non - compaction can be related to both abnormal relaxation and restrictive filling caused by the numerous prominent trabeculae ( 22 ) . the origin of systolic dysfunction in non - compaction is unclear , but there is increasing evidence that subendocardial hypoperfusion and microcirculatory dysfunction play a role in ventricular dysfunction and arrhythmogenesis . chin et al ( 1 ) suggested that subendocardial perfusion might be abnormal in lvnc despite the absence of epicardial coronary artery disease . due to the numerous prominent trabeculae , subendocardial ischemia can be the result of isometric contraction of the endocardium and myocardium within the deep intertrabecular recesses . positron emission tomography ( pet ) ( 24 ) and scintigraphy with thallium-201 ( 21 ) have shown transmural perfusion defects correlating with areas of non - compacted myocardium in lvnc . abnormalities in resting ecg are found in the majority of patients with lvnc , but findings are non - specific and include left ventricular ( lv ) hypertrophy , repolarization changes , inverted t waves , st segment changes , axis shifts , intraventricular conduction abnormalities and av block ( 1 , 5 , 20 , 21 ) . electrocardiographic findings of the wolff - parkinson - white syndrome have been described in up to 15% of pediatric patients ( 21 , 25 ) , but it was not observed in the two largest series of adults with isolated non - compaction ( 5 , 20 ) . arrhythmias are common in patients with ventricular non - compaction . sudden cardiac death accounted for half of the deaths in the larger series of patients with lvnc ( 1 , 20 ) . an association between non - compaction and neuromuscular disorders has also been described ( 3 , 26 ) , as many as 82% of patients having some form of neuromuscular disorder . the combined echocardiographic features essential for making a diagnosis include : a two - layered ventricular myocardium consisting of an subendocardial compact layer and a thick non - compact endocardial layer with prominent trabeculations and intratrabecular recesses ; continuity between the lv cavity and the recesses with blood flowing in and out of the ventricular cavity and absence of secondary causes of increased trabeculations ( fig . there is continuity between the lv cavity and the deep intratrabecular recesses that are filled with blood from the ventricular cavity , without evidence of communication with the epicardial coronary artery system ( 1 , 20 ) . in the largest series of patients with lvnc ( 5 ) , in addition to the apical and mid - ventricular inferior wall segments , hypokinesis was observed occasionally in normally compacted segments as well as in the non - compacted segments of the left ventricle ( 5 ) , which may correlate with the observation of microcirculatory dysfunction in both non - compacted and normal segments in patients with lvnc . chin et al ( 1 ) described a quantitative approach for diagnosing non - compaction using a trabeculation peak to trough ratio , but it has not been used widely in clinical practice . oechslin et al ( 5 ) and jenni et al ( 4 ) described the abnormally thickened myocardium as a two - layered structure , with a normally compacted epicardial layer and a thickened endocardial layer . they proposed a quantitative evaluation for the diagnosis of lvnc by determining the ratio of maximal thickness of the non - compacted to compacted layers ( measured at end systole in a parasternal short axis view ) , with a ratio of 2.0 diagnostic of lvnc . based on echocardiographic studies , its prevalence has been estimated at 0.05% in the general population ( 20 ) , and the finding of a ratio of 2.0 between the thickness of the non - compacted and compacted myocardial layers in systole is considered diagnostic ( 4 ) . contrast echocardiography can be helpful when standard echocardiographic image quality is limited or the diagnosis is questionable ( 27 ) . prominent trabeculations , apical hypertrophic cardiomyopathy , dilated cardiomyopathy , arrhythmogenic right ventricular dysplasia , endocardial fibroelastosis , cardiac metastasis , and lv thrombus are important differential diagnostic considerations . echocardiography can easily diagnose isolated ventricular non - compaction if the echocardiographer is familiar with this congenital disorder , and if clear - cut diagnostic criteria are used . due to the risk of familial occurrence based on the ratio of end - diastolic thickness of the non - compacted to compacted layers of the myocardium , cmr imaging is accurate when diagnosing pathological lvnc ( 28 ) . the literature supports the clinical use of cmr in diagnosing lvnc , especially for patients with poor echocardiographic windows ( 29 ) . ventriculography and computed tomography can also show the typical morphological features of non - compaction : pathognomonic combination of multiple prominent ventricular trabeculations and of multiple deep intertrabecular recesses communicating with the ventricular cavity . hypothetically , both morphology and vasomotion of the coronary vessels feeding the hypertrophic segments can be abnormal with subsequent ischemia . there is no specific option for the treatment of non - compaction cardiomyopathy , which is the same as for heart failure . some findings have pointed to the efficacy of carvedilol in improving lv function , hypertrophy , and both metabolic and adrenergic abnormalities in isolated lvnc ( 31 ) . a more aggressive approach to the diagnosis and treatment of ventricular arrhythmias may be justified , and assessment for atrial and ventricular arrhythmias by ambulatory ecg monitoring should be performed annually . biventricular pacemakers could play a role in the treatment of lvnc patients with heart failure , reduced lv function , and prolonged intraventricular conduction . prophylactic anticoagulation may be warranted because of the higher risk of thrombus formation within the intratrabecular recesses . isolated lvnc is a rare disorder of endomyocardial morphogenesis characterized by numerous prominent ventricular trabeculations and deep intratrabecular recesses ( 1 ) . during normal embryonic development , endomyocardial trabeculations emerge from the apical region of the primitive ventricles at day 32 of fetal life ( 2 ) , and involute by day 70 through a process of resorption and remodeling . it is thought to result from failure of trabecular compaction of the loose mesh of muscle fibers seen in the early stages of myocardial development . normally this process is more pronounced in the left than in the right ventricle , resulting in a smooth , flat endocardial surface . in this disorder , however , there are abnormal trabeculations resulting from non - compaction scattered all over the myocardium , but most evident near the attachment of the papillary muscles of the mitral valve ( fig . right ventricular non - compaction may accompany lvnc in < 50% of patients . due to the difficulty in distinguishing normal variants in the highly trabeculated right ventricle from the pathological non - compacted ventricle , several authors dispute the existence of right ventricular non - compaction ( 4 , 5 ) . isolated non - compaction of the ventricular myocardium , first described by chin et al ( 1 ) in 1990 , is characterized by persistent embryonic myocardial morphology found in the absence of other cardiac anomalies to explain the abnormal development . some reports have observed necrotic myocytes within the prominent trabeculations of patients with non - compaction ( 5 , 6 ) . many cases of lvnc are caused by associated anomalies that generate intraventricular pressure overload , as in the case of pulmonary atresia with intact ventricular septum ( 8) or anomalous origin of the left coronary artery from the pulmonary trunk ( 9 ) . the disorder can be sporadic , but familial recurrence and associated dysmorphism has been reported ( 8 , 9 ) . in the x - linked form of the disease , the locus has been found on q28 , and mutations have been reported in the g4.5 gene ( 10 , 11 ) . this gene is in close proximity to other genes responsible for myopathies , and a number of patients with hypertrabeculations have been found to suffer from neuromuscular disorders such as emery - dreifuss muscular dystrophy , barth s syndrome , becker s muscular dystrophy , metabolic myopathies , and roifmann syndrome ( 12 ) , skeletal abnormalities such as melnick - needles syndrome and nail - patella syndrome ( 13 ) . mutations in this gene in adult lvnc are rare , however , but mutations have been described in adult lvnc in the genes encoding a - dystrobrevin and cypher / zasp ( 14 , 16 ) , integral parts of the complex that link the extracellular matrix of the myocardial cell to the cytoskeleton . in the initial case series of isolated non - compaction ( 1 ) , the median age at diagnosis was 7 yrs ( range 11 months to 22 yrs ) . in the largest series of patients with lvnc ( 5 ) , the true prevalence is unclear since the correct diagnosis is often missed or delayed due to a lack of knowledge concerning this uncommon disease , and its similarity to other diseases of the myocardium and endocardium . clinical severity depends on the extent of non - compacted cardiac segments . diastolic dysfunction in ventricular non - compaction can be related to both abnormal relaxation and restrictive filling caused by the numerous prominent trabeculae ( 22 ) . the origin of systolic dysfunction in non - compaction is unclear , but there is increasing evidence that subendocardial hypoperfusion and microcirculatory dysfunction play a role in ventricular dysfunction and arrhythmogenesis . chin et al ( 1 ) suggested that subendocardial perfusion might be abnormal in lvnc despite the absence of epicardial coronary artery disease . due to the numerous prominent trabeculae , subendocardial ischemia can be the result of isometric contraction of the endocardium and myocardium within the deep intertrabecular recesses . abnormalities in resting ecg are found in the majority of patients with lvnc , but findings are non - specific and include left ventricular ( lv ) hypertrophy , repolarization changes , inverted t waves , st segment changes , axis shifts , intraventricular conduction abnormalities and av block ( 1 , 5 , 20 , 21 ) . electrocardiographic findings of the wolff - parkinson - white syndrome have been described in up to 15% of pediatric patients ( 21 , 25 ) , but it was not observed in the two largest series of adults with isolated non - compaction ( 5 , 20 ) . sudden cardiac death accounted for half of the deaths in the larger series of patients with lvnc ( 1 , 20 ) . embolic complications may be related to the development of thrombi in the extensively trabeculated ventricle , depressed systolic function , or the development of atrial fibrillation ( 20 ) . an association between non - compaction and neuromuscular disorders has also been described ( 3 , 26 ) , as many as 82% of patients having some form of neuromuscular disorder . the combined echocardiographic features essential for making a diagnosis include : a two - layered ventricular myocardium consisting of an subendocardial compact layer and a thick non - compact endocardial layer with prominent trabeculations and intratrabecular recesses ; continuity between the lv cavity and the recesses with blood flowing in and out of the ventricular cavity and absence of secondary causes of increased trabeculations ( fig . there is continuity between the lv cavity and the deep intratrabecular recesses that are filled with blood from the ventricular cavity , without evidence of communication with the epicardial coronary artery system ( 1 , 20 ) . in the largest series of patients with lvnc ( 5 ) , in addition to the apical and mid - ventricular inferior wall segments , hypokinesis was observed occasionally in normally compacted segments as well as in the non - compacted segments of the left ventricle ( 5 ) , which may correlate with the observation of microcirculatory dysfunction in both non - compacted and normal segments in patients with lvnc . chin et al ( 1 ) described a quantitative approach for diagnosing non - compaction using a trabeculation peak to trough ratio , but it has not been used widely in clinical practice . oechslin et al ( 5 ) and jenni et al ( 4 ) described the abnormally thickened myocardium as a two - layered structure , with a normally compacted epicardial layer and a thickened endocardial layer . they proposed a quantitative evaluation for the diagnosis of lvnc by determining the ratio of maximal thickness of the non - compacted to compacted layers ( measured at end systole in a parasternal short axis view ) , with a ratio of 2.0 diagnostic of lvnc . based on echocardiographic studies , its prevalence has been estimated at 0.05% in the general population ( 20 ) , and the finding of a ratio of 2.0 between the thickness of the non - compacted and compacted myocardial layers in systole is considered diagnostic ( 4 ) . contrast echocardiography can be helpful when standard echocardiographic image quality is limited or the diagnosis is questionable ( 27 ) . prominent trabeculations , apical hypertrophic cardiomyopathy , dilated cardiomyopathy , arrhythmogenic right ventricular dysplasia , endocardial fibroelastosis , cardiac metastasis , and lv thrombus are important differential diagnostic considerations . echocardiography can easily diagnose isolated ventricular non - compaction if the echocardiographer is familiar with this congenital disorder , and if clear - cut diagnostic criteria are used . due to the risk of familial occurrence based on the ratio of end - diastolic thickness of the non - compacted to compacted layers of the myocardium , cmr imaging is accurate when diagnosing pathological lvnc ( 28 ) . the literature supports the clinical use of cmr in diagnosing lvnc , especially for patients with poor echocardiographic windows ( 29 ) . ventriculography and computed tomography can also show the typical morphological features of non - compaction : pathognomonic combination of multiple prominent ventricular trabeculations and of multiple deep intertrabecular recesses communicating with the ventricular cavity . there is no specific option for the treatment of non - compaction cardiomyopathy , which is the same as for heart failure . some findings have pointed to the efficacy of carvedilol in improving lv function , hypertrophy , and both metabolic and adrenergic abnormalities in isolated lvnc ( 31 ) . a more aggressive approach to the diagnosis and treatment of ventricular arrhythmias may be justified , and assessment for atrial and ventricular arrhythmias by ambulatory ecg monitoring should be performed annually . biventricular pacemakers could play a role in the treatment of lvnc patients with heart failure , reduced lv function , and prolonged intraventricular conduction . prophylactic anticoagulation may be warranted because of the higher risk of thrombus formation within the intratrabecular recesses .

the main functions of root canal sealers are ( i ) sealing off of voids , patent accessory canals , and multiple foramina , ( ii ) forming a bond between the core of the filling material and the root canal wall , and ( iii ) acting as a lubricant while facilitating the placement of the filling core and entombing any remaining bacteria . due to the relative biological and technical importance of sealers , their chemical and physical properties have been the subject of considerable attention since their initial development in the early twentieth century . sealers are categorised according to their main chemical constituents : zinc oxide eugenol , calcium hydroxide , glass ionomer , silicone , resin , and bioceramic - based sealers . root canal sealers have been reviewed across a number of studies , either collectively or based on their composition , including zinc oxide eugenol , calcium hydroxide , glass ionomer , and resin - based sealers . bioceramic - based sealers have only been available for use in endodontics for the past thirty years , their rise to prominence corresponding to the increased use of bioceramic technology in the fields of medicine and dentistry . they include alumina , zirconia , bioactive glass , glass ceramics , hydroxyapatite , and calcium phosphates . the classification of bioceramic materials into bioactive or bioinert materials is a function of their interaction with the surrounding living tissue . bioactive materials , such as glass and calcium phosphate , interact with the surrounding tissue to encourage the growth of more durable tissues . bioinert materials , such as zirconia and alumina , produce a negligible response from the surrounding tissue , effectively having no biological or physiological effect . bioceramics are commonly used for orthopaedic treatments , such as joint or tissue replacements , and for coating metal implants to improve biocompatibility . additionally , porous ceramics , such as calcium phosphate - based materials , have been used as bone graft substitutes . calcium phosphate was first used as bioceramic restorative dental cement by legeros et al . . however , the first documented use of bioceramic materials as a root canal sealer was not until two years later when krell and wefel compared the efficacy of experimental calcium phosphate cement with grossman 's sealer in extracted teeth , finding no significant difference between both sealers in terms of apical occlusion , adaptation , dentinal tubule occlusion , adhesion , cohesion , or morphological appearance . nonetheless , the experimental calcium phosphate sealer failed to provide apical sealing as effectively as grossman 's sealer . later evaluated the use of calcium phosphate as a root canal sealer in adult dog teeth . they reported that the calcium phosphate - based sealer made for a more uniform and tighter adaptation to the dentinal walls as compared to gutta - percha . calcium phosphate cement has subsequently been used successfully in endodontic treatments , including pulp capping , apical barrier formation , periapical defect repairs , and bifurcation perforation repairs . there are two major advantages associated with the use of bioceramic materials as root canal sealers . secondly , bioceramic materials contain calcium phosphate which enhances the setting properties of bioceramics and results in a chemical composition and crystalline structure similar to tooth and bone apatite materials , thereby improving sealer - to - root dentin bonding . however , one major disadvantage of these materials is in the difficulty in removing them from the root canal once they are set for later retreatment or post - space preparation . the exact mechanism of bioceramic - based sealer bonding to root dentin is unknown ; however , the following mechanisms have been suggested for calcium silicate - based sealers : diffusion of the sealer particles into the dentinal tubules ( tubular diffusion ) to produce mechanical interlocking bonds .infiltration of the sealer 's mineral content into the intertubular dentin resulting in the establishment of a mineral infiltration zone produced after denaturing the collagen fibres with a strong alkaline sealer [ 21 , 22].partial reaction of phosphate with calcium silicate hydrogel and calcium hydroxide , produced through the reaction of calcium silicates in the presence of the dentin 's moisture , resulting in the formation of hydroxyapatite along the mineral infiltration zone . diffusion of the sealer particles into the dentinal tubules ( tubular diffusion ) to produce mechanical interlocking bonds . infiltration of the sealer 's mineral content into the intertubular dentin resulting in the establishment of a mineral infiltration zone produced after denaturing the collagen fibres with a strong alkaline sealer [ 21 , 22 ] . partial reaction of phosphate with calcium silicate hydrogel and calcium hydroxide , produced through the reaction of calcium silicates in the presence of the dentin 's moisture , resulting in the formation of hydroxyapatite along the mineral infiltration zone . while various branded bioceramic - based root canal sealers are available on the market , others are still experimental , requiring further laboratory and clinical testing to ascertain their efficacy . a number of commercially available bioceramic - based root canal sealers , classified according to their major constituents , are identified in table 1 . the biological and physical properties of bioceramic - based root canal sealers were reviewed based on the ideal root canal sealer properties as described by grossman , as in the following list : it should be tacky when mixed to provide good adhesion between it and the canal wall when set.it should make a hermetic seal.it should be radiopaque so that it can be visualized on the radiograph.the particles of powder should be very fine so that they can mix easily with liquid.it should not shrink upon setting.it should not discolour tooth structure.it should be bacteriostatic or at least not encourage bacterial growth.it should set slowly.it should be insoluble in tissue fluids.it should be well tolerated by the periapical tissue.it should be soluble in common solvents if it is necessary to remove the root canal filling . it should be tacky when mixed to provide good adhesion between it and the canal wall when set . the particles of powder should be very fine so that they can mix easily with liquid . it should be soluble in common solvents if it is necessary to remove the root canal filling . biocompatibility is an essential requirement of any root canal sealer as the root filling material constitutes a true implant coming into direct contact with the vital tissue at the apical and lateral foramina of the root or indirectly via surface restoration . biocompatibility is defined as the ability of a material to achieve a proper and advantageous host response in specific applications . in other words , a material is said to be biocompatible when the material coming into contact with the tissue fails to trigger an adverse reaction , such as toxicity , irritation , inflammation , allergy , or carcinogenicity . most studies assess biocompatibility through investigations of cytotoxicity , in reference to the effect of the material on cell survival . the cytotoxicity of bioceramic - based sealers has been evaluated in vitro using mouse and human osteoblast cells [ 28 , 29 ] and human periodontal ligaments cells . calcium phosphate also happens to be the main inorganic component of the hard tissues ( teeth and bone ) . consequently , the literature notes that many bioceramic sealers have the potential to promote bone regeneration when unintentionally extruded through the apical foramen during root canal filling or repairs of root perforations [ 30 , 31 ] . sankin apatite has been shown by telli et al . to be biocompatible in in vitro studies . however , kim et al . showed that sankin apatite exerts a tissue response when implanted subcutaneously in rats and that this response began to subside within two weeks . the biocompatibility of sankin apatite root canal sealer was also evaluated in comparison to an experimental calcium phosphate - based sealer composed of tetracalcium phosphate , dicalcium phosphate dihydrate , and modified mcilvaine 's buffer solution . found that sankin apatite caused severe inflammatory reactions in both the dorsal subcutaneous and the periapical tissue of rats . however , the experimental sealer produced no inflammatory response in the subcutaneous tissue and only a mild reaction in the periapical tissue . the cytotoxicity of the sankin apatite root canal sealer is the result of the presence of iodoform and polyacrylic acids in the sealer . however , sankin apatite type ii and type iii were found to be more biocompatible than either type i or grossman 's sealer . endosequence bc , iroot sp , and mta - fillapex showed moderate toxicity when freshly mixed ; however , cytotoxicity reduced over time until being completely set [ 29 , 36 , 37 ] . although in vitro evaluations of biocompatibility can be an indicator of the cytotoxicity of a material , in vitro immunological deficiencies should be taken into consideration . some sealers have been shown to have severe cytotoxicity in vitro , such as zinc oxide eugenol - based sealers ; however , such toxicity is not necessarily clinically significant . capseal i and capseal ii sealers have been shown to produce less tissue irritation and less inflammation compared to other sealers [ 30 , 31 , 33 ] . studied the effects of capseal i and capseal ii in comparison to sankin apatite root sealer ( type i and type iii ) and a zinc oxide eugenol - based sealer ( pulp canal sealer ) . investigators exposed human periodontal fibroblast cells to the various sealers before measuring the inflammatory response by way of inflammatory mediators and the viability and osteogenic potential of osteoblast mg63 cells . they found capseal i and capseal ii to possess low cytotoxicity and to facilitate periapical dentoalveolar healing by regulating cellular mediators from periodontal ligaments cells and osteoblast differentiation . mta - fillapex was found to have a severe cytotoxic effect on fibroblast cells when freshly mixed . the ideal root canal sealer setting time should permit adequate working time . however , a slow setting time can result in tissue irritation , with most root canal sealers producing some degree of toxicity until being completely set . according to the manufacturers of endosequence bc sealer or iroot sp , the setting reaction is catalysed by the presence of moisture in the dentinal tubules . while the normal setting time is four hours , in patients with particularly dry canals , the setting time might be considerably longer . the amount of moisture present in the dentinal tubules of the canal walls can be affected by absorption with paper points , the presence of smear plugs , or tubular sclerosis . . reported that endosequence bc sealer requires at least 168 hours before being completely set under different humidity conditions , as evaluated using the gilmore needle method . in phase i , monobasic calcium phosphate reacts with calcium hydroxide in the presence of water to produce water and hydroxyapatite . in phase ii , the water derived from the dentin humidity , as well as that produced by the phase i reaction , contributes to the hydration of calcium silicate particles to trigger a calcium silicate hydrate phase . the manufacturer of mta - fillapex claims that their product will set in a minimum of two hours and this setting time has been confirmed in at least two studies [ 44 , 51 ] . however , even shorter setting times for mta - fillapex ( 66 min ) have been reported . the setting reaction of mta material is complicated and has been discussed by darvell and wu ; however , the setting reaction of mta - based sealers has not been described in the literature . flow is an essential property that allows the sealer to fill difficult - to - access areas , such as the narrow irregularities of the dentin , isthmus , accessory canals , and voids between the master and accessory cones . according to iso 6786/2001 , a root canal sealer should have a flow rate of not less than 20 mm . factors that influence the flow rate of the sealer include particle size , temperature , shear rate , and time from mixing . the internal diameter of the tubes and rate of insertion are considered when assessing flow rate via the rheometer method . the flow rate for endosequence bc sealer has been variously reported as 23.1 mm and 26.96 mm [ 44 , 54 ] . similarly , the flow rate of mta - fillapex has been variously reported as 22 mm , 24.9 mm , and 29.04 mm [ 44 , 51 , 52 ] . while most of the bioceramic - based root sealer manufacturers included in table 1 claim that the flow rate of their sealers meets iso requirements , the literature does not support such claims . root filling materials provide a mechanical barrier for the isolation of necrotic tissue or bacteria responsible for the persistence of periapical inflammation or postoperative pain [ 56 , 57 ] . therefore , the complete removal of the sealer is essential during endodontic retreatment to establish healthy periapical tissues . endosequence bc sealer is difficult to remove from the root canal using conventional retreatment techniques , including heat , chloroform , rotary instruments , and hand files . a number of cases have been reported in which obstruction of the apical foramen has resulted in a loss of patency . by contrast , ersev et al . reported that the removability of endosequence bc sealer from the root canal is comparable to ah plus . sankin apatite root canal sealer is easily removed during retreatment with and without the use of solvents . retreatability with mta - fillapex is comparable to that of ah plus in terms of material remaining in the canal , dentin removal , and time taken to reach working length . solubility is the mass loss of a material during a period of immersion in water . according to ansi / ada specification 57 a highly soluble root canal sealer would invariably permit the formation of gaps within and between the material and the root dentin , thereby providing avenues for leakage from the oral cavity and periapical tissues . both iroot sp and mta - fillapex are highly soluble , 20.64% and 14.89% , respectively , which does not meet ansi / ada requirements [ 52 , 64 ] . this high solubility is the result of hydrophilic nanosized particles being present in both sealers which increases their surface area and allows more liquid molecules to come into contact with the sealer . however , the literature contains conflicting accounts , with viapiana et al . finding mta - fillapex to be highly soluble and vitti et al . reporting the solubility of mta - fillapex to be < 3% , consistent with iso 6876/2001 . similarly , the solubility of endosequence bc is reported to be consistent with iso 6876/2001 . this discrepancy between the findings of these studies might be attributed to variations in the methods used to dry the samples after having subjected them to solubility testing . the low solubility of mta - angelus , consistent with ansi / ada requirements , is the result of an insoluble matrix of crystalline silica present within the sealer that maintains its integrity even in the presence of water . for reasons of aesthetic appearance , a root canal sealer should not stain the tooth . the chromogenic effects of root sealers are increased when excess sealer is not removed from the coronal dentin of the pulp chamber . observe that sankin apatite iii results in the least discolouration nine months after application as compared with ah26 , endofill , tubli - seal , and zinc oxide eugenol sealers . the greatest degree of discolouration was observed following treatment of the cervical third of the crown . mta - fillapex was found to cause the least crown discolouration to the extent of not being clinically perceptible . root canal sealers should be sufficiently radiopaque so as to be distinguishable from adjacent anatomical structures . this allows the quality of the root filling to be evaluated through radiographic examination . according to iso 6876/2001 , the minimum radiopacity for a root canal sealer endo cpm sealer was found to have a radiopacity of 6 mm due to the presence of bismuth trioxide and barium sulphate . similarly , the presence of bismuth trioxide in mta - fillapex gives it a radiopacity of 7 mm [ 52 , 70 ] . the antimicrobial activity of a root canal sealer increases the success rate of endodontic treatments by eliminating residual intraradicular infections that might have survived root canal treatment or have invaded the canal later through microleakage [ 71 , 72 ] . according to the literature , the key antimicrobial properties of root canal sealers lie in their alkalinity and release of calcium ions which stimulates repair via the deposition of mineralised tissue . two methods are commonly used to evaluate the antibacterial activity of bioceramic - based root canal sealers : the agar diffusion test [ 74 , 75 ] and direct contact testing [ 23 , 75 ] . endosequence bc sealer has been shown to have high ph ( > 11 ) as well as high tendency to release calcium ions . zhang et al . tested the antibacterial activity of iroot sp sealer in vitro against enterococcus faecalis through a modified direct contact test , finding that iroot sp sealer had a high ph value ( 11.5 ) even after setting but that its antibacterial effect was greatly diminished after seven days . the investigators suggested two additional mechanisms associated with the antibacterial efficacy of iroot sp : hydrophilicity and active calcium hydroxide diffusion . hydrophilicity reduces the contact angle of the sealer and facilitates penetration of the sealer into the fine areas of the root canal system to enhance the antibacterial effectiveness of iroot sp in vivo . morgental et al . evaluated the antibacterial activity of mta - fillapex and endo cpm against enterococcus faecalis using an agar diffusion test after mixing and a direct contact test after setting . the ph of the endo cpm suspension was greater than that of mta - fillapex ( > 11 ) ; however , the bacterial inhibition zone produced by mta - fillapex was greater than that produced by endo cpm . the investigators attributed the antibacterial activity of mta - fillapex to the presence of resin as a core ingredient . nevertheless , neither sealer was able to sustain its antibacterial activity after setting despite their initial high ph levels . enterococcus faecalis is the most common intraradicular microbe isolated from periapical periodontitis [ 76 , 77 ] and is therefore commonly used to test the antibacterial activity of root canal sealers . other microorganisms , such as micrococcus luteus , staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , candida albicans , and streptococcus mutans , have also been used to test the antibacterial effects of bioceramic - based sealers [ 74 , 78 ] . freshly mixed endo cpm exhibits antibacterial activity against staphylococcus aureus and streptococcus mutans with no significant reduction of the inhibition zone after setting . mta - angelus has an antibacterial effect against micrococcus luteus , staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , and candida albicans . root canal sealer adhesion is defined as its capacity to adhere to the root canal dentin and promote gp cone adhesion to each other and the dentin . argued that the term adhesion should be replaced with bonding in the case of root canal sealers because the attachment between the substances involves mechanical interlocking forces rather than molecular attraction . there is no standard method used to measure the adhesion of a sealer to the root dentin ; therefore , the adhesion potential of the root filling material is commonly tested using microleakage and bond strength tests . the sealing ability of a sealer is related to its solubility and to its bonding to the dentin and root canal filling cones . several studies have evaluated the sealing abilities of different bioceramic - based sealers in vitro . regardless of the different methodologies used , the sealing ability of bioceramic - based sealers has been found to be satisfactory and comparable to other commercially available sealers . however , until recently , there had been a paucity of literature concerning the long - term sealing ability or clinical outcomes associated with bioceramic - based sealers . bond strength is the force per unit area required to debond the adhesive material from the dentin . although no correlation has been identified between leakage and bond strength , the bond strength test has received significant attention due to the development of the monoblock concept in which a sealer bonds to both the core material and the dentinal wall to create a singular unit that enhances sealing and strengthens the root - filled tooth against fracture . a strong bond between the root canal sealer and the root dentin is essential for maintaining the integrity of the sealer - dentin interface during the preparation of post - spaces and during tooth flexure . bioceramic - based sealers have the ability to create bonds between the dentin and core filling materials . the bonding of iroot sp to root dentin is comparable to that of ah plus and stronger than either sealapex or endorez sealers . shokouhinejad et al . evaluated the bond strength of endosequence bc sealer compared to ah plus in the presence and absence of a smear layer , finding that the dislocation resistance of endosequence bc sealer was equal to that of ah plus and with no significant effect on the smear layer . studied the bond strengths of several sealers under various moisture conditions present in the root canal , concluding that a sealer 's bond strength is greatest in moist and wet canals , the presence of residual moisture positively affecting the adhesion of the root canal sealers to radicular dentin . as compared with ah plus , epiphany , and mta - fillapex , iroot sp had the highest dislodgment resistance from the root dentin . moreover , the prior placement of intracanal calcium hydroxide improved the bonding of iroot sp to the root dentin ; however , the bonding was less than that of ah plus and comparable to mta - fillapex in the absence of calcium hydroxide . this improvement in bonding is explained by way of the chemical interaction between calcium hydroxide and the iroot sp sealer increasing the frictional resistance and/or micromechanical retention of the sealer . endo cpm has a significantly higher bond strength compared to mta - fillapex or ah plus . testing the bond strength at the coronal third of the root canal shows no significant difference between mta - fillapex , iroot sp , and ah plus . however , in middle and apical thirds , iroot sp and ah plus have equivalent bond strengths superior to mta - fillapex . huffman et al . tested the dislocation resistance of proroot endo sealer , ah plus jet , and pulp canal sealer from root dentin with and without immersion in a simulated body fluid ( sbf ) . the investigators concluded that proroot endo sealer possesses greater bond strength than the other two sealers , especially after sbf immersion . according to huffman et al . , the greater bonding of the proroot endo sealer is due to the presence of spherical amorphous calcium phosphate and apatite - like phases enhancing frictional resistance . there was no negative effect of the iroot sp root canal sealer on the push - out bond strength of fibre posts cemented with self - adhesive resin cement . compared to activ gp sealer ( glass ionomer - based sealer , brasseler usa , savanah , ga ) , iroot sp was found to increase the fracture resistance of endodontically treated roots in vitro , a potential indicator of the high bond strength of the sealer . however , discrepancies in the results of these studies reveal that these sealers do not fulfil all of the requirements demanded of the ideal root sealer . the biocompatibility and biomineralization effect of these sealers might avail them for alternative uses in direct pulp capping and root end filling . further studies are required to clarify the clinical outcomes associated with the use of these sealers .

bioceramic - based root canal sealers are considered to be an advantageous technology in endodontics . the aim of this review was to consider laboratory experiments and clinical studies of these sealers . an extensive search of the endodontic literature was made to identify publications related to bioceramic - based root canal sealers . the outcome of laboratory and clinical studies on the biological and physical properties of bioceramic - based sealers along with comparative studies with other sealers was assessed . several studies were evaluated covering different properties of bioceramic - based sealers including physical properties , biocompatibility , sealing ability , adhesion , solubility , and antibacterial efficacy . bioceramic - based sealers were found to be biocompatible and comparable to other commercial sealers . the clinical outcomes associated with the use of bioceramic - based root canal sealers are not established in the literature .

1. Introduction 2. Ideal Root Canal Sealer Properties 3. Conclusion

the main functions of root canal sealers are ( i ) sealing off of voids , patent accessory canals , and multiple foramina , ( ii ) forming a bond between the core of the filling material and the root canal wall , and ( iii ) acting as a lubricant while facilitating the placement of the filling core and entombing any remaining bacteria . due to the relative biological and technical importance of sealers , their chemical and physical properties have been the subject of considerable attention since their initial development in the early twentieth century . sealers are categorised according to their main chemical constituents : zinc oxide eugenol , calcium hydroxide , glass ionomer , silicone , resin , and bioceramic - based sealers . root canal sealers have been reviewed across a number of studies , either collectively or based on their composition , including zinc oxide eugenol , calcium hydroxide , glass ionomer , and resin - based sealers . bioceramic - based sealers have only been available for use in endodontics for the past thirty years , their rise to prominence corresponding to the increased use of bioceramic technology in the fields of medicine and dentistry . they include alumina , zirconia , bioactive glass , glass ceramics , hydroxyapatite , and calcium phosphates . the classification of bioceramic materials into bioactive or bioinert materials is a function of their interaction with the surrounding living tissue . bioactive materials , such as glass and calcium phosphate , interact with the surrounding tissue to encourage the growth of more durable tissues . bioceramics are commonly used for orthopaedic treatments , such as joint or tissue replacements , and for coating metal implants to improve biocompatibility . however , the first documented use of bioceramic materials as a root canal sealer was not until two years later when krell and wefel compared the efficacy of experimental calcium phosphate cement with grossman 's sealer in extracted teeth , finding no significant difference between both sealers in terms of apical occlusion , adaptation , dentinal tubule occlusion , adhesion , cohesion , or morphological appearance . later evaluated the use of calcium phosphate as a root canal sealer in adult dog teeth . they reported that the calcium phosphate - based sealer made for a more uniform and tighter adaptation to the dentinal walls as compared to gutta - percha . calcium phosphate cement has subsequently been used successfully in endodontic treatments , including pulp capping , apical barrier formation , periapical defect repairs , and bifurcation perforation repairs . there are two major advantages associated with the use of bioceramic materials as root canal sealers . secondly , bioceramic materials contain calcium phosphate which enhances the setting properties of bioceramics and results in a chemical composition and crystalline structure similar to tooth and bone apatite materials , thereby improving sealer - to - root dentin bonding . however , one major disadvantage of these materials is in the difficulty in removing them from the root canal once they are set for later retreatment or post - space preparation . the exact mechanism of bioceramic - based sealer bonding to root dentin is unknown ; however , the following mechanisms have been suggested for calcium silicate - based sealers : diffusion of the sealer particles into the dentinal tubules ( tubular diffusion ) to produce mechanical interlocking bonds .infiltration of the sealer 's mineral content into the intertubular dentin resulting in the establishment of a mineral infiltration zone produced after denaturing the collagen fibres with a strong alkaline sealer [ 21 , 22].partial reaction of phosphate with calcium silicate hydrogel and calcium hydroxide , produced through the reaction of calcium silicates in the presence of the dentin 's moisture , resulting in the formation of hydroxyapatite along the mineral infiltration zone . diffusion of the sealer particles into the dentinal tubules ( tubular diffusion ) to produce mechanical interlocking bonds . infiltration of the sealer 's mineral content into the intertubular dentin resulting in the establishment of a mineral infiltration zone produced after denaturing the collagen fibres with a strong alkaline sealer [ 21 , 22 ] . partial reaction of phosphate with calcium silicate hydrogel and calcium hydroxide , produced through the reaction of calcium silicates in the presence of the dentin 's moisture , resulting in the formation of hydroxyapatite along the mineral infiltration zone . while various branded bioceramic - based root canal sealers are available on the market , others are still experimental , requiring further laboratory and clinical testing to ascertain their efficacy . a number of commercially available bioceramic - based root canal sealers , classified according to their major constituents , are identified in table 1 . the biological and physical properties of bioceramic - based root canal sealers were reviewed based on the ideal root canal sealer properties as described by grossman , as in the following list : it should be tacky when mixed to provide good adhesion between it and the canal wall when set.it should make a hermetic seal.it should be radiopaque so that it can be visualized on the radiograph.the particles of powder should be very fine so that they can mix easily with liquid.it should not shrink upon setting.it should not discolour tooth structure.it should be bacteriostatic or at least not encourage bacterial growth.it should set slowly.it should be insoluble in tissue fluids.it should be well tolerated by the periapical tissue.it should be soluble in common solvents if it is necessary to remove the root canal filling . it should be soluble in common solvents if it is necessary to remove the root canal filling . biocompatibility is an essential requirement of any root canal sealer as the root filling material constitutes a true implant coming into direct contact with the vital tissue at the apical and lateral foramina of the root or indirectly via surface restoration . in other words , a material is said to be biocompatible when the material coming into contact with the tissue fails to trigger an adverse reaction , such as toxicity , irritation , inflammation , allergy , or carcinogenicity . most studies assess biocompatibility through investigations of cytotoxicity , in reference to the effect of the material on cell survival . the cytotoxicity of bioceramic - based sealers has been evaluated in vitro using mouse and human osteoblast cells [ 28 , 29 ] and human periodontal ligaments cells . calcium phosphate also happens to be the main inorganic component of the hard tissues ( teeth and bone ) . consequently , the literature notes that many bioceramic sealers have the potential to promote bone regeneration when unintentionally extruded through the apical foramen during root canal filling or repairs of root perforations [ 30 , 31 ] . to be biocompatible in in vitro studies . the biocompatibility of sankin apatite root canal sealer was also evaluated in comparison to an experimental calcium phosphate - based sealer composed of tetracalcium phosphate , dicalcium phosphate dihydrate , and modified mcilvaine 's buffer solution . however , the experimental sealer produced no inflammatory response in the subcutaneous tissue and only a mild reaction in the periapical tissue . the cytotoxicity of the sankin apatite root canal sealer is the result of the presence of iodoform and polyacrylic acids in the sealer . however , sankin apatite type ii and type iii were found to be more biocompatible than either type i or grossman 's sealer . endosequence bc , iroot sp , and mta - fillapex showed moderate toxicity when freshly mixed ; however , cytotoxicity reduced over time until being completely set [ 29 , 36 , 37 ] . although in vitro evaluations of biocompatibility can be an indicator of the cytotoxicity of a material , in vitro immunological deficiencies should be taken into consideration . some sealers have been shown to have severe cytotoxicity in vitro , such as zinc oxide eugenol - based sealers ; however , such toxicity is not necessarily clinically significant . capseal i and capseal ii sealers have been shown to produce less tissue irritation and less inflammation compared to other sealers [ 30 , 31 , 33 ] . mta - fillapex was found to have a severe cytotoxic effect on fibroblast cells when freshly mixed . the ideal root canal sealer setting time should permit adequate working time . however , a slow setting time can result in tissue irritation , with most root canal sealers producing some degree of toxicity until being completely set . the amount of moisture present in the dentinal tubules of the canal walls can be affected by absorption with paper points , the presence of smear plugs , or tubular sclerosis . in phase i , monobasic calcium phosphate reacts with calcium hydroxide in the presence of water to produce water and hydroxyapatite . the setting reaction of mta material is complicated and has been discussed by darvell and wu ; however , the setting reaction of mta - based sealers has not been described in the literature . flow is an essential property that allows the sealer to fill difficult - to - access areas , such as the narrow irregularities of the dentin , isthmus , accessory canals , and voids between the master and accessory cones . according to iso 6786/2001 , a root canal sealer should have a flow rate of not less than 20 mm . factors that influence the flow rate of the sealer include particle size , temperature , shear rate , and time from mixing . the internal diameter of the tubes and rate of insertion are considered when assessing flow rate via the rheometer method . similarly , the flow rate of mta - fillapex has been variously reported as 22 mm , 24.9 mm , and 29.04 mm [ 44 , 51 , 52 ] . while most of the bioceramic - based root sealer manufacturers included in table 1 claim that the flow rate of their sealers meets iso requirements , the literature does not support such claims . therefore , the complete removal of the sealer is essential during endodontic retreatment to establish healthy periapical tissues . endosequence bc sealer is difficult to remove from the root canal using conventional retreatment techniques , including heat , chloroform , rotary instruments , and hand files . reported that the removability of endosequence bc sealer from the root canal is comparable to ah plus . sankin apatite root canal sealer is easily removed during retreatment with and without the use of solvents . retreatability with mta - fillapex is comparable to that of ah plus in terms of material remaining in the canal , dentin removal , and time taken to reach working length . according to ansi / ada specification 57 a highly soluble root canal sealer would invariably permit the formation of gaps within and between the material and the root dentin , thereby providing avenues for leakage from the oral cavity and periapical tissues . this high solubility is the result of hydrophilic nanosized particles being present in both sealers which increases their surface area and allows more liquid molecules to come into contact with the sealer . however , the literature contains conflicting accounts , with viapiana et al . finding mta - fillapex to be highly soluble and vitti et al . reporting the solubility of mta - fillapex to be < 3% , consistent with iso 6876/2001 . similarly , the solubility of endosequence bc is reported to be consistent with iso 6876/2001 . this discrepancy between the findings of these studies might be attributed to variations in the methods used to dry the samples after having subjected them to solubility testing . the low solubility of mta - angelus , consistent with ansi / ada requirements , is the result of an insoluble matrix of crystalline silica present within the sealer that maintains its integrity even in the presence of water . for reasons of aesthetic appearance , a root canal sealer should not stain the tooth . the chromogenic effects of root sealers are increased when excess sealer is not removed from the coronal dentin of the pulp chamber . observe that sankin apatite iii results in the least discolouration nine months after application as compared with ah26 , endofill , tubli - seal , and zinc oxide eugenol sealers . the greatest degree of discolouration was observed following treatment of the cervical third of the crown . mta - fillapex was found to cause the least crown discolouration to the extent of not being clinically perceptible . root canal sealers should be sufficiently radiopaque so as to be distinguishable from adjacent anatomical structures . this allows the quality of the root filling to be evaluated through radiographic examination . according to iso 6876/2001 , the minimum radiopacity for a root canal sealer endo cpm sealer was found to have a radiopacity of 6 mm due to the presence of bismuth trioxide and barium sulphate . the antimicrobial activity of a root canal sealer increases the success rate of endodontic treatments by eliminating residual intraradicular infections that might have survived root canal treatment or have invaded the canal later through microleakage [ 71 , 72 ] . according to the literature , the key antimicrobial properties of root canal sealers lie in their alkalinity and release of calcium ions which stimulates repair via the deposition of mineralised tissue . two methods are commonly used to evaluate the antibacterial activity of bioceramic - based root canal sealers : the agar diffusion test [ 74 , 75 ] and direct contact testing [ 23 , 75 ] . the investigators suggested two additional mechanisms associated with the antibacterial efficacy of iroot sp : hydrophilicity and active calcium hydroxide diffusion . hydrophilicity reduces the contact angle of the sealer and facilitates penetration of the sealer into the fine areas of the root canal system to enhance the antibacterial effectiveness of iroot sp in vivo . the ph of the endo cpm suspension was greater than that of mta - fillapex ( > 11 ) ; however , the bacterial inhibition zone produced by mta - fillapex was greater than that produced by endo cpm . enterococcus faecalis is the most common intraradicular microbe isolated from periapical periodontitis [ 76 , 77 ] and is therefore commonly used to test the antibacterial activity of root canal sealers . other microorganisms , such as micrococcus luteus , staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , candida albicans , and streptococcus mutans , have also been used to test the antibacterial effects of bioceramic - based sealers [ 74 , 78 ] . freshly mixed endo cpm exhibits antibacterial activity against staphylococcus aureus and streptococcus mutans with no significant reduction of the inhibition zone after setting . mta - angelus has an antibacterial effect against micrococcus luteus , staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , and candida albicans . root canal sealer adhesion is defined as its capacity to adhere to the root canal dentin and promote gp cone adhesion to each other and the dentin . argued that the term adhesion should be replaced with bonding in the case of root canal sealers because the attachment between the substances involves mechanical interlocking forces rather than molecular attraction . there is no standard method used to measure the adhesion of a sealer to the root dentin ; therefore , the adhesion potential of the root filling material is commonly tested using microleakage and bond strength tests . the sealing ability of a sealer is related to its solubility and to its bonding to the dentin and root canal filling cones . several studies have evaluated the sealing abilities of different bioceramic - based sealers in vitro . regardless of the different methodologies used , the sealing ability of bioceramic - based sealers has been found to be satisfactory and comparable to other commercially available sealers . however , until recently , there had been a paucity of literature concerning the long - term sealing ability or clinical outcomes associated with bioceramic - based sealers . although no correlation has been identified between leakage and bond strength , the bond strength test has received significant attention due to the development of the monoblock concept in which a sealer bonds to both the core material and the dentinal wall to create a singular unit that enhances sealing and strengthens the root - filled tooth against fracture . a strong bond between the root canal sealer and the root dentin is essential for maintaining the integrity of the sealer - dentin interface during the preparation of post - spaces and during tooth flexure . bioceramic - based sealers have the ability to create bonds between the dentin and core filling materials . the bonding of iroot sp to root dentin is comparable to that of ah plus and stronger than either sealapex or endorez sealers . evaluated the bond strength of endosequence bc sealer compared to ah plus in the presence and absence of a smear layer , finding that the dislocation resistance of endosequence bc sealer was equal to that of ah plus and with no significant effect on the smear layer . studied the bond strengths of several sealers under various moisture conditions present in the root canal , concluding that a sealer 's bond strength is greatest in moist and wet canals , the presence of residual moisture positively affecting the adhesion of the root canal sealers to radicular dentin . as compared with ah plus , epiphany , and mta - fillapex , iroot sp had the highest dislodgment resistance from the root dentin . moreover , the prior placement of intracanal calcium hydroxide improved the bonding of iroot sp to the root dentin ; however , the bonding was less than that of ah plus and comparable to mta - fillapex in the absence of calcium hydroxide . this improvement in bonding is explained by way of the chemical interaction between calcium hydroxide and the iroot sp sealer increasing the frictional resistance and/or micromechanical retention of the sealer . testing the bond strength at the coronal third of the root canal shows no significant difference between mta - fillapex , iroot sp , and ah plus . , the greater bonding of the proroot endo sealer is due to the presence of spherical amorphous calcium phosphate and apatite - like phases enhancing frictional resistance . there was no negative effect of the iroot sp root canal sealer on the push - out bond strength of fibre posts cemented with self - adhesive resin cement . compared to activ gp sealer ( glass ionomer - based sealer , brasseler usa , savanah , ga ) , iroot sp was found to increase the fracture resistance of endodontically treated roots in vitro , a potential indicator of the high bond strength of the sealer . however , discrepancies in the results of these studies reveal that these sealers do not fulfil all of the requirements demanded of the ideal root sealer . the biocompatibility and biomineralization effect of these sealers might avail them for alternative uses in direct pulp capping and root end filling . further studies are required to clarify the clinical outcomes associated with the use of these sealers .

the public has become increasingly aware of the wide variety of chemicals present not just in the environmental media to which they are exposed ( such as air , water , and soil ) but also in the food they eat and the products they use . as access to relevant information continues to grow , notably via the internet , many communities have voiced concerns about health effects associated with the multiple chemicals in their daily lives . to address these concerns , many organizations have responded with approaches , guidelines , focused workshops , and illustrative applications to better assess cumulative risks . these organizations include the us environmental protection agency ( epa ) , national institute for occupational safety and health ( niosh ) , agency for toxic substances and disease registry ( atsdr ) , world health organization ( who ) , california environmental protection agency ( cal / epa ) , the environmental justice ( ej ) community , and professional organizations such as the society of toxicology . cumulative risk assessment ( cra ) explicitly considers the combined fate and effects of multiple contaminants from multiple sources through multiple exposure pathways . the goal of cra is to address more realistic conditions than those addressed under the classic approach of the 1983 national research council ( nrc ) red book on risk assessment in the federal government , which agencies historically implemented by evaluating one chemical at a time . reflecting advances in scientific knowledge since that time , which made more detailed and integrated analyses possible , epa outlined its framework for cumulative risk assessment in 2003 that considered joint exposure to multiple chemical , physical , and biological stressors . a number of organizations have published additional cumulative risk reports over the past decade [ 39 ] . despite the increase in relevant analyses and reports , the translation of a more fully integrated approach to practice has lagged behind the science . with various groups and individual community members unaware of available tools that could be used to assess cumulative risk , the concept of a cumulative risk toolbox emerged soon after the epa framework was published , during the early stages of developing a companion report to support evaluations at contaminated sites . the purpose of this cra toolbox was to overcome the awareness hurdle and serve as a practical resource for cumulative risk assessors and other interested parties . recognizing that cumulative risk encompasses many different kinds of stressors ranging from chemical and physical to biological and psychological ones , the cra toolbox compilation that began in 2004 targeted a specific scope , namely , health risks from exposure to multiple anthropogenic chemicals as well as to elevated concentrations of chemicals that occur naturally . the main objectives of the cra toolbox were to ( 1 ) consolidate resources for conducting cumulative risk assessments , ( 2 ) provide coverage for the main elements of the assessment process , and ( 3 ) offer practical notes to help assessors understand the strengths and limitations of a given approach or model for cumulative risk applications . the original cra toolbox was published as an appendix to the 2007 agency report , concepts , methods , and data sources for cumulative health risk assessment of multiple chemicals , exposures , and effects : a resource document . it was recently reevaluated to determine if the descriptions and electronic web links for the individual entries were current or needed to be updated . this paper highlights relevant excerpts from the cra toolbox with interpretive context for cras at contaminated sites , with updates as indicated ; it also identifies several additional resources not included in the original cra toolbox . the cra toolbox was developed in three phases : ( 1 ) identification of information resources related to cra , ( 2 ) assessment of their relevance to contaminated sites and facilities , and ( 3 ) categorization of the relevant resources to facilitate their use in analyses . in searching the scientific literature and other online materials for information related to cra , the resources pursued included conceptual approaches , models , and other tools related to assessing health risks posed by multiple chemicals in multiple environmental media via multiple exposure routes . in addition , risk analysts from several agencies , universities , and the private sector were informally invited to identify methods and models they had found useful for assessing multiple contaminants , exposures , and effects as part of their environmental projects and programs . some information evaluated in the second phase was designed to assess ecological effects , while other material addressed acute and shorter - term human exposures ; however , the primary emphasis of the cra toolbox was on approaches and tools for evaluating health effects from chronic to lifetime exposures . an example of this type of resource is the niosh approach for worker protection that accounts for exposures to chemical mixtures . the resources considered less well suited than others to this type of application were screened out at this stage , as were resources that essentially duplicated content that had already been compiled . in the third phase , the purpose of this last phase was to define a manageable set of categories to serve as the organizational structure of the cra toolbox . this toolbox was not intended to be comprehensive ; rather , it was developed to provide a suite of resources , including guidelines , approaches , and models , that could be applied to assess cumulative health risks associated with contaminated sites . for this reason , three basic documents guided the determination of the cra toolbox categories : the 1983 nrc risk assessment paradigm , the 1989 epa risk assessment guidance for superfund , and the 2003 epa framework for cumulative risk . the online searches and interactions with risk practitioners produced more than 100 methods and tools relevant to cras at contaminated sites . that is , many were developed for general risk assessment purposes , but they were found to be either directly useful or adaptable for population - specific cras , or the underlying approach was found to offer useful insights for such analyses . many of the materials addressed one or two components of a cra , while others addressed multiple elements or the overall concept . although certain resources clearly consider multiple exposures to multiple chemicals , such as the standard agency guidance for risk assessment at contaminated sites , only a fraction are explicitly defined as cumulative risk tools . relatively few specifically focus on exposure groupings or joint toxicity , or on a given population group for cra , such as children ( e.g. , see ( 4.1 ) in table 4 and ( 6.7 ) in table 6 ) . although many focus on contaminant sources or affected environmental media , as has been the historical practice , they can also be applied to more complex cumulative risk analyses . similarly , various resources can be helpful for evaluating subsets of an overall cumulative risk assessment , such as cumulative hazards , threats , exposures , or impacts , including health impact analyses . the resources compiled for the cra toolbox are available on websites managed by a variety of organizations , including ( 1 ) federal agencies and institutes such as the epa , us nuclear regulatory commission , us department of energy ( doe ) , and us department of health and human services , including niosh and atsdr within the centers for disease control and prevention ( cdc ) , as well as the national institutes of health ( nih ) , including the national institute of environmental health sciences ( niehs ) ; ( 2 ) national scientific organizations such as the national academies , including the national academy of sciences and national research council ; ( 3 ) other federal entities such as the national environmental justice advisory council ; ( 4 ) national organizations in other countries , including canada ( health canada , environment canada , and the quebec worker protection institute irsst , institut de recherche robert - sauv en sant et en scurit du travail ) and the netherlands ( organization for applied scientific research , nederlandse organisatie voor toegepast natuurwetenschappelijk onderzoek ( tno ) nutrition and food institute ) ; ( 5 ) international bodies , such as the european commission and who ; ( 6 ) private nonprofit organizations such as the international institute of indigenous resource management ( iiirm ) ; ( 7 ) state agencies , such as the cal / epa office of environmental health hazard assessment ( oehha ) and air resources board ( arb ) ; ( 8) industry organizations such as the american chemistry council ( acc ) ; ( 9 ) scientific groups at universities and national laboratories ; and ( 10 ) local community advisory boards at contaminated sites and facilities , such as site - specific , restoration , and citizen advisory boards ( ssabs , rabs , and cabs ) . the cra toolbox is divided into five sections as described in table 1 and illustrated in figure 1 , with some sections further subdivided to identify specific content . ( the resource count in table 1 refers to the number of tools highlighted in tables for each topic . ) the resources highlighted in this section focus on those identified in the original cra toolbox ( which was developed in 2004 ) , with limited updates . while several tools apply to multiple categories , they are generally listed with the first cra toolbox category for which they are particularly useful . ( note that planning and scoping for cras continues iteratively throughout the assessment process . ) each section begins with a topical introduction , followed by text bullets highlighting selected resources , and concluding with a table that summarizes the following for a larger number of entries : ( 1 ) title , author / organization , and access information ( e.g. , web link ) , where available ; ( 2 ) purpose and scope ; ( 3 ) remarks on application to cra . nearly 80 resources are summarized across the five tables combined , with additional resources highlighted in the accompanying text . topics addressed during planning , scoping , and problem formulation include the purpose and breadth of the assessment ( considering multiple chemicals , population groups , exposures , and effects ) , the type of product needed from the assessment to inform a decision , the data to be collected and synthesized , the general assessment approach , and stakeholder involvement . cras can involve a very large number of potential combinations of chemicals and interactions inherent to the environmental setting . during this initial and iterative phase of the process , common questions include which chemicals are most likely to contribute significantly to risks , whether they might interact , and what the nature of those interactions could be . the internet has become a valuable tool for promoting and enhancing stakeholder involvement , and successful programs often combine traditional methods ( ranging from one - on - one to town hall meetings and printed newsletters or other information sent in regular mail ) with electronic approaches to take advantage of the unique benefits of each . selected resources that can be used to support planning , scoping , and problem formulation for cras , including stakeholder involvement , are presented in table 2 ; several specific resources are highlighted below . , epa released its guidance on cumulative risk assessment , part 1 , planning and scoping ( ( 2.1 ) in table 2 ) that presented broad - based approaches that considered ( 1 ) multiple endpoints , sources , pathways , and routes of exposure ; ( 2 ) community - based decision making ; ( 3 ) flexibility in achieving goals ; ( 4 ) case - specific responses ; ( 5 ) all environmental media ; and ( 6 ) holistic risk reduction . the companion lessons learned on the planning and scoping of environmental risk assessments ( ( 2.2 ) in table 2 ) followed in 2002 , presenting case studies to illustrate organizing principles for cras . the following year , the agency published its framework for cumulative risk assessment ( ( 2.3 ) in table 2 ) that outlines an umbrella structure for cras , identifies key issues , and defines common terms . neither a procedural guide nor a regulatory requirement , the framework document summarizes key elements of the cra process as part of a flexible structure rather than identifying prescriptive protocols . providing the basic information about important aspects of cumulative risk , that framework continues to serve as a key foundation for cra reports . one example of a tool based on the framework is ejview ( ( 2.4 ) in table 2 ) , a gis - based module jointly designed by the epa offices of environmental information ( oei ) and environmental justice ( oej ) . combining environmental , socioeconomic , and health indicators in statistical tables , this tool was initially developed to evaluate potential ej issues . for community - based approaches , this and other ranking and prioritization tools can help identify the problems warranting consideration in a cra . note that although it is presented here within the planning / problem formulation stage to support front - end scoping , this and other such tools are also very useful for other phases of the cra process , including risk characterization . a number of tools have been developed to support stakeholder involvement in cras , particularly for contaminated sites . ranging from guidance for epa 's superfund and ej programs to project - specific field activities ( see the second section of table 2 , ( 2.5 ) through ( 2.10 ) ) , these resources chronicle approaches taken to solicit inputs from multiple stakeholders and incorporate them into the assessment plans . a number of examples from doe legacy waste sites reflect inputs of long - standing community advisory boards , from the doe savannah river site in georgia to the doe hanford site in washington . practical insights can also be gained from the risk analysis , communication , evaluation , and reduction ( racer ) project at the doe los alamos national laboratory ( lanl ) in new mexico . led by the risk assessment corporation ( rac ) team , extensive stakeholder involvement has been a hallmark of that effort ( ( 2.8 ) in table 2 ) . the epa has developed a series of guidance documents to help ensure that all data collection , including at contaminated sites , is appropriate for the intended use , which is particularly important for cras given the typical complexity of these analyses . these documents outline a systematic process for developing performance criteria to collect , evaluate , and use environmental data . statistical and analytical tools underlie data quality objectives ( dqos ) , as highlighted in the last section of table 2 ( ( 2.11 ) through ( 2.17 ) ) . a large number of tools have been developed to assess the environmental fate and transport of chemicals , and many of these can be used to support cras , as highlighted below and in table 3 . these tools include computer models available from the epa center for subsurface modeling support ( csmos ) , as well as resources for physicochemical constants and guidance for determining background concentrations in soil . risk assessments for contaminated sites and also for urban environments and other settings impacted by multiple pollutant sources commonly simulate the behavior of multiple chemicals in the environment because of the relatively high costs ( in terms of both manpower and dollars ) to conduct measurements . hundreds of computer models have been developed to model contaminant fate and transport in the environment . some are very general and conceptual , while others address specific media characteristics and setting conditions . the use and suitability of individual models vary widely depending on the project objectives and data required , so it is important for the selected model to be appropriate for the given evaluation . csmos is a key resource for these tools ; the center maintains an online database of publicly available groundwater and vadose zone fate and transport models , a number of which are included in table 3 ( see entries ( 3.13)(3.23 ) ) . several online resources provide information on the chemical , physical , and biological properties of substances , including industrial products and byproducts . resources highlighted in the original toolbox include the epa chembiofinder database ( ( 3.1 ) in table 3 ) and soil screening guidance ( ( 3.2 ) in table 3 ) , which includes an extensive set of environmental and physical constants and parameters that can be used to model the fate and transport of chemicals in soil . the chemical - specific properties used to derive epa regional screening levels are also available online ( see related discussion for table 5 ) . information about these properties can be used to predict that chemicals will likely share a similar environmental fate to support exposure groupings for cras concentrations that represent natural background or ambient conditions are location specific and provide valuable context for assessing chemical fate and transport as well as incremental risk . the epa has outlined an approach for characterizing background concentrations , including protocols for determining whether site measurements are statistically elevated , in guidelines for characterizing background chemicals in soil at superfund sites ( ( 3.3 ) in table 3 ) . data on background concentrations of inorganic chemicals can be found in several sources that provide baseline context for community - based environmental risk assessments . information sources include atsdr toxicological profiles ( ( 5.4 ) in table 5 ) and technical reports from the us geological survey , as illustrated by a report on constituents of ambient surface soil in chicago that was prepared in cooperation with the city of chicago . similar regional data can be found in state - specific resource reports , such as from the massachusetts department of environment as well as the texas commission on environmental quality ( tceq ) , regarding background levels of polycyclic aromatic hydrocarbons ( pahs ) and other constituents in soil . ( also , see related tceq entry ( 6.5 ) in table 6 . ) vapor intrusion can be an important exposure pathway for multiple chemicals when volatile organic compounds are in the subsurface ( e.g. , soil and groundwater ) and can migrate to indoor air . contributions from vapor intrusion are commonly combined with estimates from other indoor air pathways ( e.g. , inhalation of volatiles during showering ) to quantify aggregate exposures and risks for single chemicals ( e.g. , benzene ) and cumulative risks for groups of chemicals ( e.g. , chlorinated solvents ) . this pathway has historically been evaluated using a model based on the equation published by johnson and ettinger in 1991 . the model is a one - dimensional spreadsheet that estimates convective and diffusive transport of chemical vapors to indoor air from sources near a building 's perimeter . attenuating factors such as biological degradation were not included in the original model , and the source was assumed to be infinite over the exposure duration assessed ( e.g. , 25 years for a commercial or industrial worker ) . the epa provided a detailed description of this earlier vapor intrusion model in its draft guidance issued in 2002 , and since that time , the agency and others have continued to strengthen the modeling approach ( ( 3.11 ) in table 3 ) . following the early johnson and ettinger model , several us states have adopted simple equations based on this method to conduct screening evaluations of indoor air ( ( 3.11 ) in table 3 ) . the indoor air concentrations calculated by these models across multiple chemicals can be combined to estimate cumulative exposures and corresponding risks . many exposure models are well suited to assessing multiple chemicals by multiple routes , although this is generally performed by combining predictions made for individual chemicals . tools range from relatively straightforward screening models to comprehensive multimedia models , as highlighted below and further illustrated in table 4 . for example , models for subsurface vapor migration described earlier are often tapped for multimedia exposure assessments because they consider both soil and groundwater inputs . in addition , several technical reports identify exposure factors , their bases , and parameters commonly used to estimate cumulative exposures . this category includes resources linked to fate and transport models , in some cases to account for the time lag between release and exposure considering the movement of chemicals from source to receptor . the analysis of changing chemical exposures over time is also an important concept for grouping chemicals , and models that incorporate this factor are included in table 3 ( e.g. , see ( 3.7 ) , ( 3.14 ) , and ( 3.15 ) ) . exposure factors . risk assessments commonly rely on exposure models to capture receptor - specific factors that influence chemical intakes . for example , factors that address exposure duration , time involved in certain activities , body weight and surface area , intake rates ( e.g. , inhalation , or ingestion of food , soil , or water ) , and many others parameters needed to estimate potential risks from multiple exposures are available from the epa 2011 exposure factors handbook and 2008 child - specific exposure factors handbook ( see ( 4.1 ) in table 4 ) . to support the evaluation of potentially vulnerable or susceptible subgroups , epa 's 1999 report on sociodemographic data used for identifying potentially highly exposed populations ( ( 4.2 ) in table 4 ) provides information to help identify subsets of the general population who may be at greater risk for negative health consequences , which can be incorporated into cras . an additional valuable resource is the national human exposure assessment survey ( nhexas , ( 4.3 ) in table 4 ) , which was developed by epa in the early 1990s to compile information on human exposures to chemicals at the community and regional scales , with an emphasis on associating these exposures with personal activities . multipathway releases and exposures . the 3mra model ( ( 4.4 ) in table 4 ) is a multimedia , multipathway , multireceptor exposure and risk assessment model developed by epa to assess releases from land - based waste management units . after simulating releases from disposal units , modules model fate and transport through the environment , the 3mra methodology uses a monte carlo scheme to quantify uncertainty ( e.g. , from natural variability or based on selection of representative sites ) . the exposure and fate assessment screening tool ( e - fast , ( 4.5 ) in table 4 ) is another computer - based model that can provide screening - level estimates for general population , consumer , and environmental exposures to chemicals released to air , surface water , or landfills and those released from consumer products . potential inhalation , dermal , and ingestion doses resulting from these releases are estimated , with the modeled concentrations and doses designed to reasonably overestimate exposures for use in screening - level assessments . states have also developed tools to assess exposures via the air pathway , such as california 's air toxics hot spots program that requires stationary air emission sources in the state to report the types and quantities of certain substances routinely released to air and also to estimate potential exposures to surrounding populations . a software package was developed to support these evaluations ( ( 4.10 ) in table 4 ) . the dietary exposure potential model ( depm ) ( ( 4.12 ) in table 4 ) estimates dietary exposure to multiple chemicals based on data from several national , government - sponsored food intake surveys and chemical residue monitoring programs . the depm includes recipes developed specifically for exposure analyses that link consumption survey data for prepared foods to the chemical residue information , which is normally reported for raw food ingredients , to estimate daily dietary exposure . the summary databases are aggregated in a way that allows the analyst to select appropriate demographic factors , such as age / sex groups , geographical regions , ethnic groups , and economic status . the model also includes modules for evaluating chemical exposures from residues , soil , and tap water . resources that can be used to support toxicity analyses for cras are highlighted here and summarized in table 5 . topics include ( 1 ) resources for toxicity reference values for various exposure routes and durations ; ( 2 ) development of toxicity factors , including for whole mixtures ; ( 3 ) identification of toxicity criteria for similar or surrogate compounds or mixtures to represent a mixture or its components ; and ( 4 ) joint toxicity of the components of a mixture . the epa integrated risk information system ( iris ) ( ( 5.7 ) in table 5 ) is a key source of information on chronic toxicity reference values , including reference doses ( rfds ) , reference concentrations ( rfcs ) , and oral slope factors , unit risks , and corresponding risk - based concentrations ( see ( 5.7 ) in table 5 ) . chronic toxicity values are also available in iris for certain mixtures ( such as the rfc for diesel exhaust and cancer toxicity values for polychlorinated biphenyls ) . note that if a standard toxicity value is not available in iris , a provisional peer - reviewed toxicity value ( pprtv ; derived for use in the superfund program ) may be available ( ( 5.8 ) in table 5 ) . pprtvs are derived using the same methods , data sources , and epa guidance used to derive iris values but undergo a comparatively more rapid scientific review . if a relevant pprtv is not available , some states have also developed selected toxicity values that may be found in summary tables of epa regional screening levels ( ( 5.11 ) in table 5 ) . recent iris assessments commonly include data on effects other than the critical effect used to derive a toxicity value ; these effects ( sometimes called secondary effects ) can be used to at least qualitatively assess joint toxicity ( usually via dose or response addition ) of combined chemical exposures . the information provided in epa 's rfd arrays also could be used to support estimates of target organ toxicity doses based on secondary effects . the atsdr has developed toxicological profiles for many individual chemicals that identify the effects of the given chemical , as well as primary environmental and metabolic transformation products to support grouping by specific target organs and systems . a smaller set of interaction profiles has also been developed that assesses joint toxicity ( see ( 5.4 ) in table 5 ) . , epa updated its 1986 guidelines for chemical mixtures ( ( 5.1 ) in table 5 ) . this supplemental guidance describes risk assessment approaches that depend on the type , nature , and quality of available data , and it includes equations , definitions , discussions of toxicological interactions and pharmacokinetic models , and approaches for assessing whole mixtures , surrogate mixtures , and individual mixture components . the whole - mixture discussion includes the derivation of whole - mixture toxicity values ( rfds , rfcs , cancer slope factors , and inhalation unit risks ) , as well as consideration of comparative potency and environmental transformations . the component discussion includes dose addition , the hazard index ( hi ) , interaction - based hi , relative potency factors ( rpfs ) , and response addition . in 2002 , epa published guidance for assessing the cumulative risk of pesticides with a common toxic mechanism to address requirements set forth in the food quality protection act of 1996 ( see ( 5.2 ) in table 5 ) ; that guidance was updated in 2006 . the initial guidance considered potential exposures to 30 organophosphate pesticides via food , drinking water , and residential uses , and it applied methods to account for variable exposures per different ages , seasons , and geographic factors . epa scientists in the national center for environmental assessment collaborated with colleagues in the national toxicological research center of the food and drug administration to develop additional component approaches for assessing the cumulative risk posed by exposures to multiple chemicals by evaluating three scenarios [ 22 , 23 ] . they explored a simple scenario in which it was certain that all chemicals being considered shared a common toxic mode of action , so a dose - additive approach could be applied . in the second scenario , modes of action for the chemicals in the mixture were known and could be used to divide the chemicals into independent mode - of - action subclasses ; dose addition and response addition were then integrated to assess the risk . in the third scenario , the mode or modes of action were uncertain for the chemicals in the mixture , so a joint dose - response modeling procedure was developed that created a range of risk estimates . physiologically based models and chemical mixtures toxicology research . statistically based methods and computer tools that can model interactions and effects associated with multiple chemicals continue to be developed and refined . a main area of study involves applying physiologically based pharmacokinetic / pharmacodynamic ( pbpk / pd ) models to chemical mixtures . reaction network modeling is an example of a computer - based approach that has been used in petroleum engineering to predict chemical reaction rates , products , and outcomes based on various statistical methods ( including monte carlo - type analysis ) . a molecular - based model ( biomol ) was designed to use the reaction network modeling approach to predict effects of chemicals in complex biological systems . joint toxicology studies have also improved the understanding of potential health effects of chemical mixtures with different modes of action . for example , the tno nutrition and food research institute of the netherlands has evaluated the use of mechanistic models to describe interactions between mixture components expected to act by different modes of action . in a pilot study funded by the american chemistry council long - range research initiative ( see ( 5.10 ) in table 5 ) , the tno team applied pbpk models to assess possible toxicokinetic interactions between compounds in an applied mixture and compared those estimates to empirical dose - response modeling of observed pathological changes in the liver , blood , and kidney . the aim of such research is to develop and refine methods to be applied to other chemical mixtures . other tno studies have developed and applied statistical methods combining multivariate data analysis and modeling in in vitro and in vivo studies on various chemical mixtures such as petroleum hydrocarbons , aldehydes , food contaminants , industrial solvents , and mycotoxins [ 25 , 26 ] . similarly , nih / niehs has sponsored studies on mixtures toxicology and environmental health , including as part of the national toxicology program ( ntp ) , for which related reports and fact sheets are available from the niehs website . a search engine on this website can be used to tap research and tools for specific applications , including those related to cumulative risk . niehs also publishes environmental health perspectives , a monthly journal that often reports on studies relevant to chemical mixtures , with some issues and supplements entirely dedicated to mixtures . also , nih maintains the national library of medicine and other databases ( see ( 5.9 ) in table 5 ) . in addition to the organizations highlighted in table 5 , others have also been assessing mixtures to support cras during the past decade . for example , the health canada toxic substances research initiative ( tsri ) assessed cumulative effects of environmental toxics to both human and ecological receptors [ 28 , 29 ] . cal / epa conducted a health assessment in the 1990s that focused on a representative complex mixture , diesel particulate matter ( dpm ) . the scientific review panel for this study used the analyses of epidemiological data from workers to develop a unit risk estimate for diesel particulates , which was then used to derive an inhalation slope factor . this approach offered insights not only for assessments involving diesel exhaust but also for other chemical mixtures . the epa developed the bmds to fit mathematical models to toxicological dose - response data for a particular toxic effect ( ( 5.6 ) in table 5 ) . the user evaluates results of this statistical software to select a benchmark dose ( bmd ) associated with a predetermined benchmark response ( bmr ) , such as a 10% increase in the incidence of a particular lesion or a 10% decrease in body weight . a goal of the bmd approach is to define a point of departure to derive an rfd or rfc that is more independent of study design than the traditional method based on a single experimental dose , such as the no - observed - adverse - effect level ( noael ) . the hi uses rfds or rfcs in a formula that is based on dose addition to scale the exposure levels in a mixture , producing an indicator of the extent of concern for toxicity . the bmd values used with dose addition could estimate a bmd for the mixture , allowing the mixture dose to be interpreted in terms of the risk of a particular effect . the categorical regression tool catreg was developed to conduct dose - response modeling of data on diverse endpoints across multiple toxicological studies by categorizing effects into different severity levels , such as no - effect and adverse - effect levels ( ( 5.3 ) in table 5 ) . the epa has suggested that categorical regression results for a single chemical can yield benchmark doses ( e.g. , a 10% effective dose or ed10 ) or risk estimates that reflect the probability of observing a severity level of ( nonspecific ) response . once this is done for each component of a mixture , risk assessment methods for chemical mixtures such as the hi or response addition approach can then be applied to yield an indication of risk for the mixture . for cras therefore , this tool could be helpful for both the toxicity assessment and risk characterization components of an integrated risk analysis risk - based screening concentrations have been developed for environmental media ( including soil , drinking water , and air ) by several organizations , including epa . for example , epa regions 3 , 6 , and 9 previously developed risk - based concentrations ( rbcs ) , medium - specific screening levels ( mssls ) , and preliminary remediation goals ( prgs ) , respectively ; these similar values were subsequently combined to produce regional screening levels ( rsls ) for assessing contaminated sites ( ( 5.11 ) in table 5 ) . these screening values can be used to narrow the focus of assessments on key contributors to risks ; they are based on conservative default assumptions for exposure and environmental parameters , and they reflect toxicity values from iris and other sources ( e.g. , cal / epa ) . thus , it is important for the risk estimates and associated uncertainties to be well characterized and clearly presented so this information can be interpreted appropriately to guide sound decisions . this final phase of the cra process has come to rely on issue - specific summaries and graphical tools to display statistical and spatial information , as highlighted here and in table 6 . the sada tool was jointly developed by the epa , us nuclear regulatory commission , and university of tennessee as an integrated software package to support human and ecological cras ( see ( 6.1 ) of table 6 ) . like many other tools ( including the rsls ) , the human health module of sada includes equations from the standard superfund guidance and accommodates different land use scenarios and exposure pathways . this tool emphasizes the spatial distribution of contaminant data , and individual modules cover visualization , geospatial analysis , statistical analysis , sampling design , and decision analysis . outputs can be tabular or graphical , and they can be used to identify where estimated risks exceed target values . although the input data for these pathways can be tailored to reflect site - specific conditions , interactions are not considered . risk assessments commonly present human health risks as single - point estimates ( e.g. , 1 10 ) in accordance with standard guidance for contaminated sites . for example , monte carlo simulation tools can be used to consider the effect of uncertainty and variability , with results approximating a full range of reasonably possible outcomes that are typically plotted as graphs ( e.g. , frequency distributions ) or tabulated . while these probabilistic approaches have not yet been widely implemented in environmental health risk analyses for contaminated sites , such simulations can help assessors represent uncertainty and variability in the risk results ( see ( 6.7 ) in table 6 ) . extensive research in probabilistic analysis relevant to cras has been conducted at a number of universities , including north carolina state university ( c. frey and colleagues ) and the university of washington ( a. cullen and colleagues ) . these evolving tools incorporate distributions of parameter values that may be more appropriate for a given assessment than point estimates , and they are particularly well suited for cras . the epa region 6 regional air impact modeling initiative ( raimi ) was originally designed as a gis - based system that could tap a number of emissions data sources to assess potential impacts at the community level ( ( 6.3 ) in table 6 ) . this system also supports source attribution analyses , and early findings indicated that a small number of sources were responsible for most of the impact . initially implemented at a pilot scale that focused on risks from direct inhalation exposures , rami was subsequently expanded to assess indirect exposures resulting from airborne releases , thus increasing the relevance for additional cra applications . tools identified with the exposure assessment phase that consider the spatial scale of various impacts ( e.g. , ( 4.7 ) in table 4 ) can also be valuable resources for the risk characterization phase of cras . similarly , disease registries that provide context for exposure assessments ( see ( 4.15 ) in table 4 ) also serve as resources for risk characterization by presenting health data that can be considered in concert with modeled or measured chemical data to assess potential influences of multiple exposures ( including population - specific or location - specific patterns ) and to calibrate risk models . this reevaluation of the original toolbox ( as described in epa 's 2007 report , concepts , methods , and data sources for cumulative health risk assessment of multiple chemicals , exposures , and effects : a resource document ) found that only half the web links were still valid , as most websites and models have been updated since the original toolbox was published . however , most information is still available in some form , and only a few web pages no longer exist . the web links presented in this overview of the original toolbox reflect the updated web addresses as of early 2013 . the review of the original toolbox also found that many compilations , approaches , and models have been updated , and additional resources are now available online . for example , a targeted search produced more than twice the number of resources reflected in the original toolbox . additional organizations represented in this ongoing update of the cra toolbox include the us army corps of engineers , us geological survey , and us department of agriculture . both new and updated models can be found via a number of epa ( and other ) websites , including the center for exposure assessment modeling ( ceam ) , national center for environmental assessment ( ncea ) , and national exposure research laboratory ( nerl ) , as well as the council for environmental regulatory modeling ( cerm ) . most of these additional resources focus on the broad topic of exposure , including environmental fate and transport , but some address toxicity and risk characterization resources . several cumulative risk meetings held within the last several years have produced compilations in a manner similar to the effort undertaken for the original toolbox in 2004 to support the epa cra resource document ( which was published in 2007 ) . these include workshops organized by epa that involved external scientists , as well as an internal epa workshop that convened cumulative risk experts and epa program and regional project leads in july 2009 to share insights from recent and ongoing applications . the models and other resources compiled from some of these meetings are available in the scientific literature and online [ 3133 ] . furthermore , the nrc published two studies relevant to cumulative risk [ 7 , 8 ] , one in 2008 that focused on a specific class of chemicals ( phthalates and cumulative risk assessment : the task ahead ) , and the other in 2009 that focused on how current risk analysis approaches could be improved ( science and decisions : advancing risk assessment ) . in addition , after an extensive process of collaborative workshops and soliciting input from interested parties , in late 2010 cal / epa oehha released its report on cumulative impacts : building a scientific foundation . for the exposure category , a key resource not included in the original toolbox is the cdc 's national health and nutrition examination survey ( nhanes ) database . this database contains information on the health and nutritional status of adults and children , and it is unique in its incorporation of data from both interviews and physical examinations . nhanes is also a source of information on the distribution of some contaminant concentrations in human tissues drawn from samples collected in the us population . valuable research findings have been made possible by this unique database , including recent evaluations of cadmium exposures and effects in children that inform risk assessments for contaminated products from overseas . information about the nhanes program and research publications can be found online via the cdc website . for the toxicity category , in addition to the recent online availability of the epa pprtvs , the iris database now provides additional toxicity information that can be used to assess chemical mixtures . for example , information is included from recent toxicological reviews that evaluate common modes of action and common target organs and systems , which can be considered in evaluating secondary effects and establishing toxicity groupings for cras . ncea has also developed a new database of the toxicity literature for a number of chemicals , called health and environmental research online ( hero ) , which provides information underlying recent risk - related epa analyses and can also be mined to support cras . the international toxicity estimates for risk assessment ( iter ) database is another toxicity resource , which was created and is maintained by the nonprofit scientific organization toxicology excellence for risk assessment ( tera ) . its tabulated data include toxicity values and cancer classifications for more than 600 chemicals , with synopses that explain data differences and web links to the source organizations for further information . progress also continues on the international front , spanning each key element of a cra . in 2009 , the who released its draft report for public review , risk assessment of combined exposures to multiple chemicals : a who / ipcs framework . also that year , the european commission moved forward with its ongoing initiative , nomiracle ( novel methods for integrated risk assessment of cumulative stressors in europe ) , including making a toolbox available to support these analyses . many more continue to be developed and refined to support cras , as the science evolves to meet the demands of the broader community for more realistic assessments that can guide effective risk management decisions . resource toolboxes such as this one can support more integrated compilations to help address the need for increased awareness and access to practical approaches for cumulative risk assessors . cras that tap resources in this toolbox which extend from epa 's cumulative risk framework and resource document to conceptual models , exposure factor handbooks , and toxicity databases , together with census data , specific fate models , community involvement processes , and new visualization tools are being reflected in the growing literature on this topic ( e.g. , see [ 32 , 3844 ] ) . a number of existing methods and tools can be applied to assess cumulative risks for contaminated sites , and progress continues in developing and refining such resources . most of the tools in the original cra toolbox have been updated , and others have become available . nevertheless , explicit cra applications are not yet widespread as issues related to general awareness and integration of relevant tools persist . meanwhile , programs continue to evolve that are well suited to cra approaches , which could help enhance the understanding of , and guide measures to address , combined stressors before they become a problem . thus , the need exists for a broadly accessible toolbox that can serve as a practical foundation for cumulative risk practitioners across agencies , academia , and the private sector , as well as for interested members of the general public . plans designed to address this need include the following : strengthen the online accessibility of cra toolbox resources ; provide additional practical context from case studies ; andincorporate further tools that extend to nonchemical stressors and other applications , to address emerging themes including sustainable communities . strengthen the online accessibility of cra toolbox resources ; provide additional practical context from case studies ; and incorporate further tools that extend to nonchemical stressors and other applications , to address emerging themes including sustainable communities .

the historical approach to assessing health risks of environmental chemicals has been to evaluate them one at a time . in fact , we are exposed every day to a wide variety of chemicals and are increasingly aware of potential health implications . although considerable progress has been made in the science underlying risk assessments for real - world exposures , implementation has lagged because many practitioners are unaware of methods and tools available to support these analyses . to address this issue , the us environmental protection agency developed a toolbox of cumulative risk resources for contaminated sites , as part of a resource document that was published in 2007 . this paper highlights information for nearly 80 resources from the toolbox and provides selected updates , with practical notes for cumulative risk applications . resources are organized according to the main elements of the assessment process : ( 1 ) planning , scoping , and problem formulation ; ( 2 ) environmental fate and transport ; ( 3 ) exposure analysis extending to human factors ; ( 4 ) toxicity analysis ; and ( 5 ) risk and uncertainty characterization , including presentation of results . in addition to providing online access , plans for the toolbox include addressing nonchemical stressors and applications beyond contaminated sites and further strengthening resource accessibility to support evolving analyses for cumulative risk and sustainable communities .

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

the public has become increasingly aware of the wide variety of chemicals present not just in the environmental media to which they are exposed ( such as air , water , and soil ) but also in the food they eat and the products they use . these organizations include the us environmental protection agency ( epa ) , national institute for occupational safety and health ( niosh ) , agency for toxic substances and disease registry ( atsdr ) , world health organization ( who ) , california environmental protection agency ( cal / epa ) , the environmental justice ( ej ) community , and professional organizations such as the society of toxicology . the goal of cra is to address more realistic conditions than those addressed under the classic approach of the 1983 national research council ( nrc ) red book on risk assessment in the federal government , which agencies historically implemented by evaluating one chemical at a time . despite the increase in relevant analyses and reports , the translation of a more fully integrated approach to practice has lagged behind the science . with various groups and individual community members unaware of available tools that could be used to assess cumulative risk , the concept of a cumulative risk toolbox emerged soon after the epa framework was published , during the early stages of developing a companion report to support evaluations at contaminated sites . recognizing that cumulative risk encompasses many different kinds of stressors ranging from chemical and physical to biological and psychological ones , the cra toolbox compilation that began in 2004 targeted a specific scope , namely , health risks from exposure to multiple anthropogenic chemicals as well as to elevated concentrations of chemicals that occur naturally . the main objectives of the cra toolbox were to ( 1 ) consolidate resources for conducting cumulative risk assessments , ( 2 ) provide coverage for the main elements of the assessment process , and ( 3 ) offer practical notes to help assessors understand the strengths and limitations of a given approach or model for cumulative risk applications . the original cra toolbox was published as an appendix to the 2007 agency report , concepts , methods , and data sources for cumulative health risk assessment of multiple chemicals , exposures , and effects : a resource document . this paper highlights relevant excerpts from the cra toolbox with interpretive context for cras at contaminated sites , with updates as indicated ; it also identifies several additional resources not included in the original cra toolbox . the cra toolbox was developed in three phases : ( 1 ) identification of information resources related to cra , ( 2 ) assessment of their relevance to contaminated sites and facilities , and ( 3 ) categorization of the relevant resources to facilitate their use in analyses . in searching the scientific literature and other online materials for information related to cra , the resources pursued included conceptual approaches , models , and other tools related to assessing health risks posed by multiple chemicals in multiple environmental media via multiple exposure routes . in addition , risk analysts from several agencies , universities , and the private sector were informally invited to identify methods and models they had found useful for assessing multiple contaminants , exposures , and effects as part of their environmental projects and programs . some information evaluated in the second phase was designed to assess ecological effects , while other material addressed acute and shorter - term human exposures ; however , the primary emphasis of the cra toolbox was on approaches and tools for evaluating health effects from chronic to lifetime exposures . this toolbox was not intended to be comprehensive ; rather , it was developed to provide a suite of resources , including guidelines , approaches , and models , that could be applied to assess cumulative health risks associated with contaminated sites . for this reason , three basic documents guided the determination of the cra toolbox categories : the 1983 nrc risk assessment paradigm , the 1989 epa risk assessment guidance for superfund , and the 2003 epa framework for cumulative risk . the online searches and interactions with risk practitioners produced more than 100 methods and tools relevant to cras at contaminated sites . although many focus on contaminant sources or affected environmental media , as has been the historical practice , they can also be applied to more complex cumulative risk analyses . similarly , various resources can be helpful for evaluating subsets of an overall cumulative risk assessment , such as cumulative hazards , threats , exposures , or impacts , including health impact analyses . the resources compiled for the cra toolbox are available on websites managed by a variety of organizations , including ( 1 ) federal agencies and institutes such as the epa , us nuclear regulatory commission , us department of energy ( doe ) , and us department of health and human services , including niosh and atsdr within the centers for disease control and prevention ( cdc ) , as well as the national institutes of health ( nih ) , including the national institute of environmental health sciences ( niehs ) ; ( 2 ) national scientific organizations such as the national academies , including the national academy of sciences and national research council ; ( 3 ) other federal entities such as the national environmental justice advisory council ; ( 4 ) national organizations in other countries , including canada ( health canada , environment canada , and the quebec worker protection institute irsst , institut de recherche robert - sauv en sant et en scurit du travail ) and the netherlands ( organization for applied scientific research , nederlandse organisatie voor toegepast natuurwetenschappelijk onderzoek ( tno ) nutrition and food institute ) ; ( 5 ) international bodies , such as the european commission and who ; ( 6 ) private nonprofit organizations such as the international institute of indigenous resource management ( iiirm ) ; ( 7 ) state agencies , such as the cal / epa office of environmental health hazard assessment ( oehha ) and air resources board ( arb ) ; ( 8) industry organizations such as the american chemistry council ( acc ) ; ( 9 ) scientific groups at universities and national laboratories ; and ( 10 ) local community advisory boards at contaminated sites and facilities , such as site - specific , restoration , and citizen advisory boards ( ssabs , rabs , and cabs ) . each section begins with a topical introduction , followed by text bullets highlighting selected resources , and concluding with a table that summarizes the following for a larger number of entries : ( 1 ) title , author / organization , and access information ( e.g. , web link ) , where available ; ( 2 ) purpose and scope ; ( 3 ) remarks on application to cra . nearly 80 resources are summarized across the five tables combined , with additional resources highlighted in the accompanying text . topics addressed during planning , scoping , and problem formulation include the purpose and breadth of the assessment ( considering multiple chemicals , population groups , exposures , and effects ) , the type of product needed from the assessment to inform a decision , the data to be collected and synthesized , the general assessment approach , and stakeholder involvement . cras can involve a very large number of potential combinations of chemicals and interactions inherent to the environmental setting . selected resources that can be used to support planning , scoping , and problem formulation for cras , including stakeholder involvement , are presented in table 2 ; several specific resources are highlighted below . , epa released its guidance on cumulative risk assessment , part 1 , planning and scoping ( ( 2.1 ) in table 2 ) that presented broad - based approaches that considered ( 1 ) multiple endpoints , sources , pathways , and routes of exposure ; ( 2 ) community - based decision making ; ( 3 ) flexibility in achieving goals ; ( 4 ) case - specific responses ; ( 5 ) all environmental media ; and ( 6 ) holistic risk reduction . the companion lessons learned on the planning and scoping of environmental risk assessments ( ( 2.2 ) in table 2 ) followed in 2002 , presenting case studies to illustrate organizing principles for cras . the following year , the agency published its framework for cumulative risk assessment ( ( 2.3 ) in table 2 ) that outlines an umbrella structure for cras , identifies key issues , and defines common terms . neither a procedural guide nor a regulatory requirement , the framework document summarizes key elements of the cra process as part of a flexible structure rather than identifying prescriptive protocols . one example of a tool based on the framework is ejview ( ( 2.4 ) in table 2 ) , a gis - based module jointly designed by the epa offices of environmental information ( oei ) and environmental justice ( oej ) . note that although it is presented here within the planning / problem formulation stage to support front - end scoping , this and other such tools are also very useful for other phases of the cra process , including risk characterization . a number of tools have been developed to support stakeholder involvement in cras , particularly for contaminated sites . ranging from guidance for epa 's superfund and ej programs to project - specific field activities ( see the second section of table 2 , ( 2.5 ) through ( 2.10 ) ) , these resources chronicle approaches taken to solicit inputs from multiple stakeholders and incorporate them into the assessment plans . led by the risk assessment corporation ( rac ) team , extensive stakeholder involvement has been a hallmark of that effort ( ( 2.8 ) in table 2 ) . the epa has developed a series of guidance documents to help ensure that all data collection , including at contaminated sites , is appropriate for the intended use , which is particularly important for cras given the typical complexity of these analyses . a large number of tools have been developed to assess the environmental fate and transport of chemicals , and many of these can be used to support cras , as highlighted below and in table 3 . risk assessments for contaminated sites and also for urban environments and other settings impacted by multiple pollutant sources commonly simulate the behavior of multiple chemicals in the environment because of the relatively high costs ( in terms of both manpower and dollars ) to conduct measurements . resources highlighted in the original toolbox include the epa chembiofinder database ( ( 3.1 ) in table 3 ) and soil screening guidance ( ( 3.2 ) in table 3 ) , which includes an extensive set of environmental and physical constants and parameters that can be used to model the fate and transport of chemicals in soil . information about these properties can be used to predict that chemicals will likely share a similar environmental fate to support exposure groupings for cras concentrations that represent natural background or ambient conditions are location specific and provide valuable context for assessing chemical fate and transport as well as incremental risk . information sources include atsdr toxicological profiles ( ( 5.4 ) in table 5 ) and technical reports from the us geological survey , as illustrated by a report on constituents of ambient surface soil in chicago that was prepared in cooperation with the city of chicago . the epa provided a detailed description of this earlier vapor intrusion model in its draft guidance issued in 2002 , and since that time , the agency and others have continued to strengthen the modeling approach ( ( 3.11 ) in table 3 ) . this category includes resources linked to fate and transport models , in some cases to account for the time lag between release and exposure considering the movement of chemicals from source to receptor . , see ( 3.7 ) , ( 3.14 ) , and ( 3.15 ) ) . , inhalation , or ingestion of food , soil , or water ) , and many others parameters needed to estimate potential risks from multiple exposures are available from the epa 2011 exposure factors handbook and 2008 child - specific exposure factors handbook ( see ( 4.1 ) in table 4 ) . to support the evaluation of potentially vulnerable or susceptible subgroups , epa 's 1999 report on sociodemographic data used for identifying potentially highly exposed populations ( ( 4.2 ) in table 4 ) provides information to help identify subsets of the general population who may be at greater risk for negative health consequences , which can be incorporated into cras . an additional valuable resource is the national human exposure assessment survey ( nhexas , ( 4.3 ) in table 4 ) , which was developed by epa in the early 1990s to compile information on human exposures to chemicals at the community and regional scales , with an emphasis on associating these exposures with personal activities . the exposure and fate assessment screening tool ( e - fast , ( 4.5 ) in table 4 ) is another computer - based model that can provide screening - level estimates for general population , consumer , and environmental exposures to chemicals released to air , surface water , or landfills and those released from consumer products . a software package was developed to support these evaluations ( ( 4.10 ) in table 4 ) . topics include ( 1 ) resources for toxicity reference values for various exposure routes and durations ; ( 2 ) development of toxicity factors , including for whole mixtures ; ( 3 ) identification of toxicity criteria for similar or surrogate compounds or mixtures to represent a mixture or its components ; and ( 4 ) joint toxicity of the components of a mixture . the epa integrated risk information system ( iris ) ( ( 5.7 ) in table 5 ) is a key source of information on chronic toxicity reference values , including reference doses ( rfds ) , reference concentrations ( rfcs ) , and oral slope factors , unit risks , and corresponding risk - based concentrations ( see ( 5.7 ) in table 5 ) . note that if a standard toxicity value is not available in iris , a provisional peer - reviewed toxicity value ( pprtv ; derived for use in the superfund program ) may be available ( ( 5.8 ) in table 5 ) . the atsdr has developed toxicological profiles for many individual chemicals that identify the effects of the given chemical , as well as primary environmental and metabolic transformation products to support grouping by specific target organs and systems . a smaller set of interaction profiles has also been developed that assesses joint toxicity ( see ( 5.4 ) in table 5 ) . , epa updated its 1986 guidelines for chemical mixtures ( ( 5.1 ) in table 5 ) . in 2002 , epa published guidance for assessing the cumulative risk of pesticides with a common toxic mechanism to address requirements set forth in the food quality protection act of 1996 ( see ( 5.2 ) in table 5 ) ; that guidance was updated in 2006 . in the third scenario , the mode or modes of action were uncertain for the chemicals in the mixture , so a joint dose - response modeling procedure was developed that created a range of risk estimates . in a pilot study funded by the american chemistry council long - range research initiative ( see ( 5.10 ) in table 5 ) , the tno team applied pbpk models to assess possible toxicokinetic interactions between compounds in an applied mixture and compared those estimates to empirical dose - response modeling of observed pathological changes in the liver , blood , and kidney . similarly , nih / niehs has sponsored studies on mixtures toxicology and environmental health , including as part of the national toxicology program ( ntp ) , for which related reports and fact sheets are available from the niehs website . in addition to the organizations highlighted in table 5 , others have also been assessing mixtures to support cras during the past decade . for example , epa regions 3 , 6 , and 9 previously developed risk - based concentrations ( rbcs ) , medium - specific screening levels ( mssls ) , and preliminary remediation goals ( prgs ) , respectively ; these similar values were subsequently combined to produce regional screening levels ( rsls ) for assessing contaminated sites ( ( 5.11 ) in table 5 ) . while these probabilistic approaches have not yet been widely implemented in environmental health risk analyses for contaminated sites , such simulations can help assessors represent uncertainty and variability in the risk results ( see ( 6.7 ) in table 6 ) . extensive research in probabilistic analysis relevant to cras has been conducted at a number of universities , including north carolina state university ( c. frey and colleagues ) and the university of washington ( a. cullen and colleagues ) . , ( 4.7 ) in table 4 ) can also be valuable resources for the risk characterization phase of cras . similarly , disease registries that provide context for exposure assessments ( see ( 4.15 ) in table 4 ) also serve as resources for risk characterization by presenting health data that can be considered in concert with modeled or measured chemical data to assess potential influences of multiple exposures ( including population - specific or location - specific patterns ) and to calibrate risk models . this reevaluation of the original toolbox ( as described in epa 's 2007 report , concepts , methods , and data sources for cumulative health risk assessment of multiple chemicals , exposures , and effects : a resource document ) found that only half the web links were still valid , as most websites and models have been updated since the original toolbox was published . the review of the original toolbox also found that many compilations , approaches , and models have been updated , and additional resources are now available online . additional organizations represented in this ongoing update of the cra toolbox include the us army corps of engineers , us geological survey , and us department of agriculture . most of these additional resources focus on the broad topic of exposure , including environmental fate and transport , but some address toxicity and risk characterization resources . several cumulative risk meetings held within the last several years have produced compilations in a manner similar to the effort undertaken for the original toolbox in 2004 to support the epa cra resource document ( which was published in 2007 ) . furthermore , the nrc published two studies relevant to cumulative risk [ 7 , 8 ] , one in 2008 that focused on a specific class of chemicals ( phthalates and cumulative risk assessment : the task ahead ) , and the other in 2009 that focused on how current risk analysis approaches could be improved ( science and decisions : advancing risk assessment ) . valuable research findings have been made possible by this unique database , including recent evaluations of cadmium exposures and effects in children that inform risk assessments for contaminated products from overseas . for the toxicity category , in addition to the recent online availability of the epa pprtvs , the iris database now provides additional toxicity information that can be used to assess chemical mixtures . ncea has also developed a new database of the toxicity literature for a number of chemicals , called health and environmental research online ( hero ) , which provides information underlying recent risk - related epa analyses and can also be mined to support cras . also that year , the european commission moved forward with its ongoing initiative , nomiracle ( novel methods for integrated risk assessment of cumulative stressors in europe ) , including making a toolbox available to support these analyses . many more continue to be developed and refined to support cras , as the science evolves to meet the demands of the broader community for more realistic assessments that can guide effective risk management decisions . cras that tap resources in this toolbox which extend from epa 's cumulative risk framework and resource document to conceptual models , exposure factor handbooks , and toxicity databases , together with census data , specific fate models , community involvement processes , and new visualization tools are being reflected in the growing literature on this topic ( e.g. a number of existing methods and tools can be applied to assess cumulative risks for contaminated sites , and progress continues in developing and refining such resources . most of the tools in the original cra toolbox have been updated , and others have become available . thus , the need exists for a broadly accessible toolbox that can serve as a practical foundation for cumulative risk practitioners across agencies , academia , and the private sector , as well as for interested members of the general public . plans designed to address this need include the following : strengthen the online accessibility of cra toolbox resources ; provide additional practical context from case studies ; andincorporate further tools that extend to nonchemical stressors and other applications , to address emerging themes including sustainable communities . strengthen the online accessibility of cra toolbox resources ; provide additional practical context from case studies ; and incorporate further tools that extend to nonchemical stressors and other applications , to address emerging themes including sustainable communities .

our primary objective was to investigate whether an mdi regimen using insulin glargine as basal insulin with insulin lispro at mealtimes can achieve glycemic control ( a1c ) equivalent to csii ( insulin lispro ) in people with type 1 diabetes . secondary objectives were to compare other measures of blood glucose control ( pre- and postprandial , bedtime and 0300 h levels , and within - day glucose variability ) , hypoglycemia , adverse events ( aes ) , treatment costs , and treatment satisfaction . this was a randomized , parallel - group , open - label , multicenter ( n = 5 ) study performed in three european countries with a 1-week run - in period followed by a 24-week treatment period ( including 4 weeks of active dose titration ) , with clinic visits at 0 , 2 , 8 , 16 , and 24 weeks and 2 weeks follow - up . an open - label design was necessary owing to the different insulin administration regimens . the study was performed according to good clinical ( research ) practice / declaration of helsinki . participants were aged 1870 years , with a bmi 27.0 kg / m and diabetes for > 1 year , a1c 6.59.0% , fasting plasma glucose ( fpg ) > 7.0 mmol / l ( > 126 mg / dl ) , and fasting plasma c - peptide < 0.10 nmol / l at the screening visit . participants with a mean duration of treatment of 18.4 ( csii ) and 20.7 ( mdi ) years , who were currently using an mdi regimen with nph insulin , were recruited and randomized . participants were excluded if they were prior users of csii or insulin glargine , were unwilling or unable to use csii or mdis , had more than two severe hypoglycemic events in the previous 6 months , or had recent diabetic ketoacidosis or impaired hepatic / renal function . randomization was to insulin glargine ( lantus , once daily in the evening ; sanofi - aventis , paris , france ) plus mealtime insulin lispro ( humalog , three - times daily ; eli lilly , indianapolis , in ) or to insulin lispro administered subcutaneously using a minimed 508 pump ( minimed technologies , northridge , ca ) . mmol / l ( 80120 mg / dl ) , other preprandial blood glucose 5.07.7 mmol / l ( 90140 mg / dl ) , 2-h postprandial blood glucose < 7.7 mmol / l ( < 140 mg / dl ) , and bedtime blood glucose 6.18.3 mmol / l ( 110150 mg / dl ) . titration was based on the mean fpg over at least 3 days , adjusted by + 2 units if fpg was 6.68.8 mmol / l ( 120160 mg / dl ) , + 4 units if fpg was 8.811 if fpg was < 4.4 mmol / l ( < 80 mg / dl ) . further titration of the dose was performed at investigator discretion if symptoms consistent with severe hypoglycemia occurred . the initial insulin lispro dose was investigator selected , based on prior mealtime doses , and was titrated once basal insulin dose was optimized . initiation of csii was based on the pump manufacturer 's recommendations , using the normal bolus option ; a bolus wizard calculator was not used . infusion sites were advised to be changed every 23 days in accordance with investigators ' routine clinical practice and manufacturer 's recommendations . analysis of a1c was performed at a central laboratory ( diabetes control and complications trial [ dcct ] research group aligned ) at screening and weeks 8 , 16 , and 24 . participants were asked to perform self - monitored plasma glucose measurements four times daily ( bedtime and preprandial ) using a plasma - calibrated memory glucose meter ( onetouch ultra ; lifescan , milpitas , ca ) . eight - point profiles ( pre-/postprandial , bedtime , 0300 h ) were requested on 3 days ( including 1 weekend day ) in the 2-week period prior to each study visit . participants were asked to complete diabetes treatment satisfaction questionnaires ( dtsqs ) ( status dtsq and change dtsq ) ( 18 ) at randomization and week 24 . the costs of treatment were calculated in euros ( ) based on the cost of therapies , devices , and consumables . equipment costs include the glucose meters and consumables ( lancets and test strips ) , insulin pens and consumables ( needles ) , and insulin pumps and consumables ( batteries , and infusion sets ) . the costs of insulin were calculated by two methods : insulin dispensed and insulin used . insulin dispensed was based on the entire cost of treatment , including wastage according to dispensing records ( based on pharmacy and investigator dispensing records ) ; insulin used was based on insulin doses recorded in treatment diaries by participants . nonsevere hypoglycemia was defined as symptoms consistent with hypoglycemia not requiring the assistance of another person and confirmed by plasma glucose < 4.0 mmol / l ( < 72 mg / dl ) ; severe hypoglycemia was defined as similar symptoms but requiring management assistance and either plasma glucose < 2.0 mmol / l ( < 36 mg / dl ) or prompt recovery after oral or intravenous carbohydrate or glucagon ; nocturnal hypoglycemia was defined as between bedtime and rising . sixty randomized individuals were estimated to give 80% power , after 15% dropout , to demonstrate equivalence at 0.6% a1c at a mean a1c of 7.0% and an sd of 0.7% . ancova , with center and group as the fixed variables and baseline values as covariates , was used for continuous variables . the cochran - mantel - haenszel test , stratified by center , was used for discrete end points . two - sided 95% cis for treatment differences were calculated without adjustment for multiple end points . the mean amplitude of glycemic excursion was calculated from the eight - point plasma glucose profiles . all results are presented for the per - protocol population as is appropriate for an equivalence study to minimize bias . this population included all randomized participants treated with at least one dose of study insulin and without a major protocol deviation that would interfere with treatment efficacy . an intent - to - treat ( itt ) analysis ( all randomized patients ) was also performed as a secondary analysis . safety and hypoglycemia were reported for all participants who took at least one dose of study insulin and provided follow - up data ( safety population ) . randomization was to insulin glargine ( lantus , once daily in the evening ; sanofi - aventis , paris , france ) plus mealtime insulin lispro ( humalog , three - times daily ; eli lilly , indianapolis , in ) or to insulin lispro administered subcutaneously using a minimed 508 pump ( minimed technologies , northridge , ca ) . 5.07.7 mmol / l ( 90140 mg / dl ) , 2-h postprandial blood glucose < 7.7 mmol / l ( < 140 mg / dl ) , and bedtime blood glucose 6.18.3 mmol / l ( 110150 mg / dl ) . insulin glargine was started according to prior basal insulin dose . titration was based on the mean fpg over at least 3 days , adjusted by + 2 units if fpg was 6.68.8 mmol / l ( 120160 mg / dl ) , + 4 units if fpg was 8.811 mmol / l ( 160200 mg / dl ) , or 2 units if fpg was < 4.4 mmol / l ( < 80 mg / dl ) . further titration of the dose was performed at investigator discretion if symptoms consistent with severe hypoglycemia occurred . the initial insulin lispro dose was investigator selected , based on prior mealtime doses , and was titrated once basal insulin dose was optimized . initiation of csii was based on the pump manufacturer 's recommendations , using the normal bolus option ; a bolus wizard calculator was not used . infusion sites were advised to be changed every 23 days in accordance with investigators ' routine clinical practice and manufacturer 's recommendations . analysis of a1c was performed at a central laboratory ( diabetes control and complications trial [ dcct ] research group aligned ) at screening and weeks 8 , 16 , and 24 . participants were asked to perform self - monitored plasma glucose measurements four times daily ( bedtime and preprandial ) using a plasma - calibrated memory glucose meter ( onetouch ultra ; lifescan , milpitas , ca ) . eight - point profiles ( pre-/postprandial , bedtime , 0300 h ) were requested on 3 days ( including 1 weekend day ) in the 2-week period prior to each study visit . participants were asked to complete diabetes treatment satisfaction questionnaires ( dtsqs ) ( status dtsq and change dtsq ) ( 18 ) at randomization and week 24 . the costs of treatment were calculated in euros ( ) based on the cost of therapies , devices , and consumables . equipment costs include the glucose meters and consumables ( lancets and test strips ) , insulin pens and consumables ( needles ) , and insulin pumps and consumables ( batteries , and infusion sets ) . the costs of insulin were calculated by two methods : insulin dispensed and insulin used . insulin dispensed was based on the entire cost of treatment , including wastage according to dispensing records ( based on pharmacy and investigator dispensing records ) ; insulin used was based on insulin doses recorded in treatment diaries by participants . nonsevere hypoglycemia was defined as symptoms consistent with hypoglycemia not requiring the assistance of another person and confirmed by plasma glucose < 4.0 mmol / l ( < 72 mg / dl ) ; severe hypoglycemia was defined as similar symptoms but requiring management assistance and either plasma glucose < 2.0 mmol / l ( < 36 mg / dl ) or prompt recovery after oral or intravenous carbohydrate or glucagon ; nocturnal hypoglycemia was defined as between bedtime and rising . sixty randomized individuals were estimated to give 80% power , after 15% dropout , to demonstrate equivalence at 0.6% a1c at a mean a1c of 7.0% and an sd of 0.7% . ancova , with center and group as the fixed variables and baseline values as covariates , was used for continuous variables . the cochran - mantel - haenszel test , stratified by center , was used for discrete end points . two - sided 95% cis for treatment differences were calculated without adjustment for multiple end points . the mean amplitude of glycemic excursion was calculated from the eight - point plasma glucose profiles . all results are presented for the per - protocol population as is appropriate for an equivalence study to minimize bias . this population included all randomized participants treated with at least one dose of study insulin and without a major protocol deviation that would interfere with treatment efficacy . an intent - to - treat ( itt ) analysis ( all randomized patients ) was also performed as a secondary analysis . safety and hypoglycemia were reported for all participants who took at least one dose of study insulin and provided follow - up data ( safety population ) . of 67 people screened , 58 met inclusion criteria and were randomized : 28 to csii and 30 to mdi . of 57 patients treated , 7 were protocol violators , 1 had a baseline a1c > 9.0% , 1 had previously used insulin glargine / csii , 1 had no c - peptide status , and 4 had used corticosteroids / glucocorticoids . therefore , the population analyzed ( per - protocol population , n = 50 ) included 24 patients in the csii group and 26 in the mdi group ( table 1 ) ; the itt and safety analysis comprised 57 patients ( screening failures excluded ) . three withdrawing consent , one due to an ae , one due to pregnancy , and two due to protocol noncompliance . baseline characteristics of the people with type 1 diabetes studied ( per - protocol population ) data are means sd . the per - protocol population consisted of patients who were correctly randomized and receiving study insulin . after randomization , basal and prandial doses were 17.2 8.5 and 17.4 6.7 units / day , respectively ( total : 34.6 13.0 units / day ) , and at 24 weeks 18.2 8.0 and 18.0 6.1 units / day , respectively ( total : 36.2 11.5 units / day ) . in the mdi group after randomization , basal and prandial doses were 19.6 6.3 and 25.7 12.0 units / day , respectively ( total : 45.3 15.0 units / day ) , and at 24 weeks 22.5 7.0 and 20.1 9.8 units / day , respectively ( total : 42.6 15.5 units / day ) . a similar decrease in a1c from baseline to end point was observed in the two groups ( fig . 1a ) : 0.7 0.7% on csii and 0.6 0.8% on mdi , with a baseline - adjusted difference of 0.1% ( 95% ci 0.5 to 0.3 ) . mean blood glucose decreased in both treatment groups ( table 2 ) ( fig . 1 ) , with a difference of 0.03 mmol / l ( 0.8 to 0.8 ) ( 0.6 mg / dl [ 13.5 to 14.7 ] ) . a : data are the change in a1c ( % ) from baseline over the course of the study in the per - protocol population with type 1 diabetes managed with csii with insulin lispro ( insulin pump therapy ) or insulin glargine plus insulin lispro mdi therapy . b : data are the mean eight - point blood glucose profiles at baseline and end point over the course of the study for people in the per - protocol population randomized to insulin glargine plus insulin lispro mdi therapy . c : data are the mean eight - point blood glucose profiles at baseline and end point over the course of the study for people in the per - protocol population randomized to csii with insulin lispro ( insulin pump therapy ) . d : data are the number of hypoglycemic events by visit for the two insulin regimens ( safety population ) . summary of blood glucose control results ( per - protocol population ) data are means sd unless otherwise indicated . the per - protocol population consisted of patients who were correctly randomized and receiving study insulin . mage , mean amplitude of glycemic excursion ; smbg , self - monitored blood glucose . fasting blood glucose levels improved in both groups ( table 2 ) , with a difference of 0.7 mmol / l ( 95% ci 1.8 to 0.5 ) ( 12.3 mg / dl [ 32.9 to 8.2 ] ) . coefficient of variation ( cv ) of fasting blood glucose was unchanged ( csii from 41 12 to 42 13% ; mdi from 43 18 to 45 12% ) and did not differ between groups ( difference : 2.4% [ 9.5 to 4.7 ] ) . preprandial blood glucose levels decreased in both groups ( table 2 ) , with a treatment difference of 0.9 mmol / l ( 2.3 to 0.4 ) ( 17.1 mg / dl [ 42.1 to 8.0 ] ) . postprandial blood glucose levels decreased in the csii group and increased in the mdi group ( table 2 ) but with no statistically significant differences between groups ( difference : 0.3 mmol / l [ 1.1 to 1.7 ] ) ( 5.5 mg / dl [ 18.9 to 29.9 ] ) . at 0300 h , plasma glucose increased with both csii and mdi treatment ( table 2 ) , with a treatment difference of 3.0 mmol / l ( 0.4 to 6.5 ) ( 54.8 mg / dl [ 7.2 to 116.7 ] ) . 1b and c. the cv of the eight - point blood glucose profile decreased from 53 10 to 46 8% ( csii ) and from 52 12 to 47 11% ( mdi ) , a treatment difference of 1.4% ( 95% ci 6.6 to 3.7 ) . the mean amplitude of glycemic excursion decreased similarly in both groups ( table 2 ) , with a treatment difference of 0.4 mmol / l ( 1.8 to 1.0 ) ( 7.1 mg / dl [ 31.9 to 17.8 ] ) . the itt population was comparable with the per - protocol results for all secondary end points of blood glucose control . on csii , 1,152 hypoglycemic events were recorded by 23 of 28 participants ( 82% ) , and in the mdi group 1,022 hypoglycemic events were recorded by 27 of 29 participants ( 93% ) . the incidence of hypoglycemia was similar with csii and mdi for overall ( 41 43 vs. 35 35 events / patient ; p = 0.93 ) , nonsevere ( 35 37 vs. 31 32 events / patient ; p = 0.97 ) , nocturnal ( 3 5 vs. 5 7 events / patient ; p = 0.34 ) , symptomatic ( 13 12 vs. 14 15 events / patient ; p = 0.84 ) , and asymptomatic ( 1.2 2.0 vs. 1.4 2.3 events / patient ; p = 0.95 ) hypoglycemia . the dtsq treatment satisfaction score ( sd ) increased from 22.8 8.1 at baseline to 31.5 4.9 at 24 weeks in the csii group and from 24.0 6.3 to 28.8 5.4 in the mdi group ( treatment difference : 3.1 [ 95% ci 0.16.1 ] ; p = 0.042 ) . differences between treatment groups regarding perception of hyperglycemia and hypoglycemia were not statistically significant . at 24 weeks , change dtsq values of 13.3 5.3 ( csii ) and 12.9 4.9 ( mdi ) were observed ( difference : 0.4 [ 2.4 to 3.3 ] ) , with no statistical difference between groups in perception of hyperglycemia and hypoglycemia . results of the itt analyses were comparable . at the time of study conduct , the unit cost of insulin for csii was 0.021 and for prandial and basal insulin on mdi was 0.024 and 0.025 , respectively . the average cost of insulin dispensed per subject was 295 for csii ( range 131657 ) and 293 for mdi ( 127 prandial and 166 basal ) ( range 211755 ) . the average cost of insulin used per participant in the study based on diaries was 140 for csii ( range 76283 ) and 212 for mdi ( 96 prandial and 116 basal ) ( range 135444 ) . the average cost per treatment during the study , including all items of equipment and dispensed insulin , was 3.9 times higher for insulin lispro based csii versus mdi ( 3,020 vs. 778 , respectively ) , including the cost of the pump and cannula insertion kits ( see online appendix table 1 [ available at http://dx.doi.org/10.2337/dc08-1874 ] ) . a total of 115 aes were reported , 1 of which was not treatment emergent . therefore , a total of 18 patients experienced 59 treatment - emergent aes in the csii group , and 22 patients experienced 56 treatment - emergent aes in the mdi group . one patient in the mdi group withdrew due to an ae ( skin rash ) and one due to pregnancy . two individuals on csii each reported one serious ae ( both of severe hypoglycemia ) . three participants from three centers had pumps replaced once , as soon as failure was suspected . seven patients had one occlusion reported ; one of these later experienced two additional events and had six changes of the giving set as a preventative measure due to an episode of infection at the infusion site ( not recorded as occlusion events ) ; one participant had two events , and one reported nine events . of 67 people screened , 58 met inclusion criteria and were randomized : 28 to csii and 30 to mdi . of 57 patients treated , 7 were protocol violators , 1 had a baseline a1c > 9.0% , 1 had previously used insulin glargine / csii , 1 had no c - peptide status , and 4 had used corticosteroids / glucocorticoids . therefore , the population analyzed ( per - protocol population , n = 50 ) included 24 patients in the csii group and 26 in the mdi group ( table 1 ) ; the itt and safety analysis comprised 57 patients ( screening failures excluded ) . three withdrawing consent , one due to an ae , one due to pregnancy , and two due to protocol noncompliance . baseline characteristics of the people with type 1 diabetes studied ( per - protocol population ) data are means sd . the per - protocol population consisted of patients who were correctly randomized and receiving study insulin . in the csii group after randomization , basal and prandial doses were 17.2 8.5 and 17.4 6.7 units / day , respectively ( total : 34.6 13.0 units / day ) , and at 24 weeks 18.2 8.0 and 18.0 6.1 units / day , respectively ( total : 36.2 11.5 units / day ) . in the mdi group after randomization , basal and prandial doses were 19.6 6.3 and 25.7 12.0 units / day , respectively ( total : 45.3 15.0 units / day ) , and at 24 weeks 22.5 7.0 and 20.1 9.8 units / day , respectively ( total : 42.6 15.5 units / day ) . a similar decrease in a1c from baseline to end point was observed in the two groups ( fig . 1a ) : 0.7 0.7% on csii and 0.6 0.8% on mdi , with a baseline - adjusted difference of 0.1% ( 95% ci 0.5 to 0.3 ) . mean blood glucose decreased in both treatment groups ( table 2 ) ( fig . 1 ) , with a difference of 0.03 mmol / l ( 0.8 to 0.8 ) ( 0.6 mg / dl [ 13.5 to 14.7 ] ) . a : data are the change in a1c ( % ) from baseline over the course of the study in the per - protocol population with type 1 diabetes managed with csii with insulin lispro ( insulin pump therapy ) or insulin glargine plus insulin lispro mdi therapy . b : data are the mean eight - point blood glucose profiles at baseline and end point over the course of the study for people in the per - protocol population randomized to insulin glargine plus insulin lispro mdi therapy . c : data are the mean eight - point blood glucose profiles at baseline and end point over the course of the study for people in the per - protocol population randomized to csii with insulin lispro ( insulin pump therapy ) . d : data are the number of hypoglycemic events by visit for the two insulin regimens ( safety population ) . summary of blood glucose control results ( per - protocol population ) data are means sd unless otherwise indicated . the per - protocol population consisted of patients who were correctly randomized and receiving study insulin . mage , mean amplitude of glycemic excursion ; smbg , self - monitored blood glucose . fasting blood glucose levels improved in both groups ( table 2 ) , with a difference of 0.7 mmol / l ( 95% ci 1.8 to 0.5 ) ( 12.3 mg / dl [ 32.9 to 8.2 ] ) . coefficient of variation ( cv ) of fasting blood glucose was unchanged ( csii from 41 12 to 42 13% ; mdi from 43 18 to 45 12% ) and did not differ between groups ( difference : 2.4% [ 9.5 to 4.7 ] ) . preprandial blood glucose levels decreased in both groups ( table 2 ) , with a treatment difference of 0.9 mmol / l ( 2.3 to 0.4 ) ( 17.1 mg / dl [ 42.1 to 8.0 ] ) . postprandial blood glucose levels decreased in the csii group and increased in the mdi group ( table 2 ) but with no statistically significant differences between groups ( difference : 0.3 mmol / l [ 1.1 to 1.7 ] ) ( 5.5 mg / dl [ 18.9 to 29.9 ] ) . at 0300 h , plasma glucose increased with both csii and mdi treatment ( table 2 ) , with a treatment difference of 3.0 mmol / l ( 0.4 to 6.5 ) ( 54.8 mg / dl [ 7.2 to 116.7 ] ) . baseline and end point eight - point blood glucose profiles are shown in fig . 1b and c. the cv of the eight - point blood glucose profile decreased from 53 10 to 46 8% ( csii ) and from 52 12 to 47 11% ( mdi ) , a treatment difference of 1.4% ( 95% ci 6.6 to 3.7 ) . the mean amplitude of glycemic excursion decreased similarly in both groups ( table 2 ) , with a treatment difference of 0.4 mmol / l ( 1.8 to 1.0 ) ( 7.1 mg / dl [ 31.9 to 17.8 ] ) . the itt population was comparable with the per - protocol results for all secondary end points of blood glucose control . on csii , 1,152 hypoglycemic events were recorded by 23 of 28 participants ( 82% ) , and in the mdi group 1,022 hypoglycemic events were recorded by 27 of 29 participants ( 93% ) . the incidence of hypoglycemia was similar with csii and mdi for overall ( 41 43 vs. 35 35 events / patient ; p = 0.93 ) , nonsevere ( 35 37 vs. 31 32 events / patient ; p = 0.97 ) , nocturnal ( 3 5 vs. 5 7 events / patient ; p = 0.34 ) , symptomatic ( 13 12 vs. 14 15 events / patient ; p = 0.84 ) , and asymptomatic ( 1.2 2.0 vs. 1.4 2.3 events / patient ; p = 0.95 ) hypoglycemia . the dtsq treatment satisfaction score ( sd ) increased from 22.8 8.1 at baseline to 31.5 4.9 at 24 weeks in the csii group and from 24.0 6.3 to 28.8 5.4 in the mdi group ( treatment difference : 3.1 [ 95% ci 0.16.1 ] ; p = 0.042 ) . differences between treatment groups regarding perception of hyperglycemia and hypoglycemia were not statistically significant . at 24 weeks , change dtsq values of 13.3 5.3 ( csii ) and 12.9 4.9 ( mdi ) were observed ( difference : 0.4 [ 2.4 to 3.3 ] ) , with no statistical difference between groups in perception of hyperglycemia and hypoglycemia . at the time of study conduct , the unit cost of insulin for csii was 0.021 and for prandial and basal insulin on mdi was 0.024 and 0.025 , respectively . the average cost of insulin dispensed per subject was 295 for csii ( range 131657 ) and 293 for mdi ( 127 prandial and 166 basal ) ( range 211755 ) . the average cost of insulin used per participant in the study based on diaries was 140 for csii ( range 76283 ) and 212 for mdi ( 96 prandial and 116 basal ) ( range 135444 ) . the average cost per treatment during the study , including all items of equipment and dispensed insulin , was 3.9 times higher for insulin lispro based csii versus mdi ( 3,020 vs. 778 , respectively ) , including the cost of the pump and cannula insertion kits ( see online appendix table 1 [ available at http://dx.doi.org/10.2337/dc08-1874 ] ) . a total of 115 aes were reported , 1 of which was not treatment emergent . therefore , a total of 18 patients experienced 59 treatment - emergent aes in the csii group , and 22 patients experienced 56 treatment - emergent aes in the mdi group . one patient in the mdi group withdrew due to an ae ( skin rash ) and one due to pregnancy . two individuals on csii each reported one serious ae ( both of severe hypoglycemia ) . three participants from three centers had pumps replaced once , as soon as failure was suspected . seven patients had one occlusion reported ; one of these later experienced two additional events and had six changes of the giving set as a preventative measure due to an episode of infection at the infusion site ( not recorded as occlusion events ) ; one participant had two events , and one reported nine events . this study suggests that the optimization of basal nph insulin replacement with insulin lispro by csii or an mdi - based regimen of once - daily insulin glargine plus insulin lispro results in similar improvement of glycemic control in people with type 1 diabetes who are nave to either treatment regimen . both regimens achieved similar improvements in a1c , self - monitored plasma glucose , and hypoglycemia . the best estimate of difference between the regimens is 0.1% a1c , but study power implies that the actual difference could be between a csii advantage of 0.5% and an mdi advantage of 0.3% . the present study is limited by the number of participants and a duration of 6 months but has the advantage of being the first prospective , multicenter study demonstrating noninferiority of the insulin glargine based regimen versus csii . another possible limitation of the present study is that the minimed 508 pump , rather than the recent paradigm 522 or 722 pump with the bolus wizard , was used . the latter pumps might be helpful to estimate bolus doses by calculating the insulin - to - carbohydrate ratio , insulin sensitivity factor , target blood glucose , and insulin on board ( putative length of action of insulin bolus ) . however , randomized , controlled trials are required in the adult population to prove these benefits . the mean end - of - study a1c levels for both regimens were close to american diabetes association targets and were similar to those in the intensive arm of the dcct trial ( 1,19 ) . although the baseline levels were higher on nph insulin regimens , the present study did not examine the use of nph insulin and the fall from baseline may have been a study effect , though consistent with previous comparisons of csii and insulin glargine based mdi with nph insulin based mdi ( 16,17 ) . however , nph insulin regimens can be optimized by multiple nph insulin injections ( 20 ) . as expected , intensification of blood glucose control and careful recording resulted in increased reported rates of nonsevere hypoglycemia with no difference between regimens . severe hypoglycemia was rare , but the inclusion criteria excluded people prone to this problem . others have noted that the dcct - derived conclusion of an increase in severe hypoglycemia is not necessarily reproduced when intensive insulin therapy is based on insulin analogs ( 9,10 ) . although blood glucose control with csii and insulin glargine based mdi was similar by other measures , plasma glucose predinner was higher with the latter regimen . this elevation in blood glucose in the late afternoon hours on an insulin glargine based mdi regimen has been noted previously ( 21 ) . in some people with type 1 diabetes , the duration of action of insulin glargine appears to be < 24 h. however , continued food absorption beyond the duration of action of the lunchtime insulin may be another cause of predinner hyperglycemia , as the phenomenon is attenuated when lunch is skipped ( 21 ) . with csii it is possible that this was counteracted by higher basal rates in the afternoon , but these data were not collected . with insulin glargine , an additional bolus of rapid - acting analog 23 h after lunch can be given ( 21 ) ; this was not part of the protocol of the present study . interestingly , the theoretical advantage of multiple basal rates with csii did not translate into lower basal plasma glucose levels at other times of day ( fig . furthermore , this study confirms that the magnitude of any dawn phenomenon is modest when basal insulin is optimally replaced with either csii or an insulin glargine regimen ( 22 ) . in the present study , glucose variability with csii and insulin glargine based mdi was not different . a previous study ( 23 ) that compared people on long - term csii with those switched to insulin glargine based mdi found a small advantage of csii in glucose variability , despite no difference in overall glycemic control . however , the importance of variability of blood glucose when a1c , hypoglycemia , and plasma glucose profile are identical is doubtful . other data support the importance of glucose variability for vascular complications in diabetes , but clinical studies have yet to confirm this ( 24 ) . that the costs of csii are higher than those of mdi is perhaps obvious given the costs of pumps and infusion sets . there will also be a small saving in any market where insulin glargine is more expensive than insulin lispro . in economic terms , with similar efficacy but higher costs , csii is dominated by mdi . the national institute for health and clinical excellence ( u.k . ) calculated a higher cost in the order of 1,392 to 1,772 per year , compatible with our results ( 25 ) . the present study adds to previous observations ( 14,15,17 ) of the noninferiority of insulin glargine based mdi versus csii in different study designs . a similar conclusion to ours was recently reported ( 23 ) in a study in which people with type 1 diabetes were transferred from long - term csii initiated primarily in the nph insulin era to insulin glargine based mdi . thus , it is likely that the majority of people with type 1 diabetes who started csii in the nph insulin era did so due to the poor performance of basal nph insulin . similarly , it is likely that the majority of these people might today switch to mdi based on insulin glargine with no deterioration of blood glucose control ( 23 ) . while the conclusions of the present study establish noninferiority of insulin glargine based mdi against csii in unselected ( except for study purposes ) people with type 1 diabetes , they can not be applied to selected people with type 1 diabetes who may have special indications to csii , such as long duration of disease with low insulin requirements or hypoglycemia unawareness with frequent , severe hypoglycemia on long - acting insulin analog based mdi ( 6 ) . additional studies are required in these groups of selected people with type 1 diabetes to establish the possible equivalence or otherwise of insulin glargine based mdi against csii with regard to glycemic control and the incidence and awareness of hypoglycemia .

objectiveinsulin pump therapy ( continuous subcutaneous insulin infusion [ csii ] ) and multiple daily injections ( mdis ) with insulin glargine as basal insulin and mealtime insulin lispro have not been prospectively compared in people nave to either regimen in a multicenter study . we aimed to help close that deficiency.research design and methodspeople with type 1 diabetes on nph - based insulin therapy were randomized to csii or glargine - based mdi ( both otherwise using lispro ) and followed for 24 weeks in an equivalence design . fifty people were correctly randomized , and 43 completed the study.resultstotal insulin requirement ( mean sd ) at end point was 36.2 11.5 units / day on csii and 42.6 15.5 units / day on mdi . mean a1c fell similarly in the two groups ( csii 0.7 0.7% ; mdi 0.6 0.8% ) with a baseline - adjusted difference of 0.1% ( 95% ci 0.5 to 0.3 ) . similarly , fasting blood glucose and other preprandial , postprandial , and nighttime self - monitored plasma glucose levels did not differ between the regimens , nor did measures of plasma glucose variability . on csii , 1,152 hypoglycemia events were recorded by 23 of 28 participants ( 82% ) and 1,022 in the mdi group by 27 of 29 patients ( 93% ) ( all hypoglycemia differences were nonsignificant ) . treatment satisfaction score increased more with csii ; however , the change in score was similar for the groups . costs were 3.9 times higher for csii.conclusionsin unselected people with type 1 diabetes nave to csii or insulin glargine , glycemic control is no better with the more expensive csii therapy compared with glargine - based mdi therapy .

RESEARCH DESIGN AND METHODS Study treatments Study measurements Statistical analyses RESULTS Study population Insulin dose Blood glucose control Hypoglycemia Treatment satisfaction Costs Safety profile CONCLUSIONS Supplementary Material

our primary objective was to investigate whether an mdi regimen using insulin glargine as basal insulin with insulin lispro at mealtimes can achieve glycemic control ( a1c ) equivalent to csii ( insulin lispro ) in people with type 1 diabetes . secondary objectives were to compare other measures of blood glucose control ( pre- and postprandial , bedtime and 0300 h levels , and within - day glucose variability ) , hypoglycemia , adverse events ( aes ) , treatment costs , and treatment satisfaction . this was a randomized , parallel - group , open - label , multicenter ( n = 5 ) study performed in three european countries with a 1-week run - in period followed by a 24-week treatment period ( including 4 weeks of active dose titration ) , with clinic visits at 0 , 2 , 8 , 16 , and 24 weeks and 2 weeks follow - up . participants were aged 1870 years , with a bmi 27.0 kg / m and diabetes for > 1 year , a1c 6.59.0% , fasting plasma glucose ( fpg ) > 7.0 mmol / l ( > 126 mg / dl ) , and fasting plasma c - peptide < 0.10 nmol / l at the screening visit . participants were excluded if they were prior users of csii or insulin glargine , were unwilling or unable to use csii or mdis , had more than two severe hypoglycemic events in the previous 6 months , or had recent diabetic ketoacidosis or impaired hepatic / renal function . randomization was to insulin glargine ( lantus , once daily in the evening ; sanofi - aventis , paris , france ) plus mealtime insulin lispro ( humalog , three - times daily ; eli lilly , indianapolis , in ) or to insulin lispro administered subcutaneously using a minimed 508 pump ( minimed technologies , northridge , ca ) . participants were asked to perform self - monitored plasma glucose measurements four times daily ( bedtime and preprandial ) using a plasma - calibrated memory glucose meter ( onetouch ultra ; lifescan , milpitas , ca ) . participants were asked to perform self - monitored plasma glucose measurements four times daily ( bedtime and preprandial ) using a plasma - calibrated memory glucose meter ( onetouch ultra ; lifescan , milpitas , ca ) . therefore , the population analyzed ( per - protocol population , n = 50 ) included 24 patients in the csii group and 26 in the mdi group ( table 1 ) ; the itt and safety analysis comprised 57 patients ( screening failures excluded ) . baseline characteristics of the people with type 1 diabetes studied ( per - protocol population ) data are means sd . after randomization , basal and prandial doses were 17.2 8.5 and 17.4 6.7 units / day , respectively ( total : 34.6 13.0 units / day ) , and at 24 weeks 18.2 8.0 and 18.0 6.1 units / day , respectively ( total : 36.2 11.5 units / day ) . in the mdi group after randomization , basal and prandial doses were 19.6 6.3 and 25.7 12.0 units / day , respectively ( total : 45.3 15.0 units / day ) , and at 24 weeks 22.5 7.0 and 20.1 9.8 units / day , respectively ( total : 42.6 15.5 units / day ) . a similar decrease in a1c from baseline to end point was observed in the two groups ( fig . 1a ) : 0.7 0.7% on csii and 0.6 0.8% on mdi , with a baseline - adjusted difference of 0.1% ( 95% ci 0.5 to 0.3 ) . 1 ) , with a difference of 0.03 mmol / l ( 0.8 to 0.8 ) ( 0.6 mg / dl [ 13.5 to 14.7 ] ) . a : data are the change in a1c ( % ) from baseline over the course of the study in the per - protocol population with type 1 diabetes managed with csii with insulin lispro ( insulin pump therapy ) or insulin glargine plus insulin lispro mdi therapy . b : data are the mean eight - point blood glucose profiles at baseline and end point over the course of the study for people in the per - protocol population randomized to insulin glargine plus insulin lispro mdi therapy . c : data are the mean eight - point blood glucose profiles at baseline and end point over the course of the study for people in the per - protocol population randomized to csii with insulin lispro ( insulin pump therapy ) . fasting blood glucose levels improved in both groups ( table 2 ) , with a difference of 0.7 mmol / l ( 95% ci 1.8 to 0.5 ) ( 12.3 mg / dl [ 32.9 to 8.2 ] ) . coefficient of variation ( cv ) of fasting blood glucose was unchanged ( csii from 41 12 to 42 13% ; mdi from 43 18 to 45 12% ) and did not differ between groups ( difference : 2.4% [ 9.5 to 4.7 ] ) . preprandial blood glucose levels decreased in both groups ( table 2 ) , with a treatment difference of 0.9 mmol / l ( 2.3 to 0.4 ) ( 17.1 mg / dl [ 42.1 to 8.0 ] ) . postprandial blood glucose levels decreased in the csii group and increased in the mdi group ( table 2 ) but with no statistically significant differences between groups ( difference : 0.3 mmol / l [ 1.1 to 1.7 ] ) ( 5.5 mg / dl [ 18.9 to 29.9 ] ) . at 0300 h , plasma glucose increased with both csii and mdi treatment ( table 2 ) , with a treatment difference of 3.0 mmol / l ( 0.4 to 6.5 ) ( 54.8 mg / dl [ 7.2 to 116.7 ] ) . 1b and c. the cv of the eight - point blood glucose profile decreased from 53 10 to 46 8% ( csii ) and from 52 12 to 47 11% ( mdi ) , a treatment difference of 1.4% ( 95% ci 6.6 to 3.7 ) . the mean amplitude of glycemic excursion decreased similarly in both groups ( table 2 ) , with a treatment difference of 0.4 mmol / l ( 1.8 to 1.0 ) ( 7.1 mg / dl [ 31.9 to 17.8 ] ) . on csii , 1,152 hypoglycemic events were recorded by 23 of 28 participants ( 82% ) , and in the mdi group 1,022 hypoglycemic events were recorded by 27 of 29 participants ( 93% ) . the incidence of hypoglycemia was similar with csii and mdi for overall ( 41 43 vs. 35 35 events / patient ; p = 0.93 ) , nonsevere ( 35 37 vs. 31 32 events / patient ; p = 0.97 ) , nocturnal ( 3 5 vs. 5 7 events / patient ; p = 0.34 ) , symptomatic ( 13 12 vs. 14 15 events / patient ; p = 0.84 ) , and asymptomatic ( 1.2 2.0 vs. 1.4 2.3 events / patient ; p = 0.95 ) hypoglycemia . the dtsq treatment satisfaction score ( sd ) increased from 22.8 8.1 at baseline to 31.5 4.9 at 24 weeks in the csii group and from 24.0 6.3 to 28.8 5.4 in the mdi group ( treatment difference : 3.1 [ 95% ci 0.16.1 ] ; p = 0.042 ) . at 24 weeks , change dtsq values of 13.3 5.3 ( csii ) and 12.9 4.9 ( mdi ) were observed ( difference : 0.4 [ 2.4 to 3.3 ] ) , with no statistical difference between groups in perception of hyperglycemia and hypoglycemia . the average cost of insulin used per participant in the study based on diaries was 140 for csii ( range 76283 ) and 212 for mdi ( 96 prandial and 116 basal ) ( range 135444 ) . the average cost per treatment during the study , including all items of equipment and dispensed insulin , was 3.9 times higher for insulin lispro based csii versus mdi ( 3,020 vs. 778 , respectively ) , including the cost of the pump and cannula insertion kits ( see online appendix table 1 [ available at http://dx.doi.org/10.2337/dc08-1874 ] ) . one patient in the mdi group withdrew due to an ae ( skin rash ) and one due to pregnancy . of 57 patients treated , 7 were protocol violators , 1 had a baseline a1c > 9.0% , 1 had previously used insulin glargine / csii , 1 had no c - peptide status , and 4 had used corticosteroids / glucocorticoids . therefore , the population analyzed ( per - protocol population , n = 50 ) included 24 patients in the csii group and 26 in the mdi group ( table 1 ) ; the itt and safety analysis comprised 57 patients ( screening failures excluded ) . baseline characteristics of the people with type 1 diabetes studied ( per - protocol population ) data are means sd . in the csii group after randomization , basal and prandial doses were 17.2 8.5 and 17.4 6.7 units / day , respectively ( total : 34.6 13.0 units / day ) , and at 24 weeks 18.2 8.0 and 18.0 6.1 units / day , respectively ( total : 36.2 11.5 units / day ) . in the mdi group after randomization , basal and prandial doses were 19.6 6.3 and 25.7 12.0 units / day , respectively ( total : 45.3 15.0 units / day ) , and at 24 weeks 22.5 7.0 and 20.1 9.8 units / day , respectively ( total : 42.6 15.5 units / day ) . a similar decrease in a1c from baseline to end point was observed in the two groups ( fig . 1a ) : 0.7 0.7% on csii and 0.6 0.8% on mdi , with a baseline - adjusted difference of 0.1% ( 95% ci 0.5 to 0.3 ) . 1 ) , with a difference of 0.03 mmol / l ( 0.8 to 0.8 ) ( 0.6 mg / dl [ 13.5 to 14.7 ] ) . a : data are the change in a1c ( % ) from baseline over the course of the study in the per - protocol population with type 1 diabetes managed with csii with insulin lispro ( insulin pump therapy ) or insulin glargine plus insulin lispro mdi therapy . b : data are the mean eight - point blood glucose profiles at baseline and end point over the course of the study for people in the per - protocol population randomized to insulin glargine plus insulin lispro mdi therapy . c : data are the mean eight - point blood glucose profiles at baseline and end point over the course of the study for people in the per - protocol population randomized to csii with insulin lispro ( insulin pump therapy ) . fasting blood glucose levels improved in both groups ( table 2 ) , with a difference of 0.7 mmol / l ( 95% ci 1.8 to 0.5 ) ( 12.3 mg / dl [ 32.9 to 8.2 ] ) . coefficient of variation ( cv ) of fasting blood glucose was unchanged ( csii from 41 12 to 42 13% ; mdi from 43 18 to 45 12% ) and did not differ between groups ( difference : 2.4% [ 9.5 to 4.7 ] ) . preprandial blood glucose levels decreased in both groups ( table 2 ) , with a treatment difference of 0.9 mmol / l ( 2.3 to 0.4 ) ( 17.1 mg / dl [ 42.1 to 8.0 ] ) . postprandial blood glucose levels decreased in the csii group and increased in the mdi group ( table 2 ) but with no statistically significant differences between groups ( difference : 0.3 mmol / l [ 1.1 to 1.7 ] ) ( 5.5 mg / dl [ 18.9 to 29.9 ] ) . at 0300 h , plasma glucose increased with both csii and mdi treatment ( table 2 ) , with a treatment difference of 3.0 mmol / l ( 0.4 to 6.5 ) ( 54.8 mg / dl [ 7.2 to 116.7 ] ) . 1b and c. the cv of the eight - point blood glucose profile decreased from 53 10 to 46 8% ( csii ) and from 52 12 to 47 11% ( mdi ) , a treatment difference of 1.4% ( 95% ci 6.6 to 3.7 ) . the mean amplitude of glycemic excursion decreased similarly in both groups ( table 2 ) , with a treatment difference of 0.4 mmol / l ( 1.8 to 1.0 ) ( 7.1 mg / dl [ 31.9 to 17.8 ] ) . on csii , 1,152 hypoglycemic events were recorded by 23 of 28 participants ( 82% ) , and in the mdi group 1,022 hypoglycemic events were recorded by 27 of 29 participants ( 93% ) . the dtsq treatment satisfaction score ( sd ) increased from 22.8 8.1 at baseline to 31.5 4.9 at 24 weeks in the csii group and from 24.0 6.3 to 28.8 5.4 in the mdi group ( treatment difference : 3.1 [ 95% ci 0.16.1 ] ; p = 0.042 ) . at 24 weeks , change dtsq values of 13.3 5.3 ( csii ) and 12.9 4.9 ( mdi ) were observed ( difference : 0.4 [ 2.4 to 3.3 ] ) , with no statistical difference between groups in perception of hyperglycemia and hypoglycemia . at the time of study conduct , the unit cost of insulin for csii was 0.021 and for prandial and basal insulin on mdi was 0.024 and 0.025 , respectively . the average cost of insulin used per participant in the study based on diaries was 140 for csii ( range 76283 ) and 212 for mdi ( 96 prandial and 116 basal ) ( range 135444 ) . the average cost per treatment during the study , including all items of equipment and dispensed insulin , was 3.9 times higher for insulin lispro based csii versus mdi ( 3,020 vs. 778 , respectively ) , including the cost of the pump and cannula insertion kits ( see online appendix table 1 [ available at http://dx.doi.org/10.2337/dc08-1874 ] ) . therefore , a total of 18 patients experienced 59 treatment - emergent aes in the csii group , and 22 patients experienced 56 treatment - emergent aes in the mdi group . one patient in the mdi group withdrew due to an ae ( skin rash ) and one due to pregnancy . this study suggests that the optimization of basal nph insulin replacement with insulin lispro by csii or an mdi - based regimen of once - daily insulin glargine plus insulin lispro results in similar improvement of glycemic control in people with type 1 diabetes who are nave to either treatment regimen . both regimens achieved similar improvements in a1c , self - monitored plasma glucose , and hypoglycemia . the best estimate of difference between the regimens is 0.1% a1c , but study power implies that the actual difference could be between a csii advantage of 0.5% and an mdi advantage of 0.3% . although the baseline levels were higher on nph insulin regimens , the present study did not examine the use of nph insulin and the fall from baseline may have been a study effect , though consistent with previous comparisons of csii and insulin glargine based mdi with nph insulin based mdi ( 16,17 ) . although blood glucose control with csii and insulin glargine based mdi was similar by other measures , plasma glucose predinner was higher with the latter regimen . this elevation in blood glucose in the late afternoon hours on an insulin glargine based mdi regimen has been noted previously ( 21 ) . in some people with type 1 diabetes , the duration of action of insulin glargine appears to be < 24 h. however , continued food absorption beyond the duration of action of the lunchtime insulin may be another cause of predinner hyperglycemia , as the phenomenon is attenuated when lunch is skipped ( 21 ) . interestingly , the theoretical advantage of multiple basal rates with csii did not translate into lower basal plasma glucose levels at other times of day ( fig . in the present study , glucose variability with csii and insulin glargine based mdi was not different . a previous study ( 23 ) that compared people on long - term csii with those switched to insulin glargine based mdi found a small advantage of csii in glucose variability , despite no difference in overall glycemic control . however , the importance of variability of blood glucose when a1c , hypoglycemia , and plasma glucose profile are identical is doubtful . a similar conclusion to ours was recently reported ( 23 ) in a study in which people with type 1 diabetes were transferred from long - term csii initiated primarily in the nph insulin era to insulin glargine based mdi . thus , it is likely that the majority of people with type 1 diabetes who started csii in the nph insulin era did so due to the poor performance of basal nph insulin . while the conclusions of the present study establish noninferiority of insulin glargine based mdi against csii in unselected ( except for study purposes ) people with type 1 diabetes , they can not be applied to selected people with type 1 diabetes who may have special indications to csii , such as long duration of disease with low insulin requirements or hypoglycemia unawareness with frequent , severe hypoglycemia on long - acting insulin analog based mdi ( 6 ) . additional studies are required in these groups of selected people with type 1 diabetes to establish the possible equivalence or otherwise of insulin glargine based mdi against csii with regard to glycemic control and the incidence and awareness of hypoglycemia .

autism spectrum disorders ( asd ) are defined as a heterogeneous group of neurodevelopmental disorders characterized by impairments in communication and social interactions along with restrictive and repetitive behaviors . the incidence of asd in the united states is currently estimated to be 1 in 68 individuals , and it continues to rise . while the etiology of asd remains unknown , multiple interacting genetic and environmental factors are thought to contribute to the development of asd . in addition to behavioral impairments , recent studies indicate that many children with asd also exhibit impairments in energy production and redox homeostasis [ 35 ] . multiple studies have demonstrated the presence of glutathione - mediated redox imbalance and oxidative stress in individuals with asd [ 511 ] . our group has consistently reported decreased concentrations of glutathione ( gsh ) and several of its metabolic precursors as well as increased oxidized glutathione disulfide ( gssg ) and a decreased glutathione redox ratio ( gsh / gssg ) in plasma , immune cells , and postmortem brain from children with asd [ 4 , 5 , 1113 ] . oxidative stress and damage have been documented in blood and brain of individuals with asd including reports of decreased levels and activities of antioxidant enzymes and elevated levels of oxidized lipids , proteins , and dna [ 4 , 7 , 8 , 11 , 14 , 15 ] . in primary lymphocytes and in lymphoblastoid cell lines ( lcls ) derived from children with autistic disorder ( ad ) , we have found that the production of reactive oxygen species ( ros ) is elevated as compared to controls [ 12 , 13 , 16 ] . the imbalance between glutathione - mediated antioxidant capacity and ros production in autism lcls may cause these cells to be more susceptible to oxidative stress and damage from any exogenous sources of ros as compared to control lcls . recent evidence indicates that the incidence of mitochondrial dysfunction in asd may be very high , affecting up to 30% or more of children with asd . while the etiology of mitochondrial dysfunction in asd is not known , evidence suggests that oxidative stress may be a key factor driving mitochondrial dysfunction in individuals with asd [ 16 , 18 ] . recently , we demonstrated that lcls derived from children with ad exhibit abnormal mitochondrial respiration at baseline as well as a more rapid decline in mitochondrial function upon exposure to increasing ros as compared to lcls from control children . importantly , we found that these abnormal mitochondrial parameters were driven by a subgroup consisting of 32% of the ad lcls ( termed ad - a for abnormal ) , whereas the other autism lcls ( termed ad - n for normal ) had mitochondrial parameters similar to controls . furthermore , we also demonstrated that pretreatment of the ad lcls with n - acetyl cysteine ( nac ) increased intracellular gsh and the gsh / gssg redox ratio and , in the ad - a subgroup , conferred protection from mitochondrial dysfunction when ros was increased . mitochondria are both the primary producers and targets of intracellular ros due to the continuous low - level production of superoxide that accompanies electron transfer across the inner mitochondrial membrane during oxidative phosphorylation . ros are also generated from other sources such as activated immune cells [ 3 , 17 ] and prooxidant environmental toxicants such as pesticides , diesel exhaust , and mercury [ 2029 ] . mercury is one of several environmental toxicants that have been found to have an association with the development of asd [ 28 , 3034 ] . ethylmercury is an established a sulfhydryl reagent that rapidly binds to and depletes intracellular glutathione and increases intracellular ros in a dose - dependent manner [ 12 , 35 ] . we have previously demonstrated that ad lcls have increased susceptibility to oxidative stress from exposure to ethylmercury such that exposure to ethylmercury resulted in lower intracellular gsh and gsh / gssg and increased ros production in ad lcls as compared to control lcls . in the present study we tested the hypothesis that the subset of ad lcls previously found to exhibit mitochondrial dysfunction when challenged with ros would also exhibit mitochondrial dysfunction when exposed to ethylmercury ( i.e. , ethylmercury - induced mitochondrial dysfunction ) . furthermore , we hypothesized that pretreatment with nac to increase the intracellular glutathione concentration would confer protection from ethylmercury - induced mitochondrial dysfunction . to this end , we used extracellular flux analysis to measure mitochondrial oxygen consumption in ad and control lcls transiently exposed to ethylmercury . the ad lcls were also tested after pretreatment with nac to determine whether changes in mitochondrial bioenergetics after exposure to ethylmercury could be prevented by nac - induced increase in intracellular glutathione - mediated redox capacity . sixteen lcls derived from white males diagnosed with ad chosen from pedigrees with at least 1 affected male sibling ( mean / sd age 7.9 2.6 years ) were obtained from the autism genetic resource exchange ( los angeles , ca , usa ) or the national institutes of mental health ( nimh ; bethesda , md , usa ) center for collaborative genomic studies on mental disorders . table 1 denotes the ad lcl subgroups previously classified : five were classified as ad - a ( for abnormal ) and the remaining eleven were classified as ad - n ( for normal ) . sixteen control lcls derived from healthy white male donors with no documented behavioral or neurological disorder or first - degree relative with a medical disorder that could involve abnormal mitochondrial function ( mean / sd age 19.3 11.5 y ) were obtained from coriell cell repository ( camden , nj , usa ) or the nimh . on average , cells were studied at passage 12 , with a maximum passage of 15 . genomic stability is very high at this low passage number [ 36 , 37 ] . cells were maintained in rpmi 1640 culture medium with 15% fbs and 1% penicillin / streptomycin ( invitrogen , grand island , ny , usa ) in a humidified incubator at 37c with 5% co2 . we used extracellular flux analysis ( seahorse bioscience , inc . , north billerica , ma , usa ) to measure the oxygen consumption rate ( ocr ) , an indicator of mitochondrial respiration , in real - time in live intact lcls as described . upon the sequential addition of mitochondrial electron transport chain ( etc ) inhibitors and an uncoupler to the respiring cells , several parameters of mitochondrial respiration were derived , including basal respiration , atp - linked respiration , proton leak respiration , and reserve capacity ( diagramed in figure 1 ) . briefly , after measuring basal respiration , oligomycin , an inhibitor of complex v , is added , and the resulting ocr is used to derive atp - linked respiration and proton leak respiration . next carbonyl cyanide - p - trifluoromethoxyphenylhydrazone ( fccp ) , a protonophore , is added to collapse the inner membrane gradient , allowing the etc to function to its maximal rate , and maximal respiratory capacity is derived . lastly , antimycin a , a complex iii inhibitor , and rotenone , a complex i inhibitor , are added to shut down etc function , revealing the nonmitochondrial respiration , which is subtracted from the other rates to obtain the corrected basal respiration , proton leak respiration and maximal respiratory capacity . two hours prior to the assay , cells were seeded onto poly - d - lysine coated 96-well xf - ps plates at a density of 1.1 10 cells / well in dmem xf assay media ( unbuffered dmem supplemented with 11 mm glucose , 2 mm l - glutamax , and 1 mm sodium pyruvate ) . optimal concentrations of oligomycin ( 1.0 m ) , fccp ( 0.3 m ) , antimycin a ( 0.3 m ) , and rotenone ( 1.0 m ) were carefully titrated . cells were exposed to increasing concentrations of ethylmercury for 2 h prior to the mitochondrial assay . louis , mo , usa ) , a mercurial compound composed of 49.6% ethylmercury by weight , was diluted in dmem xf assay media into 10x stocks and added to cells in an xf - ps plate and incubated for 2 h at 37c in a non - co2 incubator . the final concentrations of ethylmercury were 0.063 m , 0.125 m , 0.25 m and 0.5 m , 1.25 m , and 2.5 m . to determine whether pretreatment with a glutathione precursor could rescue any atypical response to the ros challenge , ad lcls were plated in t25 flasks at a density of 5.0 10 cells / ml in culture media with or without 1 mm nac for 48 h prior to the assay . control lcls were cultured identically without nac . cells were washed twice in dmem xf media to remove any remaining nac prior to mercury treatment and mitochondrial assays . to confirm this regimen was sufficient to remove all nac from the cells , 2.0 10 nac - pretreated cells were pelleted following the two washes and analyzed by hplc , as previously described , for the presence of nac . presented in figure 2 is a representative chromatogram which demonstrates that there was no nac remaining in the nac - pretreated cells following the two washes . a mixed - model regression was conducted via sas version 9.3 ( cary , nc , usa ) glimmix the mixed - model allowed data from each ad lcl to be compared to the paired control lcl run on the same plate . the mitochondrial respiratory parameter was the response variable with a between - group dichotomous effect ( e.g. , ad versus control ) and within - group repeated factor of ethylmercury concentration ( modeled as a multilevel factor ) as well as the interaction between these effects . we present the overall difference between the two comparison groups ( group effect ) , the overall effect of the ethylmercury concentration ( ethylmercury effect ) , and whether the effect of ethylmercury concentration was different between the two groups ( ethylmercury x group interaction ) . the same analysis was used to analyze the difference in mitochondrial respiratory parameters between each ad subgroup and matched controls . atp - linked respiration was overall higher for ad lcls as compared to control lcls [ f(1 , 662 ) = 134.55 , p < 0.0001 ] ( figure 3(a ) ) . atp - linked respiration decreased significantly as ethylmercury concentration increased [ f(4 , 89 ) = 30.95 , p < 0.0001 ] and was found to be significantly lower than baseline at 0.5 m [ t(89 ) = 3.90 , p < 0.001 ] , 1.25 m [ t(89 ) = 6.94 , p < 0.0001 ] and 2.5 m [ t(89 ) = 11.53 , p < 0.0001 ] ethylmercury in both groups . however , the decrease in atp - linked respiration with increasing ethylmercury concentration was significantly different between the ad and control groups [ f(6 , 662 ) = 2.42 , p < 0.05 ] . the differences in atp - linked respiration between the two lcl groups were significant at every concentration of ethylmercury [ 0 m t(662 ) = 9.51 ; 0.063 m t(662 ) = 4.33 ; 0.125 m t(662 ) = 4.30 ; 0.25 m t(662 ) = 3.59 ; 0.5 m t(662 ) = 4.57 ; 1.25 m t(662 ) = 3.20 ; 2.5 m t(662 ) = 2.91 ] . however , the difference was smaller at high ethylmercury concentrations because the drop in atp - linked respiration was greater for the ad lcls as compared to the control lcls as ethylmercury concentration increased . proton leak respiration was overall higher in ad lcls as compared to the control lcls [ f(1 , 662 ) = 136.09 , p < 0.0001 ] ( figure 3(b ) ) . proton leak respiration changed significantly in both groups as ethylmercury concentration increased [ f(4 , 89 ) = 2.55 , p < 0.05 ] with a significantly lower proton leak respiration at 2.5 m ethylmercury as compared to baseline [ t(89 ) = 2.64 , p < 0.01 ] , but this change was not different between the two groups . maximal respiratory capacity was overall higher for ad lcls as compared to control lcls [ f(1 , 662 ) = 100.89 , p < 0.0001 ] ( figure 3(c ) ) . maximal respiratory capacity decreased significantly as ethylmercury concentration increased [ f(4 , 89 ) = 35.90 , p < 0.0001 ] and was found to be significantly lower than baseline at 0.5 m [ t(89 ) = 4.55 , p < 0.001 ] , 1.25 m [ t(89 ) = 7.88 , p < 0.0001 ] , and 2.5 m [ t(89 ) = 11.40 , p < 0.0001 ] ethylmercury in both ad and control lcls . however , the decrease in maximal respiratory capacity with increasing ethylmercury concentration was significantly different between the ad and control groups [ f(6 , 662 ) = 3.08 , p < 0.01 ] . maximal respiratory capacity was significantly higher in the ad lcls at each concentration of ethylmercury except the highest concentration [ 0 m t(662 ) = 8.62 ; 0.063 m t(662 ) = 3.75 ; 0.125 m t(662 ) = 4.66 ; 0.25 m t(662 ) = 3.44 ; 0.5 m t(662 ) = 3.84 ; 1.25 m t(662 ) = 2.46 ] . the decrease in maximal respiratory capacity was greater for the ad lcls as compared to the control lcls as ethylmercury concentration increased . reserve capacity was overall higher for ad lcls as compared to control lcls [ f(1 , 662 ) = 76.77 , p < 0.0001 ] ( figure 3(d ) ) . reserve capacity decreased significantly as ethylmercury concentration increased [ f(4 , 89 ) = 33.54 , p < 0.0001 ] and was found to be significantly lower than baseline at 0.5 m [ t(89 ) = 4.56 , p < 0.001 ] , 1.25 m [ t(89 ) = 7.66 , p < 0.0001 ] , and 2.5 m [ t(89 ) = 10.68 , p < 0.0001 ] ethylmercury in both ad and control lcls . however , the decrease in reserve capacity with increasing ethylmercury concentration was significantly different between the ad and control groups [ f(6 , 662 ) = 3.38 , p < 0.01 ] . reserve capacity was significantly higher in the ad lcls at each concentration of ethylmercury except the highest concentration [ 0 m t(662 ) = 7.81 ; 0.063 m t(662 ) = 3.39 ; 0.125 m t(662 ) = 4.50 ; 0.25 m t(662 ) = 3.17 ; 0.5 m t(662 ) = 3.32 ; 1.25 m t(662 ) = 1.93 ] . the decrease in reserve capacity was greater for the ad lcls as compared to the control lcls as ethylmercury concentration increased . to better understand the differences between the two ad lcl subgroups we previously identified , we compared the ad lcls to their paired control lcls within each subgroup . there were no significant differences found between the two subsets of control lcls matched to the subsets of ad lcls . atp - linked respiration was markedly higher for ad - a lcls as compared to control lcls [ f(1 , 213 ) = 183.13 , p < 0.0001 ] ( figure 4(a ) ) . atp - linked respiration decreased significantly as ethylmercury concentration increased [ f(6 , 24 ) = 9.11 , p < 0.0001 ] and was found to be significantly lower than baseline at 1.25 m [ t(24 ) = 2.92 , p < 0.01 ] and 2.5 m [ t(24 ) = 5.65 , p < 0.0001 ] ethylmercury in both ad - a and control lcls . however , the decrease in atp - linked respiration with increasing ethylmercury concentration was significantly different between the two lcl groups [ f(6 , 213 ) = 3.95 , p < 0.001 ] . although the differences in atp - linked respiration between the two lcl groups were significant at each concentration of ethylmercury [ 0 m t(213 ) = 10.97 ; 0.063 m t(213 ) = 5.63 ; 0.125 m t(213 ) = 5.60 ; 0.25 m t(213 ) = 4.90 ; 0.5 m t(213 ) = 5.03 ; 1.25 m t(213 ) = 3.62 ; 2.5 m t(213 ) = 2.45 ] , the difference between the lcl groups was reduced at high ethylmercury concentrations because the drop in atp - linked respiration was greater for the ad - a lcls as compared to the control lcls as ethylmercury increased . overall , proton leak respiration was markedly higher for ad - a lcls [ f(1 , 213 ) = 124.56 , p < 0.001 ] ( figure 4(b ) ) . proton leak respiration did not significantly change as ethylmercury concentration increased nor was there any difference in the change in proton leak respiration with increasing ethylmercury concentration between the ad - a and control lcl groups . maximal respiratory capacity was markedly higher for ad - a lcls as compared to control lcls [ f(1 , 213 ) = 148.63 , p < 0.0001 ] ( figure 4(c ) ) . maximal respiratory capacity decreased significantly as the ethylmercury concentration increased [ f(6 , 24 ) = 8.09 , p < 0.0001 ] and was found to be significantly lower than baseline at 1.25 m [ t(24 ) = 3.12 , p < 0.01 ] and 2.5 m [ t(24 ) = 4.94 , p < 0.0001 ] ethylmercury in both ad - a and control lcls . however , the decrease in maximal respiratory capacity with increasing ethylmercury concentration was significantly different between the two lcl groups [ f(6 , 213 ) = 5.00 , p < 0.0001 ] . the differences in maximal respiratory capacity between the two lcl groups were significant at each concentration of ethylmercury except the highest concentration [ 0 m t(213 ) = 10.32 , p < 0.0001 ; 0.063 m t(213 ) = 5.20 , p < 0.001 ; 0.125 m t(213 ) = 5.82 , p < 0.0001 ; 0.25 m t(213 ) = 4.54 , p < 0.001 ; 0.5 m t(213 ) = 4.33 , p < 0.0001 ; 1.25 m t(213 ) = 2.66 , p = 0.01 ] . this demonstrates that there was a greater drop in maximal respiratory capacity for the ad - a lcls as compared to the control lcls as ethylmercury concentration increased . reserve capacity was markedly higher for ad - a lcls as compared to control lcls [ f(1 , 213 ) = 123.94 , p < 0.0001 ] ( figure 4(d ) ) . reserve capacity decreased significantly as ethylmercury concentration increased [ f(6 , 24 ) = 7.72 , p = 0.0001 ] and was found to be significantly lower than baseline at 1.25 m [ t(24 ) = 3.11 , p < 0.01 ] and 2.5 m [ t(24 ) = 4.73 , p < 0.0001 ] ethylmercury in both ad - a and control lcls . however , the decrease in reserve capacity with increasing ethylmercury concentration was significantly different between the two lcl groups [ f(6 , 213 ) = 5.48 , p < 0.0001 ] . the differences in reserve capacity between the two lcl groups were significant at each concentration of ethylmercury except the highest concentration [ 0 m t(213 ) = 9.78 ; 0.063 m t(213 ) = 4.93 ; 0.125 m t(213 ) = 5.64 ; 0.25 m t(213 ) = 4.20 ; 0.5 m t(213 ) = 3.89 ; 1.25 m t(213 ) = 2.21 ] . this demonstrates that there was a greater drop in reserve capacity for the ad - a lcls as compared to the control lcls as ethylmercury concentration increased . as the focus of this study is on the abnormal ad - a subgroup , the detailed results for the ad - n subgroup are presented in the supplementary files ( see supplementary files in supplementary material available online at http://dx.doi.org/10.1155/2015/573701 ) . briefly , atp - linked respiration [ f(1 , 442 ) = 18.96 , p < 0.0001 ] , proton leak respiration [ f(1 , 442 ) = 38.16 , p < 0.0001 ] , and maximal respiratory capacity [ f(1 , 442 ) = 7.13 , p < 0.01 ] were all overall slightly , but significantly , higher in the ad - n subgroup as compared to control lcls , while there was no difference in reserve capacity between the ad - n and control lcls ( supplementary figure s1 ) . the magnitude of the differences between the ad - n and control lcls is much less than those observed between the ad - a and control lcls , and importantly , the mitochondrial response to increasing concentrations of ethylmercury was not different between the ad - n and control lcls . we examined the effects of pretreating the ad lcls for 48 hours with nac on baseline mitochondrial respiration as well as the change in mitochondrial respiration following ethylmercury exposure . pretreatment with nac markedly reduced atp - linked respiration in the ad - a lcls [ f(1 , 212 ) = 33.60 , p < 0.0001 ] ( figure 5(a ) ) . atp - linked respiration for both the nac pretreated and the nonpretreated ad - a lcls decreased as ethylmercury concentration increased [ f(6 , 24 ) = 8.30 , p < 0.0001 ] , but this decrease was not different between the two groups . atp - linked respiration was significantly lower than baseline at 1.25 m [ t(24 ) = 3.72 , p = 0.001 ] and 2.5 m [ t(24 ) = 5.87 , p < 0.0001 ] ethylmercury in both nac - pretreated and nonpretreated ad - a lcls . pretreatment with nac slightly but significantly decreased proton leak respiration in the ad - a lcls [ f(1 , 212 ) = 5.62 , p = 0.01 ] ( figure 5(b ) ) . overall proton leak for both the nac pretreated and the nonpretreated ad - a lcls did not change as ethylmercury concentration was increased and there was no difference in the change in proton leak respiration with the increase in ethylmercury . pretreatment with nac markedly decreased maximal respiratory capacity in the ad - a lcls [ f(1 , 212 ) = 40.86 , p < 0.0001 ] ( figure 5(c ) ) . maximal respiratory capacity for both the nac pretreated and the nonpretreated ad - a lcls decreased as ethylmercury concentration increased [ f(6 , 24 ) = 8.36 , p < 0.0001 ] , but this decrease was not different between the two groups . maximal respiratory capacity was significantly lower than baseline at 1.25 m [ t(24 ) = 3.84 , p < 0.001 ] and 2.5 m [ t(24 ) = 5.55 , p < 0.0001 ] ethylmercury in both nac - pretreated and nonpretreated ad - a lcls . pretreatment with nac markedly decreased overall reserve capacity in the ad - a lcls [ f(1 , 212 ) = 43.40 , p < 0.001 ] ( figure 5(d ) ) . reserve capacity for both the nac pretreated and the nonpretreated ad - a lcls decreased as ethylmercury concentration increased [ f(6 , 24 ) = 7.72 , p = 0.0001 ] and was significantly lower than baseline at 1.25 m [ t(24 ) = 3.72 , p = 0.001 ] and 2.5 m [ t(24 ) = 5.23 , p < 0.0001 ] ethylmercury in both groups . this decrease in reserve capacity was different between the two lcl groups [ f(6 , 212 ) = 2.14 , p = 0.05 ] . importantly , the differences in reserve capacity between the two lcl groups were significant at each concentration of ethylmercury except the two highest concentrations demonstrating that there was a greater drop in reserve capacity for the ad - a lcls which were not pretreated with nac as compared to the nac pretreated ad - a lcls as ethylmercury increased [ 0 m t(212 ) = 5.31 ; 0.063 m t(212 ) = 3.96 ; 0.125 m t(212 ) = 3.03 ; 0.25 m t(212 ) = 2.71 ; 0.5 m t(212 ) = 2.61 ] . the detailed results of nac pretreatment on the ad - n lcl subgroup are presented in the supplementary files . briefly , nac pretreatment resulted in a slight but significant increase in atp - linked respiration [ f(1 , 505 ) = 23.00 , p < 0.0001 ] , proton leak respiration [ f(1 , 505 ) = 10.74 , p = 0.001 ] , and maximal respiratory capacity [ f(1 , 505 ) = 5.20 , p < 0.05 ] in ad - n lcls at baseline . importantly , the change in mitochondrial parameters with ethylmercury exposure was not different in ad - n lcls with nac pretreatment as compared to ad - n lcls without pretreatment ( supplementary figure s2 ) . this study examined mitochondrial respiration in lymphoblastoid cell lines ( lcls ) derived from children with autism at baseline and following exposure to the environmental toxin , ethylmercury , and the protective potential of nac pretreatment . we show that lcls derived from children with autism exhibit significant abnormalities in mitochondrial respiration at baseline with these abnormalities worsening following exposure to ethylmercury . at baseline , ad lcls exhibit what appeared to be overactive mitochondria as evidenced by higher atp - linked and proton leak respiration , maximal respiratory capacity , and reserve capacity . following exposure to increasing concentrations of ethylmercury , we demonstrate a greater decrease in atp - linked respiration as well as maximal respiratory and reserve capacity in the ad lcls as compared to the control lcls . these findings were driven by the abnormal ( ad - a ) subset of ad lcls , which exhibit markedly abnormal mitochondrial parameters and have previously been shown to exhibit increased sensitivity to ros , resulting in ros - induced mitochondrial dysfunction . pretreatment of the ad - a subgroup with nac significantly decreased the abnormally high mitochondrial parameters at baseline and blunted the loss of reserve capacity in response to ethylmercury . overall , this study supports the notion that a subset of children with ad may have significant inherent physiological abnormalities in mitochondrial function and an increased vulnerability to oxidative environmental toxicants such as ethylmercury , which can induce mitochondrial dysfunction . the study also indicates that nac may mitigate mitochondrial dysfunction and attenuate the effects of ethylmercury . we previously classified our ad lcls as normal ( ad - n ) or abnormal ( ad - a ) based on reserve capacity at baseline and the change in response to increasing ros using 2,3-dimethoxy-1,4-naphthoquinone ( dmnq ) , an agent which produces hydrogen peroxide and superoxide upon entering a cell . in the present study using ethylmercury as a prooxidant environmental stressor , the ad - a lcls again exhibit a greater depletion of reserve capacity following ethylmercury exposure , as compared to control lcls , despite having increased reserve capacity at baseline . reserve capacity is a measure of the ability of the cell to increase mitochondrial oxidative phosphorylation to meet an increased atp demand , and the increased reserve capacity at baseline in the ad - a subgroup is likely representative of an abnormal adaptive mitochondrial response as the ad - n subgroup does not exhibit an increased reserve capacity at baseline . however , the ad - a lcls are unable to maintain the apparent adaptive increase in reserve capacity under conditions of acute oxidative stress . rapid loss of reserve capacity following ethylmercury exposure is significant as loss of reserve capacity has been associated with several disease states including aging , heart disease and neurodegenerative diseases [ 3942 ] , and complete depletion of reserve capacity has been shown to result in cell death [ 4245 ] . in addition to reserve capacity , atp - linked respiration and maximal respiratory capacity are also significantly elevated in the ad - a subgroup at baseline . atp - linked respiration is the portion of the electron transport chain ( etc ) function that produces atp . the remainder of etc function is measured as proton leak , a mechanism used to regulate oxidative stress at the inner mitochondrial membrane . maximal respiratory capacity is a measure of the maximal ability of the electron transport chain ( etc ) to produce energy . the increase in atp - linked respiration and maximal respiratory capacity indicates increased activity of the electron transport chain ( etc ) , which supports the notion of an increased atp demand and compensatory overactivity of the etc in the ad - a lcls which we have demonstrated previously [ 16 , 18 ] , and is consistent with reported etc overactivity in children with asd [ 46 , 47 ] . the ad - a lcls are unable to maintain these elevated mitochondrial activities following ethylmercury exposure , exhibiting a greater drop in atp - linked respiration and maximal respiratory capacity than in control cells . these data indicate that despite a background of increased ros production and decreased glutathione - mediated redox capacity , the ad - a lcls are capable of maintaining atp production under basal conditions ; however , they are more vulnerable to mitochondrial dysfunction when stressed with ethylmercury - induced oxidative stress . overall proton leak respiration is significantly higher in ad - a lcls as compared to control lcls , a finding that is consistent with increased ros production and overall increased mitochondrial activity in the ad - a lcls . proton leak is a mechanism used by the cell to reduce etc ros generation by reducing mitochondrial membrane potential . one of the major mechanisms to increase proton leak during conditions of chronic oxidative stress is the upregulation of uncoupling protein 2 ( ucp2 ) [ 4951 ] , and we previously have shown increased ucp2 protein levels in the ad - a lcls as compared to the ad - n lcls . the effect of increasing ethylmercury on proton leak was overall quite small compared to the effects on atp - linked and maximal oxygen consumption . while ethylmercury can lead to increased ros production in the lcls , our data indicate that , at the exposures examined , the primary effect of ethylmercury on mitochondrial function is reducing atp - linked and maximal respiration , likely by direct damage to the etc complexes , rather than increasing proton leak . iron - sulfur clusters are very sensitive to inactivation by mercury , and indeed methylmercury has been shown to directly damage several complexes in the etc [ 52 , 53 ] . pretreatment with nac provides the cells with cysteine , the rate - limiting amino acid for gsh synthesis . a 48 h pretreatment of the ad lcls was used to allow sufficient time for cysteine deacetylation and incorporation into gsh . while intracellular glutathione content was not evaluated in this study , we have previously demonstrated that pretreatment of the ad lcls with the same dose of nac for 48 h results in significantly increased glutathione levels and redox status ( gsh / gssg ) . pretreatment of the ad - a lcl subgroup with nac results in a significant reduction in atp - linked and proton leak respiration as well as maximal and reserve capacity , and it significantly blunts the loss of reserve capacity following ethylmercury exposure . nac pretreatment may increase cellular gsh content and thus antioxidant capacity , leading to a reduction in the abnormally high atp - linked respiration and maximal and reserve capacity and an improved ability to maintain reserve capacity during ethylmercury exposure . in the context of ethylmercury exposure , increased cellular gsh content from nac pretreatment may also chelate ethylmercury and reduce the cellular ethylmercury concentration , resulting in an improved mitochondrial reserve capacity . interestingly , nac pretreatment does not similarly affect the ad - n lcls but instead results in slightly increased atp - linked respiration , proton leak respiration , and maximal capacity , while having no effect on reserve capacity ( see supplementary figure s2 ) . we have previously described how the baseline mitochondrial parameters of the ad - a lcls likely represent a maladaptive mitochondrial response that is characterized by markedly elevated atp - linked and proton leak respiration and maximal and reserve capacity . on the other hand , the ad - n lcls represent a normal adaptive response that is characterized by only slight changes in mitochondrial respiration including slight increases in atp - linked and proton leak respiration . it is possible in this normal adaptive situation that adding nac relieves a relatively mild oxidative stress burden and actually boosts mitochondrial function , while in the maladaptive ad - a lcls it relieves a more serious oxidative stress burden , lessening the atp demand and reducing mitochondrial overactivity . we demonstrate that nac pretreatment does confer some protection from the ethylmercury - induced loss of reserve capacity in the ad - a lcls , providing preliminary in vitro support for the clinical use of nac to treat oxidative stress in autism . nac can protect against oxidative stress - induced mitochondrial dysfunction [ 18 , 5456 ] as well as mitochondrial - generated oxidative stress . in a mouse model of complex 1 deficiency importantly , a double - blind trial of nac in children with autism proved efficacious in reducing symptoms of irritability suggesting that reduced glutathione redox capacity and oxidative stress may also contribute to behavioral symptoms associated with autism . in the present study the mitochondrial respiratory response to mercury - induced oxidative stress was examined in ad and control lcls using the ethylmercury - containing compound , thimerosal . thimerosal has been shown to deplete glutathione and increase ros [ 12 , 35 ] , to induce dna strand breaks , membrane permeability and apoptosis , and to be cytotoxic at nanomolar and micromolar concentrations [ 6063 ] . evidence suggests that thimerosal induces apoptosis through a mitochondrial pathway [ 62 , 64 , 65 ] and that it is toxic to the mitochondria , reducing mitochondrial respiration and inducing mitochondrial dna damage and superoxide production [ 65 , 66 ] . until recently thimerosal was used as a preservative in vaccines and pharmaceuticals with some vaccines containing 12.5 g to 25 g of ethylmercury per 0.5 ml dose , equating to approximately 100200 m . while the concentrations of ethylmercury used in this study ( 0.0625 m to 2.5 m ) are at least 2 orders of magnitude lower than those once used in vaccines , any extrapolation of the dose response characteristics of this in vitro lcl model to the in vivo situation would be overstating and unsubstantiated . while ethylmercury was chosen in this study as an example of a prooxidant environmental toxin , another common environmental exposure to mercury is methylmercury in the diet . the toxicities of methylmercury and ethylmercury are thought to be very similar [ 67 , 68 ] . indeed , several studies have demonstrated that methylmercury increases intracellular ros production , depletes intracellular glutathione , and acts on the mitochondria to depolarize the mitochondrial membrane potential and induce apoptosis [ 21 , 69 , 70 ] . in conclusion , we have determined that a subgroup of ad lcls exhibits abnormal mitochondrial respiratory function at baseline and increased vulnerability to mitochondrial dysfunction when exposed to the environmental toxin , ethylmercury . this subgroup of ad lcls has previously been shown to exhibit increased mitochondrial susceptibility to ros , suggesting that these cells may be inherently vulnerable to a wide variety of oxidative insults . pretreatment of this subgroup with nac improved mitochondrial function at baseline and decreased the loss of mitochondrial reserve capacity in response to ethylmercury . our data suggest that the abnormal mitochondrial function and increased susceptibility to ethylmercury in the ad - a lcls may be related to impaired glutathione - mediated antioxidant capacity and chronic oxidative stress , since nac pretreatment , which could improve glutathione status , appears to partially correct the atypical mitochondrial function in the ad - a lcls and protect the cells against the toxic effects of ethylmercury . other prooxidant environmental toxicants associated with asd such as pesticides and polychlorinated biphenyls ( pcbs ) should be tested to determine whether these autism lcls exhibit hypersensitivity to a wide range of prooxidant environmental toxicants as our findings support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction .

the association of autism spectrum disorders with oxidative stress , redox imbalance , and mitochondrial dysfunction has become increasingly recognized . in this study , extracellular flux analysis was used to compare mitochondrial respiration in lymphoblastoid cell lines ( lcls ) from individuals with autism and unaffected controls exposed to ethylmercury , an environmental toxin known to deplete glutathione and induce oxidative stress and mitochondrial dysfunction . we also tested whether pretreating the autism lcls with n - acetyl cysteine ( nac ) to increase glutathione concentrations conferred protection from ethylmercury . examination of 16 autism / control lcl pairs revealed that a subgroup ( 31% ) of autism lcls exhibited a greater reduction in atp - linked respiration , maximal respiratory capacity , and reserve capacity when exposed to ethylmercury , compared to control lcls . these respiratory parameters were significantly elevated at baseline in the ethylmercury - sensitive autism subgroup as compared to control lcls . nac pretreatment of the sensitive subgroup reduced ( normalized ) baseline respiratory parameters and blunted the exaggerated ethylmercury - induced reserve capacity depletion . these findings suggest that the epidemiological link between environmental mercury exposure and an increased risk of developing autism may be mediated through mitochondrial dysfunction and support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction .

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

oxidative stress and damage have been documented in blood and brain of individuals with asd including reports of decreased levels and activities of antioxidant enzymes and elevated levels of oxidized lipids , proteins , and dna [ 4 , 7 , 8 , 11 , 14 , 15 ] . in primary lymphocytes and in lymphoblastoid cell lines ( lcls ) derived from children with autistic disorder ( ad ) , we have found that the production of reactive oxygen species ( ros ) is elevated as compared to controls [ 12 , 13 , 16 ] . the imbalance between glutathione - mediated antioxidant capacity and ros production in autism lcls may cause these cells to be more susceptible to oxidative stress and damage from any exogenous sources of ros as compared to control lcls . while the etiology of mitochondrial dysfunction in asd is not known , evidence suggests that oxidative stress may be a key factor driving mitochondrial dysfunction in individuals with asd [ 16 , 18 ] . recently , we demonstrated that lcls derived from children with ad exhibit abnormal mitochondrial respiration at baseline as well as a more rapid decline in mitochondrial function upon exposure to increasing ros as compared to lcls from control children . importantly , we found that these abnormal mitochondrial parameters were driven by a subgroup consisting of 32% of the ad lcls ( termed ad - a for abnormal ) , whereas the other autism lcls ( termed ad - n for normal ) had mitochondrial parameters similar to controls . furthermore , we also demonstrated that pretreatment of the ad lcls with n - acetyl cysteine ( nac ) increased intracellular gsh and the gsh / gssg redox ratio and , in the ad - a subgroup , conferred protection from mitochondrial dysfunction when ros was increased . we have previously demonstrated that ad lcls have increased susceptibility to oxidative stress from exposure to ethylmercury such that exposure to ethylmercury resulted in lower intracellular gsh and gsh / gssg and increased ros production in ad lcls as compared to control lcls . in the present study we tested the hypothesis that the subset of ad lcls previously found to exhibit mitochondrial dysfunction when challenged with ros would also exhibit mitochondrial dysfunction when exposed to ethylmercury ( i.e. furthermore , we hypothesized that pretreatment with nac to increase the intracellular glutathione concentration would confer protection from ethylmercury - induced mitochondrial dysfunction . to this end , we used extracellular flux analysis to measure mitochondrial oxygen consumption in ad and control lcls transiently exposed to ethylmercury . , north billerica , ma , usa ) to measure the oxygen consumption rate ( ocr ) , an indicator of mitochondrial respiration , in real - time in live intact lcls as described . upon the sequential addition of mitochondrial electron transport chain ( etc ) inhibitors and an uncoupler to the respiring cells , several parameters of mitochondrial respiration were derived , including basal respiration , atp - linked respiration , proton leak respiration , and reserve capacity ( diagramed in figure 1 ) . briefly , after measuring basal respiration , oligomycin , an inhibitor of complex v , is added , and the resulting ocr is used to derive atp - linked respiration and proton leak respiration . lastly , antimycin a , a complex iii inhibitor , and rotenone , a complex i inhibitor , are added to shut down etc function , revealing the nonmitochondrial respiration , which is subtracted from the other rates to obtain the corrected basal respiration , proton leak respiration and maximal respiratory capacity . the same analysis was used to analyze the difference in mitochondrial respiratory parameters between each ad subgroup and matched controls . atp - linked respiration was overall higher for ad lcls as compared to control lcls [ f(1 , 662 ) = 134.55 , p < 0.0001 ] ( figure 3(a ) ) . however , the decrease in atp - linked respiration with increasing ethylmercury concentration was significantly different between the ad and control groups [ f(6 , 662 ) = 2.42 , p < 0.05 ] . the differences in atp - linked respiration between the two lcl groups were significant at every concentration of ethylmercury [ 0 m t(662 ) = 9.51 ; 0.063 m t(662 ) = 4.33 ; 0.125 m t(662 ) = 4.30 ; 0.25 m t(662 ) = 3.59 ; 0.5 m t(662 ) = 4.57 ; 1.25 m t(662 ) = 3.20 ; 2.5 m t(662 ) = 2.91 ] . however , the difference was smaller at high ethylmercury concentrations because the drop in atp - linked respiration was greater for the ad lcls as compared to the control lcls as ethylmercury concentration increased . maximal respiratory capacity was overall higher for ad lcls as compared to control lcls [ f(1 , 662 ) = 100.89 , p < 0.0001 ] ( figure 3(c ) ) . maximal respiratory capacity decreased significantly as ethylmercury concentration increased [ f(4 , 89 ) = 35.90 , p < 0.0001 ] and was found to be significantly lower than baseline at 0.5 m [ t(89 ) = 4.55 , p < 0.001 ] , 1.25 m [ t(89 ) = 7.88 , p < 0.0001 ] , and 2.5 m [ t(89 ) = 11.40 , p < 0.0001 ] ethylmercury in both ad and control lcls . the decrease in maximal respiratory capacity was greater for the ad lcls as compared to the control lcls as ethylmercury concentration increased . reserve capacity was overall higher for ad lcls as compared to control lcls [ f(1 , 662 ) = 76.77 , p < 0.0001 ] ( figure 3(d ) ) . the decrease in reserve capacity was greater for the ad lcls as compared to the control lcls as ethylmercury concentration increased . atp - linked respiration was markedly higher for ad - a lcls as compared to control lcls [ f(1 , 213 ) = 183.13 , p < 0.0001 ] ( figure 4(a ) ) . atp - linked respiration decreased significantly as ethylmercury concentration increased [ f(6 , 24 ) = 9.11 , p < 0.0001 ] and was found to be significantly lower than baseline at 1.25 m [ t(24 ) = 2.92 , p < 0.01 ] and 2.5 m [ t(24 ) = 5.65 , p < 0.0001 ] ethylmercury in both ad - a and control lcls . however , the decrease in atp - linked respiration with increasing ethylmercury concentration was significantly different between the two lcl groups [ f(6 , 213 ) = 3.95 , p < 0.001 ] . although the differences in atp - linked respiration between the two lcl groups were significant at each concentration of ethylmercury [ 0 m t(213 ) = 10.97 ; 0.063 m t(213 ) = 5.63 ; 0.125 m t(213 ) = 5.60 ; 0.25 m t(213 ) = 4.90 ; 0.5 m t(213 ) = 5.03 ; 1.25 m t(213 ) = 3.62 ; 2.5 m t(213 ) = 2.45 ] , the difference between the lcl groups was reduced at high ethylmercury concentrations because the drop in atp - linked respiration was greater for the ad - a lcls as compared to the control lcls as ethylmercury increased . maximal respiratory capacity was markedly higher for ad - a lcls as compared to control lcls [ f(1 , 213 ) = 148.63 , p < 0.0001 ] ( figure 4(c ) ) . maximal respiratory capacity decreased significantly as the ethylmercury concentration increased [ f(6 , 24 ) = 8.09 , p < 0.0001 ] and was found to be significantly lower than baseline at 1.25 m [ t(24 ) = 3.12 , p < 0.01 ] and 2.5 m [ t(24 ) = 4.94 , p < 0.0001 ] ethylmercury in both ad - a and control lcls . this demonstrates that there was a greater drop in maximal respiratory capacity for the ad - a lcls as compared to the control lcls as ethylmercury concentration increased . reserve capacity was markedly higher for ad - a lcls as compared to control lcls [ f(1 , 213 ) = 123.94 , p < 0.0001 ] ( figure 4(d ) ) . this demonstrates that there was a greater drop in reserve capacity for the ad - a lcls as compared to the control lcls as ethylmercury concentration increased . briefly , atp - linked respiration [ f(1 , 442 ) = 18.96 , p < 0.0001 ] , proton leak respiration [ f(1 , 442 ) = 38.16 , p < 0.0001 ] , and maximal respiratory capacity [ f(1 , 442 ) = 7.13 , p < 0.01 ] were all overall slightly , but significantly , higher in the ad - n subgroup as compared to control lcls , while there was no difference in reserve capacity between the ad - n and control lcls ( supplementary figure s1 ) . pretreatment with nac markedly reduced atp - linked respiration in the ad - a lcls [ f(1 , 212 ) = 33.60 , p < 0.0001 ] ( figure 5(a ) ) . importantly , the differences in reserve capacity between the two lcl groups were significant at each concentration of ethylmercury except the two highest concentrations demonstrating that there was a greater drop in reserve capacity for the ad - a lcls which were not pretreated with nac as compared to the nac pretreated ad - a lcls as ethylmercury increased [ 0 m t(212 ) = 5.31 ; 0.063 m t(212 ) = 3.96 ; 0.125 m t(212 ) = 3.03 ; 0.25 m t(212 ) = 2.71 ; 0.5 m t(212 ) = 2.61 ] . briefly , nac pretreatment resulted in a slight but significant increase in atp - linked respiration [ f(1 , 505 ) = 23.00 , p < 0.0001 ] , proton leak respiration [ f(1 , 505 ) = 10.74 , p = 0.001 ] , and maximal respiratory capacity [ f(1 , 505 ) = 5.20 , p < 0.05 ] in ad - n lcls at baseline . importantly , the change in mitochondrial parameters with ethylmercury exposure was not different in ad - n lcls with nac pretreatment as compared to ad - n lcls without pretreatment ( supplementary figure s2 ) . this study examined mitochondrial respiration in lymphoblastoid cell lines ( lcls ) derived from children with autism at baseline and following exposure to the environmental toxin , ethylmercury , and the protective potential of nac pretreatment . we show that lcls derived from children with autism exhibit significant abnormalities in mitochondrial respiration at baseline with these abnormalities worsening following exposure to ethylmercury . at baseline , ad lcls exhibit what appeared to be overactive mitochondria as evidenced by higher atp - linked and proton leak respiration , maximal respiratory capacity , and reserve capacity . following exposure to increasing concentrations of ethylmercury , we demonstrate a greater decrease in atp - linked respiration as well as maximal respiratory and reserve capacity in the ad lcls as compared to the control lcls . these findings were driven by the abnormal ( ad - a ) subset of ad lcls , which exhibit markedly abnormal mitochondrial parameters and have previously been shown to exhibit increased sensitivity to ros , resulting in ros - induced mitochondrial dysfunction . pretreatment of the ad - a subgroup with nac significantly decreased the abnormally high mitochondrial parameters at baseline and blunted the loss of reserve capacity in response to ethylmercury . overall , this study supports the notion that a subset of children with ad may have significant inherent physiological abnormalities in mitochondrial function and an increased vulnerability to oxidative environmental toxicants such as ethylmercury , which can induce mitochondrial dysfunction . in the present study using ethylmercury as a prooxidant environmental stressor , the ad - a lcls again exhibit a greater depletion of reserve capacity following ethylmercury exposure , as compared to control lcls , despite having increased reserve capacity at baseline . reserve capacity is a measure of the ability of the cell to increase mitochondrial oxidative phosphorylation to meet an increased atp demand , and the increased reserve capacity at baseline in the ad - a subgroup is likely representative of an abnormal adaptive mitochondrial response as the ad - n subgroup does not exhibit an increased reserve capacity at baseline . in addition to reserve capacity , atp - linked respiration and maximal respiratory capacity are also significantly elevated in the ad - a subgroup at baseline . atp - linked respiration is the portion of the electron transport chain ( etc ) function that produces atp . maximal respiratory capacity is a measure of the maximal ability of the electron transport chain ( etc ) to produce energy . the increase in atp - linked respiration and maximal respiratory capacity indicates increased activity of the electron transport chain ( etc ) , which supports the notion of an increased atp demand and compensatory overactivity of the etc in the ad - a lcls which we have demonstrated previously [ 16 , 18 ] , and is consistent with reported etc overactivity in children with asd [ 46 , 47 ] . the ad - a lcls are unable to maintain these elevated mitochondrial activities following ethylmercury exposure , exhibiting a greater drop in atp - linked respiration and maximal respiratory capacity than in control cells . these data indicate that despite a background of increased ros production and decreased glutathione - mediated redox capacity , the ad - a lcls are capable of maintaining atp production under basal conditions ; however , they are more vulnerable to mitochondrial dysfunction when stressed with ethylmercury - induced oxidative stress . overall proton leak respiration is significantly higher in ad - a lcls as compared to control lcls , a finding that is consistent with increased ros production and overall increased mitochondrial activity in the ad - a lcls . one of the major mechanisms to increase proton leak during conditions of chronic oxidative stress is the upregulation of uncoupling protein 2 ( ucp2 ) [ 4951 ] , and we previously have shown increased ucp2 protein levels in the ad - a lcls as compared to the ad - n lcls . the effect of increasing ethylmercury on proton leak was overall quite small compared to the effects on atp - linked and maximal oxygen consumption . while ethylmercury can lead to increased ros production in the lcls , our data indicate that , at the exposures examined , the primary effect of ethylmercury on mitochondrial function is reducing atp - linked and maximal respiration , likely by direct damage to the etc complexes , rather than increasing proton leak . a 48 h pretreatment of the ad lcls was used to allow sufficient time for cysteine deacetylation and incorporation into gsh . while intracellular glutathione content was not evaluated in this study , we have previously demonstrated that pretreatment of the ad lcls with the same dose of nac for 48 h results in significantly increased glutathione levels and redox status ( gsh / gssg ) . pretreatment of the ad - a lcl subgroup with nac results in a significant reduction in atp - linked and proton leak respiration as well as maximal and reserve capacity , and it significantly blunts the loss of reserve capacity following ethylmercury exposure . nac pretreatment may increase cellular gsh content and thus antioxidant capacity , leading to a reduction in the abnormally high atp - linked respiration and maximal and reserve capacity and an improved ability to maintain reserve capacity during ethylmercury exposure . interestingly , nac pretreatment does not similarly affect the ad - n lcls but instead results in slightly increased atp - linked respiration , proton leak respiration , and maximal capacity , while having no effect on reserve capacity ( see supplementary figure s2 ) . we have previously described how the baseline mitochondrial parameters of the ad - a lcls likely represent a maladaptive mitochondrial response that is characterized by markedly elevated atp - linked and proton leak respiration and maximal and reserve capacity . on the other hand , the ad - n lcls represent a normal adaptive response that is characterized by only slight changes in mitochondrial respiration including slight increases in atp - linked and proton leak respiration . we demonstrate that nac pretreatment does confer some protection from the ethylmercury - induced loss of reserve capacity in the ad - a lcls , providing preliminary in vitro support for the clinical use of nac to treat oxidative stress in autism . in the present study the mitochondrial respiratory response to mercury - induced oxidative stress was examined in ad and control lcls using the ethylmercury - containing compound , thimerosal . thimerosal has been shown to deplete glutathione and increase ros [ 12 , 35 ] , to induce dna strand breaks , membrane permeability and apoptosis , and to be cytotoxic at nanomolar and micromolar concentrations [ 6063 ] . while ethylmercury was chosen in this study as an example of a prooxidant environmental toxin , another common environmental exposure to mercury is methylmercury in the diet . in conclusion , we have determined that a subgroup of ad lcls exhibits abnormal mitochondrial respiratory function at baseline and increased vulnerability to mitochondrial dysfunction when exposed to the environmental toxin , ethylmercury . pretreatment of this subgroup with nac improved mitochondrial function at baseline and decreased the loss of mitochondrial reserve capacity in response to ethylmercury . our data suggest that the abnormal mitochondrial function and increased susceptibility to ethylmercury in the ad - a lcls may be related to impaired glutathione - mediated antioxidant capacity and chronic oxidative stress , since nac pretreatment , which could improve glutathione status , appears to partially correct the atypical mitochondrial function in the ad - a lcls and protect the cells against the toxic effects of ethylmercury . other prooxidant environmental toxicants associated with asd such as pesticides and polychlorinated biphenyls ( pcbs ) should be tested to determine whether these autism lcls exhibit hypersensitivity to a wide range of prooxidant environmental toxicants as our findings support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction .

mild cognitive impairment ( mci ) refers to the transitional state between the cognitive changes of normal aging and very early dementia . patients with mci , who are at high risk of developing alzheimer disease ( ad ; ) , have smaller hippocampal volume than healthy elderly people [ 3 , 4 ] . medial temporal lobe ( mtl ) structures , in particular the hippocampus , show atrophy in the early stages of ad and are potential markers for detecting preclinical ad [ 57 ] . moreover , a recent study has demonstrated that atrophy of the hippocampus on mri in cognitively intact elderly people predicts dementia , in particular of alzheimer type , during a 6-year followup . hippocampus is particularly important for memory formation , for attention and for production of eeg rhythmic activity [ 10 , 11 ] . lesions of hippocampal synaptic plasticity block the memory - enhancing effects of direct hippocampal stimulation [ 12 , 13 ] . further , behavioral stress interferes with synaptic plasticity in the hippocampal formation [ 1416 ] . the associative memories involve the dorsal hippocampus , and a lesion of the area reduces the retrieval of associative tasks . the hippocampal network system seems to be well suited to receive synaptic inputs from both the anterior and posterior thalamic nuclei [ 1820 ] , becoming suitable for an association with brain rhythms activity generation . recent works showed that in subjects with mci is present , an increase of high alpha as compared to low alpha band occurs [ 21 , 22 ] . as a working hypothesis , eeg markers alpha3/alpha2 power ratio could show modifications proportional to the hippocampal atrophy . in the present study the association between hippocampal atrophy and increase of alpha3/alpha2 relative power ratio was investigated in subjects with mci . recent studies have demonstrated that the hippocampus is not a unitary structure from an anatomophysiological point of view . the hippocampus , including strictly speaking subfields ca1ca4 and the hippocampal formation , including also dentate gyrus , fimbria , subiculum , and parasubiculum , is a highly sophisticated structure . stimuli coming from the entorhinal cortex are processed by the dentate gyrus , subfields ca4 and ca3 , before being projected outside the medial temporal lobe via ca1 or subicular efferent projections . moreover , in addition to the unsurprising right - left specialization for verbal and visuospatial material , some degree of anterior - to - posterior specialization has been shown by fmri studies . as a consequence , it is conceivable that local structural changes take place in the hippocampus of patients with ad and that different hippocampal subregions are affected in ad brickman et al . , and shen et al . local changes in hippocampal subregions could be detected through a radial atrophy mapping method able to assess group , based on high resolution mri at 3 tesla differences . in this study , we tested the hypothesis that the increase of alpha3/alpha2 ratio is related with volumetric differences both in mci patients and in mapped hippocampal regions in ad patients . for the present study , 79 subjects with mci and 11 subjects with alzheimer 's disease ( ad ) were recruited from the memory clinic of the scientific institute for research and care ( irccs ) of alzheimer 's and psychiatric diseases fatebenefratelli in brescia , italy . informed consent was obtained from all participants or their caregivers , according to the code of ethics of the world medical association ( declaration of helsinki ) . the project was aimed to study the natural history of nondemented persons with apparently primary cognitive deficits , that is , deficits not due to psychic ( anxiety , depression ) or physical ( hypothyroidism , vit . b12 and folate deficiency , uncontrolled heart disease , and uncontrolled diabetes ) patients were rated with a series of standardized diagnostic and severity instruments , including the mini - mental state examination ( mmse ; ) , the clinical dementia rating scale ( cdrs ; ) , the hachinski ischemic scale ( his ; ) , and the instrumental and basic activities of daily living ( iadl , badl , ) . in addition , patients underwent diagnostic neuroimaging procedures ( magnetic resonance imaging , mri ) and laboratory testing to rule out other causes of cognitive impairment . these inclusion and exclusion criteria for mci were based on previous seminal studies [ 3238 ] . inclusion criteria of the study were all of the following : ( i ) complaint by the patient , or report by a relative or the general practitioner , of memory or other cognitive disturbances ; ( ii ) mini - mental state examination ( mmse ) score of 24 to 27/30 , or mmse of 28 and higher plus low performance ( score of 26 or higher ) on the clock drawing test ; ( iii ) sparing of instrumental and basic activities of daily living or functional impairment steadily due to causes other than cognitive impairment , such as physical impairments , sensory loss , and gait or balance disturbances . exclusion criteria were any one of the following : ( i ) patients aged 90 years and older ; ( ii ) history of depression or juvenile - onset psychosis ; ( iii ) history or neurological signs of major stroke ; ( iv ) other psychiatric diseases , epilepsy , drug addiction , and alcohol dependence ; ( v ) use of psychoactive drugs , including acetylcholinesterase inhibitors or other drugs enhancing brain cognitive functions ; ( vi ) current or previous uncontrolled or complicated systemic diseases ( including diabetes mellitus ) or traumatic brain injuries . all patients underwent ( i ) semistructured interview with the patient and , whenever possible , with another informant ( usually , the patient 's spouse or a child of the patient ) by a geriatrician or neurologist ; ( ii ) physical and neurological examinations ; ( iii ) performance - based tests of physical function , gait , and balance ; ( iv ) neuropsychological battery assessing verbal and nonverbal memory , attention and executive functions ( trail making test b - a ; clock drawing test ; ) , abstract thinking ( raven matrices ; ) , frontal functions ( inverted motor learning ; ) , language ( phonological and semantic fluency ; token test ; ) , and apraxia and visuoconstructional abilities ( rey figure copy ; ) ; ( v ) assessment of depressive symptoms by means of the center for epidemiologic studies depression scale ( ces - d ; ) . inclusion and exclusion criteria were homogeneous with previous works [ 4648 ] . as the aim of our study was to evaluate the meaning of alpha3/alpha2 power ratio and its associations with structural changes of hippocampus as diagnostic marker of cognitive impairment , we were not interested in this study in the clinical subtype of mci , that is , amnesic or nonamnesic , single or multiple domains . the diagnosis of ad was made according to nincds - adrda criteria and the diagnostic and statistical manual of mental disorders iv . the eeg activity was recorded continuously from 19 sites by using electrodes set in an elastic cap ( electro - cap international , inc . ) and positioned according to the 1020 international system ( fp1 , fp2 , f7 , f3 , fz , f4 , f8 , t3 , c3 , cz , c4 , t4 , t5 , p3 , pz , p4 , t6 , o1 , and o2 ) . the recordings were used off - line to rereference the scalp recordings to the common average . data were recorded with a band - pass filter of 0.370 hz and digitized at a sampling rate of 250 hz ( brainamp , brainproducts , germany ) . electrodes - skin impedance was set below 5 k. horizontal and vertical eye movements were detected by recording the electrooculogram ( eog ) . eog activity was recorded with cup electrodes for the control of blinking and eye movements . it was referred to another electrode placed 2 cm below suborbital ridge of the right eye . the left and right horizontal eog channels were collected from two electrodes at the left and the right lateral canthus . longer recordings would have reduced the variability of the data , but they would also have increased the possibility of slowing of eeg oscillations due to reduced vigilance and arousal . eeg data were then analyzed and fragmented off - line in consecutive epochs of 2 seconds , with a frequency resolution of 0.5 hz . the eeg epochs with ocular , muscular , and other types of artifacts were discarded . frequency bands were determiner on an individuale basis , because of the variability of the brain rhythms with age and diseases . a digital fft - based power spectrum analysis ( welch technique , hanning windowing function , and no phase shift ) computed the power density of eeg rhythms with a 0.5 hz frequency resolution , ranging from 2 to 40 hz . two anchor frequencies were selected according to literature guidelines ( 47 ) , which are the transition theta / alpha frequency ( tf ) and the individual alpha frequency ( iaf ) peak . as previously mentioned , the tf marks the transition frequency between theta and alpha bands , and it represents an estimate of the frequency at which theta and alpha spectra intersect . we computed the tf as the minimum power in the alpha frequency range since our eeg recordings were performed at rest . the iaf represents instead the frequency with the maximum power peak within the extended alpha range ( 514 hz ) . the tf and iaf could be clearly identified in 99 mci subjects whose eeg data were then statistically analyzed . based on the tf and iaf , we estimated for each subject the frequency band range as follows : delta from tf-4 to tf-2 , theta from tf-2 to tf , low alpha ( alpha1 and alpha2 ) from tf to iaf , and high alpha band ( or alpha3 ) from iaf to iaf + 2 . alpha1 and alpha2 band were computed for each subject as follows : alpha1 from tf to the middle point of the tf - iaf range and alpha2 from this middle point to iaf peak . we found that the bandwidth in alpha1 and alpha2 bands was different among the groups . in no vascular damage and severe vascular damage groups than in mild and moderate vascular damage groups ( 1.7 and 1.87 hz , resp . ) . we performed a statistical analysis to test if this difference was significant among groups in these frequency bands . it was not the case , since the analysis did not show a main group significant effect ( p = 0.06 ) . finally , in the frequency bands so determined , we computed the relative power spectra for each subject . relative power density for each frequency band was computed as the ratio between the absolute power and the mean power spectra from 2 to 40 hz . the relative band power at each band was defined as the mean of the relative band power for each frequency bin within that band . mri scans were acquired with a 1.0 tesla philips gyroscan at the neuroradiology unit of the citt di brescia hospital , brescia . the following sequences were used to measure hippocampal volumes : a high - resolution gradient echo t1-weighted sagittal 3d sequence ( tr = 20 ms , te = 5 ms , flip angle = 30 , field of view = 220 mm , acquisition matrix = 256 256 , and slice thickness = 1.3 mm ) and a fluid - attenuated inversion recovery ( flair ) sequence ( tr = 5000 ms , te = 100 ms , flip angle = 90 , field of view = 230 mm , acquisition matrix = 256 256 , and slice thickness = 5 mm ) . the hippocampal boundaries were manually traced on each hemisphere by a single tracer with the software program display ( mcgill university , montreal , canada ) on contiguous 1.5 mm slices in the coronal plane . the starting point for hippocampus tracing was defined as the hippocampal head when it first appears below the amygdala , the alveus defining the superior and anterior border of the hippocampus . the fimbria was included in the hippocampal body , while the grey matter rostral to the fimbria was excluded . the hippocampal tail was traced until it was visible as an oval shape located caudally and medially to the trigone of the lateral ventricles [ 51 , 52 ] . white matter hyperintensities ( wmhs ) were automatically segmented on the flair sequences by using previously described algorithms [ 51 , 52 ] . briefly , the procedure includes ( i ) filtering of flair images to exclude radiofrequency inhomogeneities , ( ii ) segmentation of brain tissue from cerebrospinal fluid , ( iii ) modelling of brain intensity histogram as a gaussian distribution , and ( iv ) classification of the voxels whose intensities were 3.5 sds above the mean as wmhs [ 51 , 52 ] . total wmhs volume was computed by counting the number of voxels segmented as wmhs and multiplying by the voxel size ( 5 mm ) . to correct for individual differences in head size , hippocampal and wmhs volumes were normalized to the total intracranial volume ( tiv ) , obtained by manually tracing with display the entire intracranial cavity on 7 mm thick coronal slices of the t1-weighted images . both manual and automated methods manual segmentation of the hippocampus is currently considered the gold standard technique for the measurement of such complex structures . the main disadvantages of manual tracing are that it is operator dependent and time consuming . conversely , automated techniques are more reliable and less time consuming , but may be less accurate when dealing with structures without clearly identifiable borders . this , however , is not the case for wmhs which appear as hyperintense on flair sequences . left and right hippocampal volumes were estimated and summed to obtain a total volume ( individual ) of both anatomical structures . hippocampal total volume has been divided in tertiles obtaining three groups . in each group , the 3d parametric surface mesh models were created from the manual tracings of hippocampal boundaries [ 53 , 54 ] . this procedure allows measurements to be made at corresponding surface locations in each subject , which are then compared statistically in 3d [ 53 , 54 ] . to assess hippocampal morphology , a medial curve was automatically defined as the 3d curve traced out by the centroid of the hippocampal boundary in each image slice . the radial size of each hippocampus at each boundary point was assessed by automatically measuring the radial 3d distance from the surface points to the medial curve defined for individual 's hippocampal surface model . preliminarily , the significant differences among groups in demographic variables , ( age , education , and mmse score ) and morphostructural characteristics , ( hippocampal and white matter hyperintensities , wmhs , and volume ) , were evaluated ( table 1 ) . in order to avoid a confounding effect , subsequent anovas were carried out using age , education , mmse score , and wmhs as covariates . the first analysis was performed in order to verify the difference of hippocampal volume among groups . the second anova was performed in order to check differences in alpha3/alpha2 relative power ratio in the three mci subgroups ordered by decreasing tertile values of the hippocampal volume . in each anova , group was the independent variable , the frequency ratios were the dependent variable . in ad patients , the radial atrophy mapping was chosen because it is more suitable to study subregional volume of the hippocampus . correlation maps between eeg rhythms and hippocampal surface were computed . the correlation analysis between eeg rhythms and hippocampal volume was performed only in 11 ad subjects ; we otherwise compared hippocampal gray matter distribution maps between normal controls and ad patients in order to verify that the ad found correlations between eeg rhythms and hippocampal regions were present in areas where ad is more atrophic than normal subjects . the correlation maps were generated on 3d models of the hippocampal formation where the dorsal and ventral surfaces can be appreciated . zones with significant correlations were mapped onto the models based on an atlas where these are shown together with the corresponding mr sections ( [ 55 , 56 ] ; figure 1 ) . the correlations and the associated p value maps were plotted onto a colour - coded model of the hippocampal surface . the statistical test for the correlations was computed using linear regression at each surface vertex on the hippocampus . a surface point significance threshold of p < 0.05 was used to visualize the regional specificity of gray matter changes in the cortex . set level correction for multiple comparisons permutation tests are based on measuring the total area of the hippocampus with suprathreshold statistics , after setting the threshold at p < 0.05 . to correct for multiple comparisons and assign an overall p value to each p map permutation , tests were used to determine how likely the observed level of significant atrophy ( proportion of suprathreshold statistics , with the threshold set at p < 0.05 ) within each p map would occur by chance . the number of permutations n was chosen to be 100,000 , to control the standard error sep of omnibus probability p , which follows a binomial distribution b(n , p ) with known standard error . n = 8 , 000 , the approximate margin of error ( 95% confidence interval ) for p is around 5% of p. both left and right hippocampal volumes were investigated in ad patients given the superior well - known left hemispheric involvement in declared dementia . the analysis on the ad subjects was conducted to verify the reliability of the alpha3/alpha2 ratio as factor associated with conversion of a subpopulation of mci subjects in alzheimer 's disease . this results need further confirmation in a larger size population to strength the statistical power of the analysis . of note , a larger population could be permitted to detect more precisely the volume ranges within the hippocampal subregions and their correlation with the eeg marker , ruling out other possible associations . in the two groups of patients , mci and ad patients , the mri analysis method performed was different : volumetric analysis for mci and radial atrophy mapping for ad patients . in this study , we were interested in morphofunctional ( mri - eeg ) association and not in neuropsychological issues . mmse values were provided as a general marker of the entity of the cognitive decline of patients . table 1 summarizes the anova results of demographic variables , that is , age , education , mmse score , and morphostructural characteristics , that is , hippocampal , and white matter hyperintensities volume in the whole mci cohort as well as in the three subgroups in study . significant statistical results were found in hippocampal volume ( respectively , f 2,76 = 157.27 ; p < 0.00001 and f 2,76 = 132.5 ; p < 0.00001 ) . duncan 's post - hoc test showed a significant increase ( p < 0.01 ) in all comparisons . table 2 shows the results of alpha3/alpha2 ratio in the groups based on the decrease of hippocampal volumes . anova results revealed significant main effect group in alpha3/alpha2 ratio for hippocampal ( f 2,76 = 3.38 ; p < 0.03 ) decreasing volume . table 3 summarizes sociodemographic characteristics , mmse scores , and alpha3/alpha2 power ratio in ad cohort . negative significant associations are found between same areas of ad left hippocampus and alpha3/2 eeg rhythms . correlations in right hippocampus resulted in being not significant at permutation testing ( p > 0.75 in both cases ) . the findings of the present study permit to identify a reliable association between an eeg index ( alpha3/alpha2 power ratio ) and hippocampal atrophy . this eeg marker shows his reliability both in mci and ad subjects , suggesting that it could identify some mci subjects prone to conversion in ad . the principal limitations of the study are ( 1 ) the small size of the ad group ; ( 2 ) the lack of a normal control group . the increase of alpha3/alpha2 ratio is associated with the decrease of hippocampal volume , confirming previous results of our group showing that the increase of high alpha is related to hippocampal atrophy in mci patients [ 21 , 22 ] . the results show that in ad patients , increase of alpha3/alpha2 power ratio is correlated with the decrease of left hippocampal gray matter volumes . in particular , hippocampal areas involved in correlation are presubiculum , dorsal and ventral subiculum , ca2 - 3 sectors of the body , ca1 mesial , and lateral portion of the head . the prevalence of the modification of eeg rhythms in the left hemisphere in patients with ad was found also in a recent eeg coherence study . indeed , in this study , pathologic changes of connectivity are significant on the frontotemporal region of the left hemisphere , but not on the right . our findings confirm the asymmetry between left and right planum temporale previously shown in patients with ad . a large body of literature has demonstrated the crucial role of the left hemisphere in semantic associative encoding and of the left hippocampal - medial prefrontal pathways [ 60 , 61 ] . the increase of high alpha synchronization has been found in internally - cued mechanisms of attention , associated with inhibitory top - down processes , acting as filter to irrelevant information . this filter activity could be carried out by hippocampus . indeed , a recent work has demonstrated that the mossy fiber ( mf ) pathway of the hippocampus , connecting the dentate gyrus to the autoassociative ca3 network , is controlled by a feedforward circuit combining disynaptic inhibition with monosynaptic excitation . analysis of the mf - associated circuit revealed that it could act as a highpass filter [ 63 , 64 ] . the increase in cortico - subcortical inputs to hippocampal formation determines an increase of memory retrieval effort in long - term memory system and dysregulation of divided attention , in particular when multiple stimuli have to be processed [ 6567 ] inducing behavioural dysfunction as well as subsequent memory deficits . the exchange of information between memory and attentive systems has been associated upper alpha band desynchronization [ 68 , 69 ] . the synchronization of high alpha power has been demonstrated to be involved in top - down cognitive processes . this finding could suggest that there is an the attempt to focus attention on highly selective aspect to prevent interference of irrelevant stimuli ( top - down process ) in order to maintain a good memory performance . recently , we suggest that mci subjects could fall in a hyperattentive state during the course of disease . a possible explanation should strongly consider that our results are obtained in an idling state . so , the discussion of the results has to address the default state of the brain . in this point of view , in a normal default state , large cell assemblies cooperate to keep an extensive network . this state is represented by the low alpha rhythm , typical of the eeg idling state . the increase of the alphae3/alpha2 power ration could suggest in mci and ad patients the prevalence of smaller cell assemblies in the default state , due to synaptic disfunction or brain atrophy . of note , seminal studies have demonstrated that large cell assemblies oscillates in low frequencies , whereas smaller cell assemblies develop higher frequencies . the specific involvement of alpha rhythm could suggest that the hippocampal atrophy in ad is linked to functional changes in a broader network . of note , the hypometabolism and atrophy of posterior cingulate / retrosplenial and medial temporal cortex pathway , strictly connected with both hippocampus and visual cortex , as well as with low alpha rhythm generation , are well demonstrated in ad . so , the prevalence of high alpha could underlie the disruption of extensive synaptic connection deriving in the formation of smaller cell assemblies . the impairment of the network could explain memory and cognitive symptoms of ad beyond the hippocampal atrophy itself . our findings confirm the possible diagnostic role of eeg activity when integrated with morphostructural measures in patients with ad .

we evaluated the association between hippocampal atrophy and increase of the eeg markers alpha3/alpha2 relative power ratio in mild cognitive impairment ( mci ) and alzheimer 's disease patients . seventy - nine subjects with mci and 11 patients with ad underwent eeg recording and mri scan . the mci group was subdivided in three subgroups according to growing hippocampal atrophy . the groups were characterized by alpha3/alpha2 relative power ratio . in ad patients group mapped hippocampal regions were computed and related with alpha3/alpha2 power ratio . results show that the increase of alpha3/alpha2 power ratio is correlated with atrophy of hippocampus both in mci and in alzheimer 's disease patients . this finding confirms the possible diagnostic role of eeg markers as diagnostic and prognostic factors in patient with prodromal and declared alzheimer 's disease .

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

mild cognitive impairment ( mci ) refers to the transitional state between the cognitive changes of normal aging and very early dementia . patients with mci , who are at high risk of developing alzheimer disease ( ad ; ) , have smaller hippocampal volume than healthy elderly people [ 3 , 4 ] . moreover , a recent study has demonstrated that atrophy of the hippocampus on mri in cognitively intact elderly people predicts dementia , in particular of alzheimer type , during a 6-year followup . hippocampus is particularly important for memory formation , for attention and for production of eeg rhythmic activity [ 10 , 11 ] . lesions of hippocampal synaptic plasticity block the memory - enhancing effects of direct hippocampal stimulation [ 12 , 13 ] . further , behavioral stress interferes with synaptic plasticity in the hippocampal formation [ 1416 ] . the associative memories involve the dorsal hippocampus , and a lesion of the area reduces the retrieval of associative tasks . recent works showed that in subjects with mci is present , an increase of high alpha as compared to low alpha band occurs [ 21 , 22 ] . as a working hypothesis , eeg markers alpha3/alpha2 power ratio could show modifications proportional to the hippocampal atrophy . in the present study the association between hippocampal atrophy and increase of alpha3/alpha2 relative power ratio was investigated in subjects with mci . recent studies have demonstrated that the hippocampus is not a unitary structure from an anatomophysiological point of view . the hippocampus , including strictly speaking subfields ca1ca4 and the hippocampal formation , including also dentate gyrus , fimbria , subiculum , and parasubiculum , is a highly sophisticated structure . stimuli coming from the entorhinal cortex are processed by the dentate gyrus , subfields ca4 and ca3 , before being projected outside the medial temporal lobe via ca1 or subicular efferent projections . moreover , in addition to the unsurprising right - left specialization for verbal and visuospatial material , some degree of anterior - to - posterior specialization has been shown by fmri studies . as a consequence , it is conceivable that local structural changes take place in the hippocampus of patients with ad and that different hippocampal subregions are affected in ad brickman et al . , and shen et al . local changes in hippocampal subregions could be detected through a radial atrophy mapping method able to assess group , based on high resolution mri at 3 tesla differences . in this study , we tested the hypothesis that the increase of alpha3/alpha2 ratio is related with volumetric differences both in mci patients and in mapped hippocampal regions in ad patients . for the present study , 79 subjects with mci and 11 subjects with alzheimer 's disease ( ad ) were recruited from the memory clinic of the scientific institute for research and care ( irccs ) of alzheimer 's and psychiatric diseases fatebenefratelli in brescia , italy . informed consent was obtained from all participants or their caregivers , according to the code of ethics of the world medical association ( declaration of helsinki ) . b12 and folate deficiency , uncontrolled heart disease , and uncontrolled diabetes ) patients were rated with a series of standardized diagnostic and severity instruments , including the mini - mental state examination ( mmse ; ) , the clinical dementia rating scale ( cdrs ; ) , the hachinski ischemic scale ( his ; ) , and the instrumental and basic activities of daily living ( iadl , badl , ) . in addition , patients underwent diagnostic neuroimaging procedures ( magnetic resonance imaging , mri ) and laboratory testing to rule out other causes of cognitive impairment . inclusion criteria of the study were all of the following : ( i ) complaint by the patient , or report by a relative or the general practitioner , of memory or other cognitive disturbances ; ( ii ) mini - mental state examination ( mmse ) score of 24 to 27/30 , or mmse of 28 and higher plus low performance ( score of 26 or higher ) on the clock drawing test ; ( iii ) sparing of instrumental and basic activities of daily living or functional impairment steadily due to causes other than cognitive impairment , such as physical impairments , sensory loss , and gait or balance disturbances . exclusion criteria were any one of the following : ( i ) patients aged 90 years and older ; ( ii ) history of depression or juvenile - onset psychosis ; ( iii ) history or neurological signs of major stroke ; ( iv ) other psychiatric diseases , epilepsy , drug addiction , and alcohol dependence ; ( v ) use of psychoactive drugs , including acetylcholinesterase inhibitors or other drugs enhancing brain cognitive functions ; ( vi ) current or previous uncontrolled or complicated systemic diseases ( including diabetes mellitus ) or traumatic brain injuries . all patients underwent ( i ) semistructured interview with the patient and , whenever possible , with another informant ( usually , the patient 's spouse or a child of the patient ) by a geriatrician or neurologist ; ( ii ) physical and neurological examinations ; ( iii ) performance - based tests of physical function , gait , and balance ; ( iv ) neuropsychological battery assessing verbal and nonverbal memory , attention and executive functions ( trail making test b - a ; clock drawing test ; ) , abstract thinking ( raven matrices ; ) , frontal functions ( inverted motor learning ; ) , language ( phonological and semantic fluency ; token test ; ) , and apraxia and visuoconstructional abilities ( rey figure copy ; ) ; ( v ) assessment of depressive symptoms by means of the center for epidemiologic studies depression scale ( ces - d ; ) . inclusion and exclusion criteria were homogeneous with previous works [ 4648 ] . as the aim of our study was to evaluate the meaning of alpha3/alpha2 power ratio and its associations with structural changes of hippocampus as diagnostic marker of cognitive impairment , we were not interested in this study in the clinical subtype of mci , that is , amnesic or nonamnesic , single or multiple domains . the diagnosis of ad was made according to nincds - adrda criteria and the diagnostic and statistical manual of mental disorders iv . the eeg activity was recorded continuously from 19 sites by using electrodes set in an elastic cap ( electro - cap international , inc . ) and positioned according to the 1020 international system ( fp1 , fp2 , f7 , f3 , fz , f4 , f8 , t3 , c3 , cz , c4 , t4 , t5 , p3 , pz , p4 , t6 , o1 , and o2 ) . the recordings were used off - line to rereference the scalp recordings to the common average . it was referred to another electrode placed 2 cm below suborbital ridge of the right eye . the left and right horizontal eog channels were collected from two electrodes at the left and the right lateral canthus . longer recordings would have reduced the variability of the data , but they would also have increased the possibility of slowing of eeg oscillations due to reduced vigilance and arousal . the eeg epochs with ocular , muscular , and other types of artifacts were discarded . frequency bands were determiner on an individuale basis , because of the variability of the brain rhythms with age and diseases . a digital fft - based power spectrum analysis ( welch technique , hanning windowing function , and no phase shift ) computed the power density of eeg rhythms with a 0.5 hz frequency resolution , ranging from 2 to 40 hz . two anchor frequencies were selected according to literature guidelines ( 47 ) , which are the transition theta / alpha frequency ( tf ) and the individual alpha frequency ( iaf ) peak . as previously mentioned , the tf marks the transition frequency between theta and alpha bands , and it represents an estimate of the frequency at which theta and alpha spectra intersect . we computed the tf as the minimum power in the alpha frequency range since our eeg recordings were performed at rest . the iaf represents instead the frequency with the maximum power peak within the extended alpha range ( 514 hz ) . alpha1 and alpha2 band were computed for each subject as follows : alpha1 from tf to the middle point of the tf - iaf range and alpha2 from this middle point to iaf peak . we found that the bandwidth in alpha1 and alpha2 bands was different among the groups . in no vascular damage and severe vascular damage groups than in mild and moderate vascular damage groups ( 1.7 and 1.87 hz , resp . ) we performed a statistical analysis to test if this difference was significant among groups in these frequency bands . it was not the case , since the analysis did not show a main group significant effect ( p = 0.06 ) . finally , in the frequency bands so determined , we computed the relative power spectra for each subject . relative power density for each frequency band was computed as the ratio between the absolute power and the mean power spectra from 2 to 40 hz . the relative band power at each band was defined as the mean of the relative band power for each frequency bin within that band . mri scans were acquired with a 1.0 tesla philips gyroscan at the neuroradiology unit of the citt di brescia hospital , brescia . the following sequences were used to measure hippocampal volumes : a high - resolution gradient echo t1-weighted sagittal 3d sequence ( tr = 20 ms , te = 5 ms , flip angle = 30 , field of view = 220 mm , acquisition matrix = 256 256 , and slice thickness = 1.3 mm ) and a fluid - attenuated inversion recovery ( flair ) sequence ( tr = 5000 ms , te = 100 ms , flip angle = 90 , field of view = 230 mm , acquisition matrix = 256 256 , and slice thickness = 5 mm ) . the hippocampal boundaries were manually traced on each hemisphere by a single tracer with the software program display ( mcgill university , montreal , canada ) on contiguous 1.5 mm slices in the coronal plane . the starting point for hippocampus tracing was defined as the hippocampal head when it first appears below the amygdala , the alveus defining the superior and anterior border of the hippocampus . the hippocampal tail was traced until it was visible as an oval shape located caudally and medially to the trigone of the lateral ventricles [ 51 , 52 ] . briefly , the procedure includes ( i ) filtering of flair images to exclude radiofrequency inhomogeneities , ( ii ) segmentation of brain tissue from cerebrospinal fluid , ( iii ) modelling of brain intensity histogram as a gaussian distribution , and ( iv ) classification of the voxels whose intensities were 3.5 sds above the mean as wmhs [ 51 , 52 ] . to correct for individual differences in head size , hippocampal and wmhs volumes were normalized to the total intracranial volume ( tiv ) , obtained by manually tracing with display the entire intracranial cavity on 7 mm thick coronal slices of the t1-weighted images . both manual and automated methods manual segmentation of the hippocampus is currently considered the gold standard technique for the measurement of such complex structures . left and right hippocampal volumes were estimated and summed to obtain a total volume ( individual ) of both anatomical structures . this procedure allows measurements to be made at corresponding surface locations in each subject , which are then compared statistically in 3d [ 53 , 54 ] . to assess hippocampal morphology , a medial curve was automatically defined as the 3d curve traced out by the centroid of the hippocampal boundary in each image slice . the radial size of each hippocampus at each boundary point was assessed by automatically measuring the radial 3d distance from the surface points to the medial curve defined for individual 's hippocampal surface model . preliminarily , the significant differences among groups in demographic variables , ( age , education , and mmse score ) and morphostructural characteristics , ( hippocampal and white matter hyperintensities , wmhs , and volume ) , were evaluated ( table 1 ) . the first analysis was performed in order to verify the difference of hippocampal volume among groups . the second anova was performed in order to check differences in alpha3/alpha2 relative power ratio in the three mci subgroups ordered by decreasing tertile values of the hippocampal volume . in each anova , group was the independent variable , the frequency ratios were the dependent variable . in ad patients , the radial atrophy mapping was chosen because it is more suitable to study subregional volume of the hippocampus . correlation maps between eeg rhythms and hippocampal surface were computed . the correlation analysis between eeg rhythms and hippocampal volume was performed only in 11 ad subjects ; we otherwise compared hippocampal gray matter distribution maps between normal controls and ad patients in order to verify that the ad found correlations between eeg rhythms and hippocampal regions were present in areas where ad is more atrophic than normal subjects . the correlation maps were generated on 3d models of the hippocampal formation where the dorsal and ventral surfaces can be appreciated . the correlations and the associated p value maps were plotted onto a colour - coded model of the hippocampal surface . a surface point significance threshold of p < 0.05 was used to visualize the regional specificity of gray matter changes in the cortex . set level correction for multiple comparisons permutation tests are based on measuring the total area of the hippocampus with suprathreshold statistics , after setting the threshold at p < 0.05 . n = 8 , 000 , the approximate margin of error ( 95% confidence interval ) for p is around 5% of p. both left and right hippocampal volumes were investigated in ad patients given the superior well - known left hemispheric involvement in declared dementia . the analysis on the ad subjects was conducted to verify the reliability of the alpha3/alpha2 ratio as factor associated with conversion of a subpopulation of mci subjects in alzheimer 's disease . this results need further confirmation in a larger size population to strength the statistical power of the analysis . of note , a larger population could be permitted to detect more precisely the volume ranges within the hippocampal subregions and their correlation with the eeg marker , ruling out other possible associations . in the two groups of patients , mci and ad patients , the mri analysis method performed was different : volumetric analysis for mci and radial atrophy mapping for ad patients . in this study , we were interested in morphofunctional ( mri - eeg ) association and not in neuropsychological issues . mmse values were provided as a general marker of the entity of the cognitive decline of patients . table 1 summarizes the anova results of demographic variables , that is , age , education , mmse score , and morphostructural characteristics , that is , hippocampal , and white matter hyperintensities volume in the whole mci cohort as well as in the three subgroups in study . significant statistical results were found in hippocampal volume ( respectively , f 2,76 = 157.27 ; p < 0.00001 and f 2,76 = 132.5 ; p < 0.00001 ) . table 2 shows the results of alpha3/alpha2 ratio in the groups based on the decrease of hippocampal volumes . anova results revealed significant main effect group in alpha3/alpha2 ratio for hippocampal ( f 2,76 = 3.38 ; p < 0.03 ) decreasing volume . table 3 summarizes sociodemographic characteristics , mmse scores , and alpha3/alpha2 power ratio in ad cohort . correlations in right hippocampus resulted in being not significant at permutation testing ( p > 0.75 in both cases ) . the findings of the present study permit to identify a reliable association between an eeg index ( alpha3/alpha2 power ratio ) and hippocampal atrophy . this eeg marker shows his reliability both in mci and ad subjects , suggesting that it could identify some mci subjects prone to conversion in ad . the principal limitations of the study are ( 1 ) the small size of the ad group ; ( 2 ) the lack of a normal control group . the increase of alpha3/alpha2 ratio is associated with the decrease of hippocampal volume , confirming previous results of our group showing that the increase of high alpha is related to hippocampal atrophy in mci patients [ 21 , 22 ] . the results show that in ad patients , increase of alpha3/alpha2 power ratio is correlated with the decrease of left hippocampal gray matter volumes . in particular , hippocampal areas involved in correlation are presubiculum , dorsal and ventral subiculum , ca2 - 3 sectors of the body , ca1 mesial , and lateral portion of the head . the prevalence of the modification of eeg rhythms in the left hemisphere in patients with ad was found also in a recent eeg coherence study . indeed , in this study , pathologic changes of connectivity are significant on the frontotemporal region of the left hemisphere , but not on the right . our findings confirm the asymmetry between left and right planum temporale previously shown in patients with ad . a large body of literature has demonstrated the crucial role of the left hemisphere in semantic associative encoding and of the left hippocampal - medial prefrontal pathways [ 60 , 61 ] . the increase of high alpha synchronization has been found in internally - cued mechanisms of attention , associated with inhibitory top - down processes , acting as filter to irrelevant information . this filter activity could be carried out by hippocampus . indeed , a recent work has demonstrated that the mossy fiber ( mf ) pathway of the hippocampus , connecting the dentate gyrus to the autoassociative ca3 network , is controlled by a feedforward circuit combining disynaptic inhibition with monosynaptic excitation . analysis of the mf - associated circuit revealed that it could act as a highpass filter [ 63 , 64 ] . the increase in cortico - subcortical inputs to hippocampal formation determines an increase of memory retrieval effort in long - term memory system and dysregulation of divided attention , in particular when multiple stimuli have to be processed [ 6567 ] inducing behavioural dysfunction as well as subsequent memory deficits . this finding could suggest that there is an the attempt to focus attention on highly selective aspect to prevent interference of irrelevant stimuli ( top - down process ) in order to maintain a good memory performance . so , the discussion of the results has to address the default state of the brain . in this point of view , in a normal default state , large cell assemblies cooperate to keep an extensive network . this state is represented by the low alpha rhythm , typical of the eeg idling state . the increase of the alphae3/alpha2 power ration could suggest in mci and ad patients the prevalence of smaller cell assemblies in the default state , due to synaptic disfunction or brain atrophy . of note , seminal studies have demonstrated that large cell assemblies oscillates in low frequencies , whereas smaller cell assemblies develop higher frequencies . the specific involvement of alpha rhythm could suggest that the hippocampal atrophy in ad is linked to functional changes in a broader network . of note , the hypometabolism and atrophy of posterior cingulate / retrosplenial and medial temporal cortex pathway , strictly connected with both hippocampus and visual cortex , as well as with low alpha rhythm generation , are well demonstrated in ad . the impairment of the network could explain memory and cognitive symptoms of ad beyond the hippocampal atrophy itself . our findings confirm the possible diagnostic role of eeg activity when integrated with morphostructural measures in patients with ad .

written informed consent was obtained after the nature of the research and possible consequences of the study were explained . the study was approved by the institutional review board of the national institutes of health . three healthy volunteers with no history of systemic or ocular disease were recruited to undergo ao - icg imaging in both eyes . prior to imaging , eyes were dilated with 1 drop of 2.5% phenylephrine hydrochloride and 1 drop of 1% tropicamide . a replica of a broadband adaptive optics scanning light ophthalmoscope ( aoslo ) was assembled as described previously . wavefront sensing was performed using an 850 nm light source , and imaging was performed using a broadband ( 17 nm full width at half maximum ) 790 nm light source with a beam diameter of 7.75 mm at the point of entry into the eye . specifically , an additional photomultiplier tube capable of near - infrared detection was added for icg imaging ( product h7422a-50 ; hamamatsu , shizuoka , japan ) , along with a dichroic filter ( product lpd02 - 830ru-25 ; semrock , rochester , ny , usa ) , and two additional filters in front of the detector ( products ff01 - 842/sp and ff01 - 832/37 ; semrock ) to effectively collect light between 810 and 830 nm . a bandpass filter was placed in front of the 790 nm light source to restrict the excitation wavelengths to only those wavelengths below 800 nm ( product et775/50x ; chroma technology , bellow falls , vt , usa ) , as well as in front of the 850 nm light source to prevent light less than 840 nm from reaching the eye ( product ff01 - 850/10 ; semrock ) . these additional filters slightly reduced the power of the light as measured at the cornea . imaging was performed using either a 90/10 or an 80/20 ( % transmission/% reflection ) system beam splitter . in this study , the light power levels measured at the cornea were maintained below 100 w for the 790 nm source and 35 w for the 850 nm source , which are under the maximum permissible exposure set by american national standards institute standard z136.1 2014 . twenty- to 30-second videos were acquired at various retinal locations at an imaging rate of 17 frames per second . the locations were selected with some overlap , using square field sizes ranging from 0.75 to 2.0 and included the fovea and parafoveal regions of both eyes , both prior to and after icg injection . additional peripheral regions were also selected for imaging at 5 , 10 , and 15 eccentricity in the temporal direction . indocyanine green was administered intravenously as a single bolus at a dose of 25 mg in 3 ml , according to the standard of care at the national eye institute eye clinic . during each recording , four image sequences were acquired simultaneously : ao confocal reflectance , icg fluorescence ( ao - icg ) , and two additional channels containing multiple scattered light from opposing directions of the confocal detector , which were used to generate ao split detection and ao dark - field images . in total , a computer - controlled fixation system was used to facilitate the capture of images . during imaging , the focal plane was set to that of the photoreceptors based on a previous report stating that the optimal focal plane for ao dark - field rpe imaging was the plane of optimal photoreceptor imaging . biometry measurements were also acquired from all subjects ( iol master ; carl zeiss meditec , dublin , ca , usa ) and used to determine scaling factors to convert pixels to micrometers . videos were postprocessed to correct for eye motion , using customized software , as described previously . because four videos were acquired simultaneously , the video with the ( subjectively determined ) sharpest features was selected for the computation of eye motion , which in all cases was the ao confocal reflectance channel . the computed eye motion was then applied to the other three corresponding videos , which enabled accurate registration of even those videos with little or no signal ( e.g. , icg videos acquired prior to the injection of icg dye can be registered using this strategy ) . after eye motion correction , registered frames were averaged , and a montage was manually created using photoshop software ( adobe , san jose , ca , usa ) . with the exception of quantitative analyses described below , the maximum number of successfully registered frames was included for averaging ( up to a total of 500 possible frames ) . for display purposes , ao - icg and ao dark - field image contrast ranges a subset of human ao - icg data was selected for further analysis . for each subject , a 0.9 square region of interest that showed the clearest rpe cell outlines on the ao dark - field images was selected for further analysis . md , usa ; available in the public domain , http://imagej.nih.gov/ij/ ) , we manually identified the centers of rpe cells based on the locations of cell outlines and following the assumption that rpe cells form a uniform hexagonal array in healthy volunteers . these cell centers were then used to construct voronoi neighborhoods to facilitate qualitative comparisons between ao dark - field and ao - icg images in order to determine whether there was a correspondence between the cell outlines seen using ao dark - field and the pattern seen using ao - icg . in addition , cell density was computed by dividing the number of identified cell centers by the area of the region of interest and compared to previously published values of rpe cell density based on histology . relative intensity measurements were also carried out to verify the uptake of icg dye . in human subjects , fluorescence intensity values were measured from raw data as the average across a single video acquired at the fovea both prior to the injection of icg and 2 hours afterward ( acquisition parameters were kept constant for this analysis : 1.5 field of view , 200 frames acquired at 17 hz , icg photomultiplier tube gain setting of 0.500 , focus set to the plane of best photoreceptor focus ) . the preinjection video was used as the baseline intensity , and the relative intensity was calculated by dividing the mean of the postinjection site video by the mean of the preinjection video in order to derive an estimate of the signal strength . for mouse data ( described below ) , the average intensity value of the image acquired in the icg channel was measured by drawing a line through the rpe layer and taking the average of the intensity values along the line , using imagej software . in this case the relative intensity was computed by dividing the measured intensities in an injected mouse by those in a noninjected mouse . human data were paired before and after injection , and mice data were paired by siblings ( injected and noninjected ) . a 1-tailed paired t - test was used to evaluate whether icg injection resulted in an increase in detected fluorescence intensity . experiments were conducted according to protocols approved by a local institutional animal care and use committee and in compliance with the arvo statement for the use of animals in ophthalmic and vision research . ten mice from various backgrounds were used ( 6 c57bl/6j , 2 b6-albino , 2 oto2 - 10 ) . mice were paired into five sets of 2 siblings with each pair taken from the same litter . the age of the mice ranged from 2 to 6 months ( average , 3.5 months ) . intraperitoneal injection of icg was performed in one mouse from each pair ( 200 l of 5 mg / ml solution ) . experimental animals were euthanized by carbon dioxide inhalation at a 16 hours after administration of icg dye for the icg - injected mice . immediately after enucleation , eyes were embedded in optical cutting temperature ( oct ) compound and rapidly frozen using acetone , cooled to approximately 70c by addition of dry ice . no fixation agents were used based on our own experience and prior reports that icg localization within the ocular tissue is altered due to the highly soluble nature of icg . cryosections ( 810 m ) were cut through the center of the eye , collected on super - frost plus microscope slides ( fisher scientific , waltham , ma , usa ) , vacuum dried , and mounted in immu - mount ( thermofisher scientific , rockville , md , usa ) . to prevent diffusion of icg dye into the unfixed tissue , cryosections were imaged immediately after mounting . microscopy was performed using a modified axio imager z1 microscope ( carl zeiss ) outfitted with an x - cite 200dc light source ( lumen dynamics , mississauga , on , canada ) and an icg filter kit ( product icg - b - zhe - zero ; semrock ) , which enabled near - infrared imaging of icg . all icg images were acquired using a 1.30 numerical aperture 40 objective and axiocam mrm charge - coupled device camera ( carl zeiss ) with an exposure of 4.0 seconds . autofluorescence images of cryosections were acquired sequentially ( immediately after icg imaging ) to allow for the identification of retinal layers ( 450490 nm excitation , 500550 nm detection ) . written informed consent was obtained after the nature of the research and possible consequences of the study were explained . the study was approved by the institutional review board of the national institutes of health . three healthy volunteers with no history of systemic or ocular disease were recruited to undergo ao - icg imaging in both eyes . prior to imaging , eyes were dilated with 1 drop of 2.5% phenylephrine hydrochloride and 1 drop of 1% tropicamide . a replica of a broadband adaptive optics scanning light ophthalmoscope ( aoslo ) was assembled as described previously . wavefront sensing was performed using an 850 nm light source , and imaging was performed using a broadband ( 17 nm full width at half maximum ) 790 nm light source with a beam diameter of 7.75 mm at the point of entry into the eye . specifically , an additional photomultiplier tube capable of near - infrared detection was added for icg imaging ( product h7422a-50 ; hamamatsu , shizuoka , japan ) , along with a dichroic filter ( product lpd02 - 830ru-25 ; semrock , rochester , ny , usa ) , and two additional filters in front of the detector ( products ff01 - 842/sp and ff01 - 832/37 ; semrock ) to effectively collect light between 810 and 830 nm . a pinhole with a 3 airy - disk diameter a bandpass filter was placed in front of the 790 nm light source to restrict the excitation wavelengths to only those wavelengths below 800 nm ( product et775/50x ; chroma technology , bellow falls , vt , usa ) , as well as in front of the 850 nm light source to prevent light less than 840 nm from reaching the eye ( product ff01 - 850/10 ; semrock ) . these additional filters slightly reduced the power of the light as measured at the cornea . imaging was performed using either a 90/10 or an 80/20 ( % transmission/% reflection ) system beam splitter . in this study , the light power levels measured at the cornea were maintained below 100 w for the 790 nm source and 35 w for the 850 nm source , which are under the maximum permissible exposure set by american national standards institute standard z136.1 2014 . twenty- to 30-second videos were acquired at various retinal locations at an imaging rate of 17 frames per second . the locations were selected with some overlap , using square field sizes ranging from 0.75 to 2.0 and included the fovea and parafoveal regions of both eyes , both prior to and after icg injection . additional peripheral regions were also selected for imaging at 5 , 10 , and 15 eccentricity in the temporal direction . indocyanine green was administered intravenously as a single bolus at a dose of 25 mg in 3 ml , according to the standard of care at the national eye institute eye clinic . during each recording , four image sequences were acquired simultaneously : ao confocal reflectance , icg fluorescence ( ao - icg ) , and two additional channels containing multiple scattered light from opposing directions of the confocal detector , which were used to generate ao split detection and ao dark - field images . in total , a computer - controlled fixation system was used to facilitate the capture of images . during imaging , the focal plane was set to that of the photoreceptors based on a previous report stating that the optimal focal plane for ao dark - field rpe imaging was the plane of optimal photoreceptor imaging . biometry measurements were also acquired from all subjects ( iol master ; carl zeiss meditec , dublin , ca , usa ) and used to determine scaling factors to convert pixels to micrometers . videos were postprocessed to correct for eye motion , using customized software , as described previously . because four videos were acquired simultaneously , the video with the ( subjectively determined ) sharpest features was selected for the computation of eye motion , which in all cases was the ao confocal reflectance channel . the computed eye motion was then applied to the other three corresponding videos , which enabled accurate registration of even those videos with little or no signal ( e.g. , icg videos acquired prior to the injection of icg dye can be registered using this strategy ) . after eye motion correction , registered frames were averaged , and a montage was manually created using photoshop software ( adobe , san jose , ca , usa ) . with the exception of quantitative analyses described below , the maximum number of successfully registered frames was included for averaging ( up to a total of 500 possible frames ) . for display purposes , ao - icg and ao dark - field image contrast ranges were stretched between the minimum and maximum values . a subset of human ao - icg data was selected for further analysis . for each subject , a 0.9 square region of interest that showed the clearest rpe cell outlines on the ao dark - field images was selected for further analysis . md , usa ; available in the public domain , http://imagej.nih.gov/ij/ ) , we manually identified the centers of rpe cells based on the locations of cell outlines and following the assumption that rpe cells form a uniform hexagonal array in healthy volunteers . these cell centers were then used to construct voronoi neighborhoods to facilitate qualitative comparisons between ao dark - field and ao - icg images in order to determine whether there was a correspondence between the cell outlines seen using ao dark - field and the pattern seen using ao - icg . in addition , cell density was computed by dividing the number of identified cell centers by the area of the region of interest and compared to previously published values of rpe cell density based on histology . relative intensity measurements were also carried out to verify the uptake of icg dye . in human subjects , fluorescence intensity values were measured from raw data as the average across a single video acquired at the fovea both prior to the injection of icg and 2 hours afterward ( acquisition parameters were kept constant for this analysis : 1.5 field of view , 200 frames acquired at 17 hz , icg photomultiplier tube gain setting of 0.500 , focus set to the plane of best photoreceptor focus ) . the preinjection video was used as the baseline intensity , and the relative intensity was calculated by dividing the mean of the postinjection site video by the mean of the preinjection video in order to derive an estimate of the signal strength . for mouse data ( described below ) , the average intensity value of the image acquired in the icg channel was measured by drawing a line through the rpe layer and taking the average of the intensity values along the line , using imagej software . in this case the relative intensity was computed by dividing the measured intensities in an injected mouse by those in a noninjected mouse . human data were paired before and after injection , and mice data were paired by siblings ( injected and noninjected ) . a 1-tailed paired t - test was used to evaluate whether icg injection resulted in an increase in detected fluorescence intensity . experiments were conducted according to protocols approved by a local institutional animal care and use committee and in compliance with the arvo statement for the use of animals in ophthalmic and vision research . ten mice from various backgrounds were used ( 6 c57bl/6j , 2 b6-albino , 2 oto2 - 10 ) . mice were paired into five sets of 2 siblings with each pair taken from the same litter . the age of the mice ranged from 2 to 6 months ( average , 3.5 months ) . intraperitoneal injection of icg was performed in one mouse from each pair ( 200 l of 5 mg / ml solution ) . experimental animals were euthanized by carbon dioxide inhalation at a 16 hours after administration of icg dye for the icg - injected mice . immediately after enucleation , eyes were embedded in optical cutting temperature ( oct ) compound and rapidly frozen using acetone , cooled to approximately 70c by addition of dry ice . no fixation agents were used based on our own experience and prior reports that icg localization within the ocular tissue is altered due to the highly soluble nature of icg . cryosections ( 810 m ) were cut through the center of the eye , collected on super - frost plus microscope slides ( fisher scientific , waltham , ma , usa ) , vacuum dried , and mounted in immu - mount ( thermofisher scientific , rockville , md , usa ) . to prevent diffusion of icg dye into the unfixed tissue , cryosections were imaged immediately after mounting . microscopy was performed using a modified axio imager z1 microscope ( carl zeiss ) outfitted with an x - cite 200dc light source ( lumen dynamics , mississauga , on , canada ) and an icg filter kit ( product icg - b - zhe - zero ; semrock ) , which enabled near - infrared imaging of icg . all icg images were acquired using a 1.30 numerical aperture 40 objective and axiocam mrm charge - coupled device camera ( carl zeiss ) with an exposure of 4.0 seconds . autofluorescence images of cryosections were acquired sequentially ( immediately after icg imaging ) to allow for the identification of retinal layers ( 450490 nm excitation , 500550 nm detection ) . following systemic injection of icg into three human subjects ( hv1 : 33-year - old male ; hv2 : 40-year - old female ; hv3 : 25-year - old female ) , a heterogeneous ao - icg signal was observed by using the modified aoslo that was qualitatively distinct from both ao confocal reflectance and ao dark - field images of the same retinal region ( fig . whereas only cell outlines were visible under ao dark - field illumination , entire cells appeared to be visible using ao - icg . the structures visible on ao - icg imaging were hexagonal in appearance and several - fold larger in size than cone photoreceptors , consistent with the expected size of rpe cells . three different modes of adaptive optics retinal images captured simultaneously in the foveas of three human subjects ( hv1 , hv2 , and hv3 ) . ( left to right ) adaptive optics confocal reflectance adaptive optics image of cone photoreceptors , outlines of rpe cells revealed using darkfield ao , and near - infrared fluorescence imaging of rpe cells using ao - icg . all rois were selected within 1.0 of the foveal center , which was visually identified as the area of highest cone density . adaptive optics dark - field imaging was used to confirm that these cells were indeed rpe cells . in regions where individual rpe cells could be visualized using both ao dark - field imaging and ao - icg imaging , there was good correspondence between the two techniques ( fig . a total of 2,209 rpe cells were manually identified in 3 regions of interest ( roi ) near the fovea of three eyes ( approximate eccentricity of the center of each roi was 1.1 in hvi ; 1.0 in hv2 ; and 1.4 in hv3 ; each roi was 1.5 ) . the density of the rpe cells based on manual cell labeling ( fig . cells / mm for the three subjects ( average : 5811 cells / mm ) , comparable to a previously published report of rpe cell density in humans ( 7500 cells / mm peak density at fovea , decreasing to 5000 cells / mm at an approximate eccentricity of 1 ) and in monkeys ( 5260 320 cells / mm peak density at fovea , decreasing to 5110 310 cells / mm at 1 ) . voronoi neighborhoods generated from the manually identified cells overlapped with the individual patterns seen in ao - icg , suggesting that icg accumulates in the cytoplasm of rpe cells . comparison of rpe cells imaged using different ophthalmic ao modalities ( one eye from each subject ) . ( left to right ) adaptive optics dark - field images showing cell outlines , ao - icg fluorescence images showing entire cells , ao dark - field and ao - icg fluorescence images with voronoi diagrams overlaid . the voronoi overlay was generated based on manually identified rpe cells from the dark - field image . for each subject , the same voronoi is shown in the third and fourth columns . the icg fluorescence within individual voronoi neighborhoods is relatively uniform , suggesting that there is good correspondence between the cells identified using ao - df and those using ao - icg . the axial resolution of confocal scanning systems is poor relative to its lateral resolution when imaging using the aoslo in the human eye . hence , histology of mouse eyes was used to confirm the layer - specific localization of the dye following systemic injection . in all cases , there was an increase in icg fluorescence signal that was specific to the rpe layer following systemic icg injection ( fig . measurements of fluorescence within the rpe layer were higher in the icg - injected animals than in their noninjected siblings , with an average intensity increase of 4.7-fold . the example shown in figure 3 illustrates a 2.7-fold increase in intensity from one pair of c57bl6/j mice . no icg fluorescence was detected in the inner retina . in humans , from in vivo adaptive optics imaging data , there was an average 2.5-fold increase in intensity , comparing intensity values 2 hours after injection to the same area prior to injection ( p < 0.05 ) . prior to injection , there was no detectable signal ( e.g. , no leakage of excitation or wavefront sensing light into the ao - icg detection channel ) ( fig . indocyanine green imaging in a mouse eye showing that icg dye accumulates in the rpe after systemic injection . representative images of unfixed cryosections from an ( a , b ) noninjected mouse and ( c , d ) a mouse injected with icg 16 hours earlier . ( a , c ) the outer retinal layers can be visualized based on the autofluorescence of the tissue . onl , outer nuclear layer ( photoreceptor nuclei ) ; is , photoreceptor inner segment ; os , photoreceptor outer segment ; rpe , retinal pigment epithelial cells ( area between arrows ) ; ch , choroid . ( b ) the rpe and choroidal layers exhibit near - infrared autofluorescence , as can be seen in the noninjected control mouse . ( d ) after icg injection , the near - infrared fluorescence of the rpe layer is increased relative to both the noninjected mouse rpe layer and the near - infrared fluorescence . in this figure , a 2.7 increase in fluorescence signal is shown , comparing d to b. the icg images in b and d have been increased in brightness by 4 for display purposes . illustration showing how correction of eye motion enables averaging and reconstruction of icg signal ( subject hv2 , left eye ) . ( a ) registered average of 300 images prior to the injection of icg , showing no discernable signal and ( b d ) images of the same region as a after injection of icg . ( c ) average of 10 frames ( cumulative light exposure : 400 ms ) . ( d ) average of 100 frames ( cumulative light exposure : 4 seconds ) . ( c , d ) intensities in the lower right half of the image are stretched between the minimum and maximum values . all data were acquired using the same photomultiplier gain . for display purposes , a constant brightness increase of 600% was applied equally to the images shown in a , b , and upper left halves of c and d. scale bar : 100 m . although the rpe mosaic was not visible on a single frame , it was easily visible after accumulation of as few as 10 frames , corresponding to approximately 400 ms of cumulative light exposure ( fig . we tested two different beam splitters to couple the light sources ( 10% or 20% reflection ) and the detectors ( 90% or 80% transmission , respectively ) . the use of an 80/20 beam splitter ( hv2 and hv3 ) resulted in a similar or better ao - icg image than that obtained using the 90/10 beam splitter ( hv1 ) , with no apparent differences in the other ao imaging modalities . ao - icg images were nearly identical when acquired using different field sizes and even across different time points ( fig . 5 ) . given the longevity of this signal , and to ensure maximal clearance of icg from the blood stream , we opted to acquire extensive ao - icg images at the time point of 2 hours post injection ( figs . 1 , 2 , 4 , 6 , 7 ) . the same region was imaged at two different times after intravenous injection of icg dye ( 20 and 120 minutes , respectively ) , using different fields of view ( 0.75 , 1.0 , and 1.5 ) . there is a small region that was not imaged due to small differences in fixation and eye motion from one imaging session to another ( top left , black box ) . the images were nearly identical , suggesting that the dye uptake is stable and can be imaged over at least 100 minutes . simultaneous aoslo imaging of multiple structures in the outer retina in subject hv2 , left eye . ( top to bottom ) image acquired at a retinal location of 5 , 10 , and 15 in the temporal direction . ( left to right ) confocal reflectance , split detection , icg , false color image ( split detection , red ; icg , green ) showing the imprinting effect of photoreceptors on the ao - icg image . the cone photoreceptors are visible in confocal reflectance and split detection at all retinal eccentricities . although individual rpe cells are visible at 5 temporal using both ao - icg and ao darkfield , at larger eccentricities , the cell boundaries become more difficult to distinguish due to signal imprinting from the overlying photoreceptors . adaptive optics - icg image of rpe cells generated from 43 overlapping ao - icg videos acquired in the late phase of icg , at the fovea of subject hv2 , right eye . dark shadows from overlying blood vessels can be seen ( vertical lines at the upper right and lower left ) . , we were still able to simultaneously acquire high quality ao confocal reflectance and ao split - detection images alongside ao - icg images ( fig . the ao - icg rpe signal appears to be visible over a range of retinal eccentricities within 5 of the foveal center without any noticeable drop - off in signal ( figs . however , at larger eccentricities , there is an imprinting effect which occurs from the overlying photoreceptors ( fig . 6 ) . adaptive optics - icg provides a novel method for imaging rpe cells within the living human eye . high - resolution single - photon fluorescence imaging revealed the presence of a stable , heterogeneous signal that persisted between 20 and 120 minutes after intravenous injection of icg at a standard clinical dosage . this appears to be a physiologically normal phenomenon in healthy volunteers free of any signs of systemic or ocular diseases . the mechanism for this differential fluorescence remains to be explored , as it is currently unknown whether this is due to differential uptake of icg dye or due to quenching from intrinsic pigments such as melanin , which has a broad absorption spectra that may be different in vivo than in vitro . interestingly , the heterogeneity of the prolonged icg signal that we observed in humans has also been reported in rats and considerable cell - to - cell heterogeneity within the rpe mosaic has been shown . images of the ao - icg signal presented in this paper will form the basis for future investigations to explore the interactions between icg and rpe at the cellular level . we confirmed the specific localization of icg to the rpe cells using histologic data from mice tissue samples ( fig . the selective uptake of icg into rpe cells appears to be specific , with no substantial uptake in neighboring retinal cells . this phenomenon was robust and repeatable over a range of different mouse strains , consistent with previous reports demonstrating this phenomenon in rats and nonhuman primates and could be demonstrated in human subjects as shown in this paper , suggesting that it is generalizable to a potentially larger range of animal or disease models . in contrast to previous studies that administered icg intravenously , we administered the dye intraperitoneally in mice in order to prolong the absorption time of the dye into the rpe cells and increase the strength of the icg signal under microscopy . although histologic data shows that there is instrinsic infrared autofluorescence in the outer retina ( fig . 3b ) , this signal is too weak to be captured using the current implementation of ao - icg ( fig . the icg signal was approximately 5 times larger than the background infrared autofluorescence for pigmented mice . in support of our claim that the near infrared fluorescence in the rpe was due to injected icg , and not due to background infrared autofluorescence , we also evaluated a pair of albino mice . whereas the icg fluorescence intensity in the rpe of the injected albino mouse was similar to that of the other pigmented strains , the near - infrared autofluorescence in the corresponding control ( noninjected sibling ) was largely undetectable . this confirms that there is uptake of icg in the rpe following systemic injection , independent of the background infrared autofluorescence . in particular , this suggests that the background infrared autofluorescence signal arises largely from pigmentation ( melanin ) and that the strength of the icg signal in rpe cells is many - fold higher than this background autofluorescence . our implementation of ao - icg uses only one imaging light source to record four different modalities of ao images ( confocal reflectance , split detection , dark field , and icg ) . because these modalities are recorded simultaneously and they originate from the same illumination source , they are in perfect registration with each other . importantly , we have shown that the modifications that are required for ao - icg imaging can be accomplished in a manner that does not diminish the image quality in the other channels ( figs . 1 , 2 , 6 ) . the instrumentation that we used for ao dark - field imaging is an exact replica of that described in a previous paper demonstrating ao dark - field imaging of rpe cells , and we were able to acquire high - quality ao dark - field images in the foveas of all three subjects . however , in our hands , ao dark - field imaging successfully revealed the rpe cells in only a small minority of subjects and is generally not successful in revealing rpe cells outside of the foveal region . nevertheless , direct comparison between ao dark field and ao - icg in three selected regions of interest in which ao dark field was successful established the fact that the cells visualized using ao - icg are contained within the cell borders as seen using ao dark field . based on a previous study , the cellular structures visualized using ao dark field also colocalize to rpe cells , revealed by ao visible - light autofluorescence imaging , consistent with our observation that ao - icg can be used to visualize rpe cells in the living human eye first , at larger eccentricities , there is signal imprinting from overlying photoreceptors visible in the ao - icg images ( fig . this imprinting could represent a method with which to visualize the interaction between photoreceptors and rpe cells , as it likely arises from the excitation light being wave guided by the overlying photoreceptors . notably , whereas rpe cells could not be distinguished using ao dark - field imaging at any of the larger eccentricities ( 5 , 10 , and 15 ) , combined color images of ao - icg / ao split - detection suggest that with further optimization , ao - icg may be a more robust method for imaging rpe cells outside of the fovea ( fig . second , the ao - icg signal is weak , with an average signal increase of 2.5 after injection compared to before injection in humans . despite the relatively weak signal , signal integration through eye motion compensation provides a reliable method for generation of images , and an ao - icg signal can be obtained using as few as 10 frames , in the best case scenario . for comparison , the initial reports of ao autofluorescence imaging of rpe cells used between 1000 and 1700 frames . although we performed a number of optimization experiments , additional improvements in instrumentation could lead to improved signal and may also enable infrared autofluorescence imaging of rpe cells . that said , the risk for adverse reactions is very low , and administration of icg dye is a standard clinical procedure and one of only a few examples of an extrinsic dye that is approved by the us food and drug administration for use in the human body . taking into consideration both the clinical and the preclinical data , we demonstrate that in the late phase of icg imaging , rpe cells contribute to the fluorescence signal that is imaged . although icg plays a relatively limited role in the clinic in comparison to other techniques , our finding may lead to a new clinical method to indirectly assess the health or status of the rpe layer as a whole using conventional methods for imaging icg in the clinic , even without the use of ao technology . it should be noted that even though icg undergoes rapid clearance from the blood plasma , the persistence of icg in the rpe may reflect a longer time course for the clearance in cells that have internalized this fluorophore . importantly , this persistence of icg provides an extended period of time for ao imaging to occur , which is generally a lengthy process since a large number of overlapping images need to be acquired in order to reconstruct a larger montage . the icg clearance time remains to be explored . in the future , implementation of real - time eye tracking capabilities , in combination with streamlined imaging protocols , should lead to dramatic improvements in imaging throughput . adaptive optics - icg complements existing ao techniques for imaging the rpe and has the added advantage that it uses near - infrared wavelength light for imaging , which is a safer alternative to visible sources of light due to the avoidance of photochemical damage associated with wavelengths of light shorter than 600 nm . in addition , it does not rely on any intrinsic fluorophores such as lipofuscin , which varies with age and from person to person and which may be significantly altered in diseases such as age - related macular degeneration . instead , the use of an extrinsic fluorophore could provide a more objective way to probe the interface between the systemic circulation and rpe cells independent of unknown intrinsic signals . because instrumentation for imaging icg shares similarities with near - infrared autofluorescence ( iraf ) instrumentation , the methods developed in this paper may contribute to future ao - enhanced iraf imaging . future studies will also explore the potential use of ao - icg for imaging the retinal and choroidal vasculature . although oct angiography ( including ao - based oct angiography ) is becoming an attractive alternative to conventional fluorescein and icg angiography for vascular imaging , this does not preclude the use of ao - based angiography techniques such as aoslo - based fluorescein angiography when additional vascular detail is desired . it is also possible to use aoslo - based angiography to obtain detailed images of the capillary network without the use of injected dyes , to directly measure and track individual blood cells , to measure pulsatility and to assess vascular walls . application of ao - icg to vascular imaging would further broaden the range of complementary approaches for assessing the vasculature for improving our understanding of disease . in summary , ao - icg provides a novel view of the rpe cell mosaic in the living human eye . the use of multimodal ao imaging will enable new investigations of relationships between photoreceptors and rpe cells in health and disease and will lead to valuable information about disease progression and the efficacy of current and future therapies for eye disease .

purposethe purpose of this study was to establish that retinal pigment epithelial ( rpe ) cells take up indocyanine green ( icg ) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy ( ao - icg ) enables direct visualization of the rpe mosaic in the living human eye.methodsa customized adaptive optics scanning light ophthalmoscope ( aoslo ) was used to acquire high - resolution retinal fluorescence images of residual icg dye in human subjects after intravenous injection at the standard clinical dose . simultaneously , multimodal aoslo images were also acquired , which included confocal reflectance , nonconfocal split detection , and darkfield . imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases . in addition , histologic studies in mice were carried out.resultsthe ao - icg channel successfully resolved individual rpe cells in human subjects at various time points , including 20 minutes and 2 hours after dye administration . adaptive optics - icg images of rpe revealed detail which could be correlated with ao dark - field images of the same cells . interestingly , there was a marked heterogeneity in the fluorescence of individual rpe cells . confirmatory histologic studies in mice corroborated the specific uptake of icg by the rpe layer at a late time point after systemic icg injection.conclusionsadaptive optics - enhanced imaging of icg dye provides a novel way to visualize and assess the rpe mosaic in the living human eye alongside images of the overlying photoreceptors and other cells .

Methods Human Subjects AO Instrumentation AO Imaging AO Image Processing Quantitative Analysis Experimental Animals Histology Microscopy Results Discussion

three healthy volunteers with no history of systemic or ocular disease were recruited to undergo ao - icg imaging in both eyes . a replica of a broadband adaptive optics scanning light ophthalmoscope ( aoslo ) was assembled as described previously . wavefront sensing was performed using an 850 nm light source , and imaging was performed using a broadband ( 17 nm full width at half maximum ) 790 nm light source with a beam diameter of 7.75 mm at the point of entry into the eye . in this study , the light power levels measured at the cornea were maintained below 100 w for the 790 nm source and 35 w for the 850 nm source , which are under the maximum permissible exposure set by american national standards institute standard z136.1 2014 . indocyanine green was administered intravenously as a single bolus at a dose of 25 mg in 3 ml , according to the standard of care at the national eye institute eye clinic . during each recording , four image sequences were acquired simultaneously : ao confocal reflectance , icg fluorescence ( ao - icg ) , and two additional channels containing multiple scattered light from opposing directions of the confocal detector , which were used to generate ao split detection and ao dark - field images . during imaging , the focal plane was set to that of the photoreceptors based on a previous report stating that the optimal focal plane for ao dark - field rpe imaging was the plane of optimal photoreceptor imaging . biometry measurements were also acquired from all subjects ( iol master ; carl zeiss meditec , dublin , ca , usa ) and used to determine scaling factors to convert pixels to micrometers . because four videos were acquired simultaneously , the video with the ( subjectively determined ) sharpest features was selected for the computation of eye motion , which in all cases was the ao confocal reflectance channel . for display purposes , ao - icg and ao dark - field image contrast ranges a subset of human ao - icg data was selected for further analysis . for each subject , a 0.9 square region of interest that showed the clearest rpe cell outlines on the ao dark - field images was selected for further analysis . md , usa ; available in the public domain , http://imagej.nih.gov/ij/ ) , we manually identified the centers of rpe cells based on the locations of cell outlines and following the assumption that rpe cells form a uniform hexagonal array in healthy volunteers . these cell centers were then used to construct voronoi neighborhoods to facilitate qualitative comparisons between ao dark - field and ao - icg images in order to determine whether there was a correspondence between the cell outlines seen using ao dark - field and the pattern seen using ao - icg . in addition , cell density was computed by dividing the number of identified cell centers by the area of the region of interest and compared to previously published values of rpe cell density based on histology . relative intensity measurements were also carried out to verify the uptake of icg dye . in human subjects , fluorescence intensity values were measured from raw data as the average across a single video acquired at the fovea both prior to the injection of icg and 2 hours afterward ( acquisition parameters were kept constant for this analysis : 1.5 field of view , 200 frames acquired at 17 hz , icg photomultiplier tube gain setting of 0.500 , focus set to the plane of best photoreceptor focus ) . the preinjection video was used as the baseline intensity , and the relative intensity was calculated by dividing the mean of the postinjection site video by the mean of the preinjection video in order to derive an estimate of the signal strength . for mouse data ( described below ) , the average intensity value of the image acquired in the icg channel was measured by drawing a line through the rpe layer and taking the average of the intensity values along the line , using imagej software . intraperitoneal injection of icg was performed in one mouse from each pair ( 200 l of 5 mg / ml solution ) . experimental animals were euthanized by carbon dioxide inhalation at a 16 hours after administration of icg dye for the icg - injected mice . microscopy was performed using a modified axio imager z1 microscope ( carl zeiss ) outfitted with an x - cite 200dc light source ( lumen dynamics , mississauga , on , canada ) and an icg filter kit ( product icg - b - zhe - zero ; semrock ) , which enabled near - infrared imaging of icg . three healthy volunteers with no history of systemic or ocular disease were recruited to undergo ao - icg imaging in both eyes . a replica of a broadband adaptive optics scanning light ophthalmoscope ( aoslo ) was assembled as described previously . wavefront sensing was performed using an 850 nm light source , and imaging was performed using a broadband ( 17 nm full width at half maximum ) 790 nm light source with a beam diameter of 7.75 mm at the point of entry into the eye . indocyanine green was administered intravenously as a single bolus at a dose of 25 mg in 3 ml , according to the standard of care at the national eye institute eye clinic . during each recording , four image sequences were acquired simultaneously : ao confocal reflectance , icg fluorescence ( ao - icg ) , and two additional channels containing multiple scattered light from opposing directions of the confocal detector , which were used to generate ao split detection and ao dark - field images . during imaging , the focal plane was set to that of the photoreceptors based on a previous report stating that the optimal focal plane for ao dark - field rpe imaging was the plane of optimal photoreceptor imaging . because four videos were acquired simultaneously , the video with the ( subjectively determined ) sharpest features was selected for the computation of eye motion , which in all cases was the ao confocal reflectance channel . for display purposes , ao - icg and ao dark - field image contrast ranges were stretched between the minimum and maximum values . for each subject , a 0.9 square region of interest that showed the clearest rpe cell outlines on the ao dark - field images was selected for further analysis . md , usa ; available in the public domain , http://imagej.nih.gov/ij/ ) , we manually identified the centers of rpe cells based on the locations of cell outlines and following the assumption that rpe cells form a uniform hexagonal array in healthy volunteers . these cell centers were then used to construct voronoi neighborhoods to facilitate qualitative comparisons between ao dark - field and ao - icg images in order to determine whether there was a correspondence between the cell outlines seen using ao dark - field and the pattern seen using ao - icg . in addition , cell density was computed by dividing the number of identified cell centers by the area of the region of interest and compared to previously published values of rpe cell density based on histology . relative intensity measurements were also carried out to verify the uptake of icg dye . in human subjects , fluorescence intensity values were measured from raw data as the average across a single video acquired at the fovea both prior to the injection of icg and 2 hours afterward ( acquisition parameters were kept constant for this analysis : 1.5 field of view , 200 frames acquired at 17 hz , icg photomultiplier tube gain setting of 0.500 , focus set to the plane of best photoreceptor focus ) . the preinjection video was used as the baseline intensity , and the relative intensity was calculated by dividing the mean of the postinjection site video by the mean of the preinjection video in order to derive an estimate of the signal strength . for mouse data ( described below ) , the average intensity value of the image acquired in the icg channel was measured by drawing a line through the rpe layer and taking the average of the intensity values along the line , using imagej software . experimental animals were euthanized by carbon dioxide inhalation at a 16 hours after administration of icg dye for the icg - injected mice . microscopy was performed using a modified axio imager z1 microscope ( carl zeiss ) outfitted with an x - cite 200dc light source ( lumen dynamics , mississauga , on , canada ) and an icg filter kit ( product icg - b - zhe - zero ; semrock ) , which enabled near - infrared imaging of icg . following systemic injection of icg into three human subjects ( hv1 : 33-year - old male ; hv2 : 40-year - old female ; hv3 : 25-year - old female ) , a heterogeneous ao - icg signal was observed by using the modified aoslo that was qualitatively distinct from both ao confocal reflectance and ao dark - field images of the same retinal region ( fig . whereas only cell outlines were visible under ao dark - field illumination , entire cells appeared to be visible using ao - icg . the structures visible on ao - icg imaging were hexagonal in appearance and several - fold larger in size than cone photoreceptors , consistent with the expected size of rpe cells . three different modes of adaptive optics retinal images captured simultaneously in the foveas of three human subjects ( hv1 , hv2 , and hv3 ) . ( left to right ) adaptive optics confocal reflectance adaptive optics image of cone photoreceptors , outlines of rpe cells revealed using darkfield ao , and near - infrared fluorescence imaging of rpe cells using ao - icg . adaptive optics dark - field imaging was used to confirm that these cells were indeed rpe cells . in regions where individual rpe cells could be visualized using both ao dark - field imaging and ao - icg imaging , there was good correspondence between the two techniques ( fig . a total of 2,209 rpe cells were manually identified in 3 regions of interest ( roi ) near the fovea of three eyes ( approximate eccentricity of the center of each roi was 1.1 in hvi ; 1.0 in hv2 ; and 1.4 in hv3 ; each roi was 1.5 ) . the density of the rpe cells based on manual cell labeling ( fig . voronoi neighborhoods generated from the manually identified cells overlapped with the individual patterns seen in ao - icg , suggesting that icg accumulates in the cytoplasm of rpe cells . ( left to right ) adaptive optics dark - field images showing cell outlines , ao - icg fluorescence images showing entire cells , ao dark - field and ao - icg fluorescence images with voronoi diagrams overlaid . hence , histology of mouse eyes was used to confirm the layer - specific localization of the dye following systemic injection . in all cases , there was an increase in icg fluorescence signal that was specific to the rpe layer following systemic icg injection ( fig . measurements of fluorescence within the rpe layer were higher in the icg - injected animals than in their noninjected siblings , with an average intensity increase of 4.7-fold . in humans , from in vivo adaptive optics imaging data , there was an average 2.5-fold increase in intensity , comparing intensity values 2 hours after injection to the same area prior to injection ( p < 0.05 ) . indocyanine green imaging in a mouse eye showing that icg dye accumulates in the rpe after systemic injection . ( d ) after icg injection , the near - infrared fluorescence of the rpe layer is increased relative to both the noninjected mouse rpe layer and the near - infrared fluorescence . ( a ) registered average of 300 images prior to the injection of icg , showing no discernable signal and ( b d ) images of the same region as a after injection of icg . the use of an 80/20 beam splitter ( hv2 and hv3 ) resulted in a similar or better ao - icg image than that obtained using the 90/10 beam splitter ( hv1 ) , with no apparent differences in the other ao imaging modalities . ao - icg images were nearly identical when acquired using different field sizes and even across different time points ( fig . given the longevity of this signal , and to ensure maximal clearance of icg from the blood stream , we opted to acquire extensive ao - icg images at the time point of 2 hours post injection ( figs . the same region was imaged at two different times after intravenous injection of icg dye ( 20 and 120 minutes , respectively ) , using different fields of view ( 0.75 , 1.0 , and 1.5 ) . ( left to right ) confocal reflectance , split detection , icg , false color image ( split detection , red ; icg , green ) showing the imprinting effect of photoreceptors on the ao - icg image . although individual rpe cells are visible at 5 temporal using both ao - icg and ao darkfield , at larger eccentricities , the cell boundaries become more difficult to distinguish due to signal imprinting from the overlying photoreceptors . adaptive optics - icg image of rpe cells generated from 43 overlapping ao - icg videos acquired in the late phase of icg , at the fovea of subject hv2 , right eye . , we were still able to simultaneously acquire high quality ao confocal reflectance and ao split - detection images alongside ao - icg images ( fig . the ao - icg rpe signal appears to be visible over a range of retinal eccentricities within 5 of the foveal center without any noticeable drop - off in signal ( figs . adaptive optics - icg provides a novel method for imaging rpe cells within the living human eye . high - resolution single - photon fluorescence imaging revealed the presence of a stable , heterogeneous signal that persisted between 20 and 120 minutes after intravenous injection of icg at a standard clinical dosage . the mechanism for this differential fluorescence remains to be explored , as it is currently unknown whether this is due to differential uptake of icg dye or due to quenching from intrinsic pigments such as melanin , which has a broad absorption spectra that may be different in vivo than in vitro . interestingly , the heterogeneity of the prolonged icg signal that we observed in humans has also been reported in rats and considerable cell - to - cell heterogeneity within the rpe mosaic has been shown . images of the ao - icg signal presented in this paper will form the basis for future investigations to explore the interactions between icg and rpe at the cellular level . we confirmed the specific localization of icg to the rpe cells using histologic data from mice tissue samples ( fig . the selective uptake of icg into rpe cells appears to be specific , with no substantial uptake in neighboring retinal cells . this phenomenon was robust and repeatable over a range of different mouse strains , consistent with previous reports demonstrating this phenomenon in rats and nonhuman primates and could be demonstrated in human subjects as shown in this paper , suggesting that it is generalizable to a potentially larger range of animal or disease models . in contrast to previous studies that administered icg intravenously , we administered the dye intraperitoneally in mice in order to prolong the absorption time of the dye into the rpe cells and increase the strength of the icg signal under microscopy . in support of our claim that the near infrared fluorescence in the rpe was due to injected icg , and not due to background infrared autofluorescence , we also evaluated a pair of albino mice . whereas the icg fluorescence intensity in the rpe of the injected albino mouse was similar to that of the other pigmented strains , the near - infrared autofluorescence in the corresponding control ( noninjected sibling ) was largely undetectable . this confirms that there is uptake of icg in the rpe following systemic injection , independent of the background infrared autofluorescence . our implementation of ao - icg uses only one imaging light source to record four different modalities of ao images ( confocal reflectance , split detection , dark field , and icg ) . the instrumentation that we used for ao dark - field imaging is an exact replica of that described in a previous paper demonstrating ao dark - field imaging of rpe cells , and we were able to acquire high - quality ao dark - field images in the foveas of all three subjects . however , in our hands , ao dark - field imaging successfully revealed the rpe cells in only a small minority of subjects and is generally not successful in revealing rpe cells outside of the foveal region . nevertheless , direct comparison between ao dark field and ao - icg in three selected regions of interest in which ao dark field was successful established the fact that the cells visualized using ao - icg are contained within the cell borders as seen using ao dark field . based on a previous study , the cellular structures visualized using ao dark field also colocalize to rpe cells , revealed by ao visible - light autofluorescence imaging , consistent with our observation that ao - icg can be used to visualize rpe cells in the living human eye first , at larger eccentricities , there is signal imprinting from overlying photoreceptors visible in the ao - icg images ( fig . this imprinting could represent a method with which to visualize the interaction between photoreceptors and rpe cells , as it likely arises from the excitation light being wave guided by the overlying photoreceptors . notably , whereas rpe cells could not be distinguished using ao dark - field imaging at any of the larger eccentricities ( 5 , 10 , and 15 ) , combined color images of ao - icg / ao split - detection suggest that with further optimization , ao - icg may be a more robust method for imaging rpe cells outside of the fovea ( fig . despite the relatively weak signal , signal integration through eye motion compensation provides a reliable method for generation of images , and an ao - icg signal can be obtained using as few as 10 frames , in the best case scenario . that said , the risk for adverse reactions is very low , and administration of icg dye is a standard clinical procedure and one of only a few examples of an extrinsic dye that is approved by the us food and drug administration for use in the human body . taking into consideration both the clinical and the preclinical data , we demonstrate that in the late phase of icg imaging , rpe cells contribute to the fluorescence signal that is imaged . although icg plays a relatively limited role in the clinic in comparison to other techniques , our finding may lead to a new clinical method to indirectly assess the health or status of the rpe layer as a whole using conventional methods for imaging icg in the clinic , even without the use of ao technology . adaptive optics - icg complements existing ao techniques for imaging the rpe and has the added advantage that it uses near - infrared wavelength light for imaging , which is a safer alternative to visible sources of light due to the avoidance of photochemical damage associated with wavelengths of light shorter than 600 nm . in addition , it does not rely on any intrinsic fluorophores such as lipofuscin , which varies with age and from person to person and which may be significantly altered in diseases such as age - related macular degeneration . in summary , ao - icg provides a novel view of the rpe cell mosaic in the living human eye .

family members and other close loved ones participate in patients ' daily routines and are often present for self - care activities ( e.g. , food selection , meal preparation , and disease - related problem solving and coping ) [ 15 ] . for adults with type 2 diabetes ( t2d ) , the receipt of helpful actions ( i.e. , instrumental support ) is more predictive of adherence to self - care than other types of support ( e.g. , emotional support ) [ 2 , 6 , 7 ] . according to both social control theory [ 8 , 9 ] and family systems theory [ 10 , 11 ] , family members are well - positioned to provide instrumental support for diabetes self - care and to create contexts valuing and supporting self - care adherence . however , patients who experience more harmful actions ( e.g. , nagging , arguing , and sabotaging self - care efforts ) are less adherent to self - care [ 2 , 12 , 13 ] and have worse glycemic control . moreover , helpful and harmful actions are each strongly associated with patients ' being more or less adherent to diet and exercise recommendations , respectively . according to social control theory , certain actions are harmful because they are misaligned with the types of support patients need [ 8 , 14 ] and/or infringe on their autonomy and create resentment ( e.g. , nagging , arguing about self - care ) . according to family systems theory , harmful actions undermine patients ' efforts to initiate and sustain self - care efforts ( i.e. , undermining or sabotaging self - care efforts , such as bringing tempting unhealthy foods into patients ' homes ) [ 10 , 11 ] . families who become involved in adults ' self - care perform both helpful and harmful actions [ 2 , 12 , 1618 ] , but family interventions for adults with t2d have not adequately addressed the harmful aspects of family involvement . family interventions targeting both helpful and harmful actions may be particularly useful for racial / ethnic minorities and persons with low socioeconomic status ( ses ) with t2d . these groups have high rates of limited health literacy , more life stressors [ 2123 ] , and depression [ 24 , 25 ] , which may make them more vulnerable to the detrimental effects of others ' harmful actions on their diabetes self - care and control [ 12 , 26 ] . family interventions may also be challenging for patients with t2d who ( a ) have diverse living and family situations [ 1 , 27 , 28 ] , ( b ) live apart from the person(s ) providing the most support [ 27 , 28 ] , ( c ) have difficulty attending and/or bringing family members to in - person interventions due to competing priorities and limited resources [ 4 , 27 , 29 ] , and ( d ) are concerned family involvement will undermine their autonomy and self - efficacy . on the contrary , family members want to be more involved in adults ' diabetes management and often feel frustrated when they do not know how to best help . thus , patients may appreciate and benefit from one - on - one guidance on how to identify and communicate their desires and needs for specific supportive actions from family members . in other chronic disease contexts ( e.g. , cardiovascular diseases , cancer , and arthritis ) , family interventions have significant and stable effects on health over and above patient - only interventions but have been less successful in t2d [ 19 , 32 ] . therefore , we developed fams ( family - focused add - on for motivating self - care ) to help patients identify a diet or exercise goal , get desired support from family members or close loved ones , and redirect or cope with undesired or harmful actions without compromising their own health goals . fams seeks to improve patients ' adherence to diet and exercise recommendations via increased self - efficacy , increased receipt of helpful actions , and reduced receipt of harmful actions . fams is delivered via basic mobile phone technology ( i.e. , phone calls and text messages ) , which is ubiquitously available in the usa , even among adults with the lowest ses and racial / ethnic minority groups [ 34 , 35 ] . a robust , multistep approach is recommended to develop effective mhealth interventions for patients with diabetes , particularly for underserved or vulnerable patients . therefore , fams was developed from ( a ) front - end qualitative and quantitative formative research with adults with t2d and low ses [ 12 , 27 ] , alongside ( b ) early feedback from potential users ( adults with t2d and low ses ) and ( c ) members of our research team ( who used and critiqued fams during internal beta testing ) , followed by ( d ) iterative usability and feasibility testing with potential users . our objectives were to develop a family - focused intervention acceptable to patients receiving care from federally qualified health centers ( fqhcs ) , obtain feedback and data to improve the intervention , and ensure our research processes were sound prior to an evaluative trial . for mhealth interventions in diabetes [ 36 , 37 ] , user - centered design entails formative research prior to and during intervention development to understand the needs , values , and abilities of users , as well as iteratively assessing the design to improve users ' perceptions of and interactions with the technology and content . following these principles , we developed fams to harness universal text messaging ( i.e. , not requiring a smartphone or the internet ) . to accommodate the diversity of family types among adults with t2d [ 1 , 27 , 28 , 38 ] , we designed fams to acknowledge that patients ' families include whomever the patient considers included , regardless of legal / biological relationships . fams content is sensitive and applicable to various living arrangements ( e.g. , living alone , with children , or with friends / roommates ) . fams text messages were designed to be 6th - grade reading level and tested with the flesh - kincaid grade level and automated readability index ( tested with https://readability-score.com/ ) . to ensure plain language and accommodate those with literacy limitations , we ensured each sentence expressed one thought , simplified sentence structure , and used active voice . we then replaced or plainly defined multisyllabic ( three syllables ) and uncommon words and medical jargon . we worked with a digital content developer to shorten messages to 160 characters ( a common limit for text messages ) while maintaining their meaning . phone coaching seeks to improve the patient 's ability to identify family members ' actions that support or impede his / her self - care goals and his / her skills and self - efficacy to ask for needed support and manage harmful actions to meet these goals . authors lsm and kjh , who have graduate degrees in counseling , developed fams phone coaching to be deliverable by counselors / counselors - in - training or health coaches / health coaches - in - training ( i.e. , persons with training in helping skills ) . fams coaching occurs with the pp alone and combines family therapy with basic health coaching . among adults with t2d , health coaching has successfully improved adherence to exercise recommendations , psychological functioning , and illness - related coping . fams coaching employs evidence - based techniques from goal setting theory ( assessing current behaviors and problem solving and assessing confidence to achieve a goal and revising goals with a confidence rating < 7 on a 10-point scale ) , cognitive behavioral therapy [ 43 , 44 ] ( role playing , homework ) , and best practices in health communication ( teach back ) . fams sends the pp one - way and two - way / interactive text message support tailored to his / her self - care goal selected during coaching and preferred time of day ( table 1 ) . patients can choose to invite a support person ( sp ) regardless of relationship type or living arrangement to receive text messages ( table 1 ) . pps were encouraged to select someone who is part of their daily life and routine and not someone with whom they have a lot of conflict . the sp does not have to be the identified family member in the coaching session . an enrolled sp receives messages tailored to the pp 's name , gender , and goal type ( table 1 ) . these aim to help sps be thoughtful about the support they provide and to increase communication about diabetes and the pp 's goal . the sp text messages do not provide information on the pp 's goal achievement to avoid encouraging nagging / arguing . before developing fams functionality , we shared fams design and content with adults with t2d through a community engaged research studio ( ces ) and made improvements based on their recommendations . the ces , conducted through the meharry - vanderbilt community engaged research core , employs direct feedback from community members ( experts ) who share similar demographic characteristics of a researcher 's desired sample to identify and address concerns ( e.g. , cultural appropriateness of study materials , fair compensation , and intervention / survey design ) . our ces experts ( ~12 ) were diagnosed with t2d , predominantly african american ( aa ) , and with low ses - no family members were included . the ces facilitator read each text message and participants shared feedback ranging from good , like it to discussions with multiple view - points expressed . authors lsm and kjh asked follow - up or clarifying questions as needed during the ces ; afterwards , the facilitators compiled and provided notes . they did not like messages presuming patients were struggling to meet their goal or that evoked strong negative language . they recommended it be changed to the following : traveling can cause you not to meet your diet goals . ces experts also recommended defining self - care to avoid confusion , so we replaced this phrase with a specific self - care behavior ( e.g. , diet or exercise ) or taking care of your diabetes . community experts said they would feel comfortable inviting a sp and provided feedback on text frequency ( recommended daily ) and coaching frequency ( most recommended every two weeks or monthly ) and duration ( most recommended 2030 minutes ) . fams functionality was developed and tested for usability alongside reach ( rapid education / encouragement and communications for health ) , a text messaging intervention supporting diabetes self - care . reach ( a ) sends tailored messages addressing users ' barriers to diabetes medication adherence , ( b ) sends nontailored messages addressing adherence to diet , exercise , and self - monitoring of blood glucose , ( c ) sends daily adherence assessment messages with weekly feedback and support , ( d ) sends messages with hipaa - compliant access to a1c test results , and ( e ) provides access to a helpline tethered to a clinical pharmacist . in addition to the reach elements , fams users received phone coaching and had the option to invite a support person ( sp ) to receive messages . fams also replaced reach 's general diet and exercise messages with messages tailored to users ' coaching goal . text messages were tailored and sent by memotext , an algorithmic communications and data management platform supporting personalized user outputs and inputs via text messaging . we worked with memotext to design fams functionality and conduct three rounds of internal beta testing during which research team members served as pseudousers to identify technical bugs and improve the user experience before usability and feasibility testing . costs owed to memotext included the initial development , a monthly maintenance fee of $ 200 , and ~$0.07 per message sent ( per - message costs decrease when more messages are sent ) . we conducted three iterative rounds of testing , each with a new sample of users . each round included a single phone coaching session with the pp followed by two weeks of text message support for the pp and ( if enrolled ) the sp . pps were adults with t2d recruited from three fqhcs in nashville , tn , via flyers , interest cards , and referrals from clinic staff . eligible pps were receiving care at one of the clinic sites and taking diabetes medications ( not caregiver administered ) . both pps and sps had to be adults ( 18 years old ) , speak and read english , have a cell phone with text messaging , and provide a social security number ( to process compensation ) . pps and sps were excluded if they had an existing diagnosis of dementia , were unable to orally communicate , or had an auditory limitation ( for interviews ) . we aimed to enroll n = 6 pps per round to satisfy the recommendation of three small studies with five participants in each round to identify all usability problems . study procedures were approved by the vanderbilt university irb . trained research assistants ( ras ) conducted eligibility screening and administered informed consent and survey measures in a private room at the pp 's clinic . after enrollment , coaches ( authors lsm and kjh ) called the pp to complete phone coaching . coaches then called and invited the sp to participate , asking if they would like to receive text messages related to being a continued support for the pp and complete an interview telling the research team what they thought about fams and how to improve it . interested sps gave their consent via phone and provided a preferred time of day to receive text messages . data from pps ' enrollment survey , coaching , and enrolled sps were entered into redcap and transferred automatically to memotext via an application - programming interface for the tailoring and delivery of text messages . authors lsm and kjh met between rounds to discuss successes and shortcomings of the coaching protocols and sp enrollment process . following each round of testing , we compiled pp- and sp - user feedback to improve fams and research processes . we received irb approval before recruiting participants for the next round of testing . during the enrollment survey , pps self - reported demographic and diabetes information and completed validated survey instruments to characterize the sample ( health literacy , family involvement , and adherence to self - care behaviors [ 51 , 52 ] ) . we also asked pps if they were comfortable using their cell phone and text messaging . glycemic control ( a1c ) was assessed via lab test with a blood sample drawn at enrollment . immediately after each coaching session , coaches recorded the pp 's goal and type of family action helpful or harmful identified and addressed with psychoeducation and assessed the success of each phase of the coaching protocol using a coaching assessment we developed . technical data collected by memotext captured user engagement : we assessed pps engagement as the number of text responses received out of the number of two - way texts sent . the fams user experience was assessed via semistructured interviews with the pp and , separately , the sp . interviews were conducted by phone and included open - ended questions and closed- and open - ended question pairs ( e.g. , yes / no followed by why / why not ? and 10-point scales followed by can you tell me why you chose that number ? ) . we asked participants to rate different aspects of the intervention ( e.g. , how easy the text messages were to understand , how helpful they were , and how much each fams component motivated them to reach their goal ) on a scale from 1 ( least favorable ) to 10 ( most favorable ) . interviews with pps and sps were audiorecorded with transcribed verbatim by an independent professional transcriber . we exported survey and quantitative interview data from redcap and conducted all descriptive analyses using stata v. 13.1 . nine pps invited a sp and seven sps enrolled , including four boyfriends / girlfriends / fiancs , one spouse , one ex - spouse , and one son . there were no differences ( significant or meaningful nonsignificant ) between participants who chose to invite a sp and those who did not in any variable in table 2 . findings are described below with corresponding participant quotes in text and additional quotes in table 3 . changes made between rounds and prior to a longer trial are described below and in table 4 . most participants focused on a helpful family action during skill building ( examples in table 5 ) . all but one of the pps who completed skill building were very confident ( 7 on a 10-point scale ) they could apply the skill with an identified family member and , separately , doing so would result in a desired change ( table 5 ) . in interviews , pps said coaching helped them set a goal that was important to them ( 8.8 1.4 , range 610 , example goals in table 5 ) . several explained the coach helped them learn how to set a realistic and attainable goal : oh , i thought [ coaching ] was helpful because it pinpointed what i could do and then helped me keep it doable or obtainable . it helped me focus on what i want to do , made me vocalize what i want to do , and then the coach part helped me make it - choose something that was obtainable . also with the help , it was explained to me that it did n't need to be 15 solid minutes [ of exercise ] . so , it made me take a break at work , go walk in the mall for my 15 today . and tomorrow , take a break at work , or i could walk an entire floor , which would take about 5 minutes . you know , and then i 'd do that 3 times and i 'd have my 15 minutes . so , i could come home and watch a show and stand up and move around all the time it was on . hey , it was movement and it 's a start . ( pp , 50-year - old male , race unknown)two - thirds ( 67% ) said coaching improved communication about diabetes with family members : i talk to my husband , and i explain about the goal , and then he helped me more . like , now he probably is going to understand when i tell him , if we go out , what kind of food to order instead [ of our regular order ] , because i already talked to him about what i talked about [ in coaching ] . ( pp , 36-year - old hispanic female)participants said they would want coaching once ( 33% ) or twice ( 33% ) per month in a longer intervention , and 65% said each session should last 1530 minutes . oh , i thought [ coaching ] was helpful because it pinpointed what i could do and then helped me keep it doable or obtainable . it helped me focus on what i want to do , made me vocalize what i want to do , and then the coach part helped me make it - choose something that was obtainable . also with the help , it was explained to me that it did n't need to be 15 solid minutes [ of exercise ] . so , it made me take a break at work , go walk in the mall for my 15 today . and tomorrow , take a break at work , or i could walk an entire floor , which would take about 5 minutes . you know , and then i 'd do that 3 times and i 'd have my 15 minutes . so , i could come home and watch a show and stand up and move around all the time it was on . hey , it was movement and it 's a start . ( pp , 50-year - old male , race unknown ) i talk to my husband , and i explain about the goal , and then he helped me more . like , now he probably is going to understand when i tell him , if we go out , what kind of food to order instead [ of our regular order ] , because i already talked to him about what i talked about [ in coaching ] . ( pp , 36-year - old hispanic female ) however , more than one in three participants ( 37% ) did not buy into the coaching session ( table 5 ) , and these same participants rated the coaching as less helpful in interviews . the most common reason ( n = 4 ; 21% ) was that the participant did not accept a connection between his / her goal and the actions of close loved ones . coaches reported they had an insufficient amount of time to discuss family actions before asking pps to think of how they would want such actions to change . buy into the coaching session because the skill in the coaching protocol was not a good fit for the identified family actions or his / her family relationships ( table 5 ) . for example , one participant had unsuccessfully tried assertive communication with his wife about the type of food she cooked and felt doing so again would not be productive ( in his interview , this pp explained , because my family members , they ai n't going to do that for me . finally , coaches reported spending a disproportionate amount of time on goal setting and rushing through the family - focused portions of the coaching protocol . pps rated the helpfulness of the goal support messages 8.4 2.7 ( range 110 ) and 80% said these messages motivated them to meet their goal . pps liked that messages were short and easy to read and provided encouragement and support . one pp saved messages she wanted to memorize or apply to review in her free time . three described using receipt of message as a cue to action to meet their goal and three others reported sharing messages with loved ones : you know , when i got the texts , i took a few minutes and did some arm rows and leg lifts and stretched my leg out , bring it to my stomach as much as i could . ( pp , 46-year - old caucasian female)i mean , a lot of stuff i can explain to my wife that - she might understand it but seeing it for herself [ in the messages ] , she understand it different . ( pp , 37-year - old aa male)two pps and one sp wanted messages to be more interactive . one pp said texts that were not interactive make people feel like they 're being preached at . often , responses to one - way messages were received from pps ( e.g. , that 's right ! and i will , thank you ) and sps ( e.g. , we can work on it together ) . others appreciated not having to respond and said they were used to receiving text messages without responding . you know , when i got the texts , i took a few minutes and did some arm rows and leg lifts and stretched my leg out , bring it to my stomach as much as i could . ( pp , 46-year - old caucasian female ) i mean , a lot of stuff i can explain to my wife that - she might understand it but seeing it for herself [ in the messages ] , she understand it different . ( pp , 37-year - old aa male ) pps responded to 65% of the goal assessment messages and rated the helpfulness of these messages in keeping them on track with their goal 8.7 2.1 ( range 410 ) . pps appreciated getting feedback on their progress , and said the encouraging tone motivated them to keep trying to meet their goal : i would get a message : did you do it ? and then i said , oh , only 3 times . and it said , ooh , you 're halfway to your goal . what could - think about what would help you , you know , maybe get closer ? so , i did n't feel ashamed or i like ugh , i need to think of why did n't i do that ? you know ? ( pp , 50-year - old male , race unknown ) i would get a message : did you do it ? and then i said , oh , only 3 times . and it said , ooh , you 're halfway to your goal . what could - think about what would help you , you know , maybe get closer ? so , i did n't feel ashamed or i like ugh , i need to think of why did n't i do that ? you know ? ( pp , 50-year - old male , race unknown ) we made three changes to improve the goal assessment messages during testing ( table 4 ) . first , we created functional flexibility to allow pps to set a goal for 4 days . we altered feedback messages and functionality so a pp 's response of 03 implied the participant had not met their goal , 46 implied success , and 7 received especially congratulatory feedback . third , we initially developed feedback to reference progress relative to the prior week ( i.e. , improvement , decline , or consistency ) which resulted in nonresponse affecting feedback for the week in question and the subsequent week . therefore , we changed the functionality of the feedback text to reference progress only when two weeks ' responses were provided and to default to more basic feedback if not ( e.g. , of the 19 pps , nine ( 47% ) invited a sp to enroll in the study and seven sps were enrolled ( we could not contact one ; one declined ) . six more pps wanted to have a sp receive text messages but did not provide the sp 's contact information to study staff . reasons included the following : sp was ineligible ( having no cell phone or being non - english - speaking ) , pp could not identify a sp , and sp expressed disinterest to the pp . only four pps ( 21% ) did not want to invite a sp : well , i guess i do have family members . but i just felt like this was my deal and i just wanted to own it myself because that really drives me more . ( pp , 50-year - old male , race unknown)my family members are really busy . ( pp , 56-year - old caucasian female ) well , i guess i do have family members . but i just felt like this was my deal and i just wanted to own it myself because that really drives me more . ( pp , 50-year - old male , race unknown ) my family members are really busy . ( pp , 56-year - old caucasian female ) enrolled sps reported the messages motivated them to talk with the pp about diabetes ( 9.3 1.2 , range 710 ) and improved their support of the pp ( 9.3 1.2 , range 710 ) . when asked how many messages per week they would want to receive in a six - month intervention , sps said three times per week ( n = 3 ) , daily ( n = 2 ) , or every other day ( n = 1 ) . of the six sps who completed an interview , four said fams increased their knowledge about diabetes and all reported discussing message content with pps.i think it was a great experience for me because i had never known that much about diabetes and i was able to learn a great deal about it , you know , through those texts and then talking with [ the pp ] . i learned quite a bit and in ways that i can actually help someone else . you know , if they 're going through the same things i also tried to , you know , watch what i eat as well . and , in turn , you know , we talk to each other about always trying to eat healthy and try to make the right choices and , you know , just try not to overdo things that would hurt her health - wise . i mean , i kind of looked forward to getting to see what it was going to be the next time , you know ? well , it 's going to come through here in a minute . you know , because it gave me an idea of what to - kind of get it in my head , you know , what i need to ask him for that evening , you know , just to throw it out there . i usually try to call him on my way home , and we discuss things . and that just kind of gives me an idea of something to actually throw out there , you know , and get his input on it . and ( sp , ex - wife ) i think it was a great experience for me because i had never known that much about diabetes and i was able to learn a great deal about it , you know , through those texts and then talking with [ the pp ] . i learned quite a bit and in ways that i can actually help someone else . you know , if they 're going through the same things i also tried to , you know , watch what i eat as well . and , in turn , you know , we talk to each other about always trying to eat healthy and try to make the right choices and , you know , just try not to overdo things that would hurt her health - wise . i mean , i kind of looked forward to getting to see what it was going to be the next time , you know ? well , it 's going to come through here in a minute . you know , because it gave me an idea of what to - kind of get it in my head , you know , what i need to ask him for that evening , you know , just to throw it out there . i usually try to call him on my way home , and we discuss things . and that just kind of gives me an idea of something to actually throw out there , you know , and get his input on it . ( sp , ex - wife ) in round one , a sp - pp dyad reported the sp 's text messages led to interpersonal conflict . the sp received this text message : do you argue with george about his diabetes ? take a step back . the sp thought this message was sent to her because of something the pp told us : but there was one that kind of got me a little bit it was kind of like i was being aggressive with him or something and that 'd never been the case . i do n't understand this one because i do n't do that to him - pressure him about anything . but anyway , that was the only one that was kind of strange that one really needs to be worked on because that was disturbing ( laughs ) . i mean , it just made me feel like i 'd done something that maybe he had told you or something that i did.the pp was very upset about this : y'all sent her text messages asking her why she want to argue with me when she 's not arguing with me . she has never been negative about this ; she 's the one who wanted me to do this [ study ] . you all make her feel like i 'm telling you bad shit about her and i 'm not that thoroughly pissed me off , and i texted back some pretty hot shit to them . i 'm not sure if the computer understood it or not , but it made me feel better.we reported this unexpected adverse event to the irb and fixed it for subsequent rounds . with family intervention experts , we reviewed and edited all sp messages to avoid insinuating sps were performing a harmful action or experiencing conflict with the pp ( table 5 ) . we also added this to the informed consent process for both the pp ( i.e. , conflict with friends / family members as a risk ) and the sp ( i.e. , explained all of the sp text messages were sent randomly to all sps in the study and not based on any information from pps ) . but there was one that kind of got me a little bit it was kind of like i was being aggressive with him or something and that 'd never been the case . i do n't understand this one because i do n't do that to him - pressure him about anything . but anyway , that was the only one that was kind of strange that one really needs to be worked on because that was disturbing ( laughs ) . i mean , it just made me feel like i 'd done something that maybe he had told you or something that i did . y' all sent her text messages asking her why she want to argue with me when she 's not arguing with me . she has never been negative about this ; she 's the one who wanted me to do this [ study ] . you all make her feel like i 'm telling you bad shit about her and i 'm not that thoroughly pissed me off , and i texted back some pretty hot shit to them . i 'm not sure if the computer understood it or not , but it made me feel better . in addition to described changes made between rounds of testing , we made several decisions and changes to fams for an upcoming evaluative trial based on these results ( table 4 ) . the first session will allocate half of the session to assessing current diet or exercise activities and collaboratively setting a smart goal , and the other half will be allocated to discussing the role of family actions / support in meeting and sustaining health goals . this session ends with a brief family behavior observation skill building exercise , requesting that the pp observe the role of close loved ones as he / she tries to meet the goal for the first month . we hope this change will allow sufficient time for goal setting and increase the pp 's acceptance of family members ' role prior to asking pps to identify desired family changes in subsequent sessions . we also created flexibility in the coaching protocol so coaches can tailor the skill to the pp in vivo . each session involves the same elements ( table 1 ) , but coaches choose the skill most applicable to pps during sessions 25 ( table 4 ) . we added the skill building exercise cognitive behavioral coping for pps who report not having supportive family members or experiencing persistent harmful family actions . we also made changes to our process for enrolling and inviting sps to receive text messages ( table 4 ) . with these changes , we hope pps will understand the sp does not need to be a biological / legal family member and will have opportunity to ask the sp 's permission to give study staff his / her contact information . we designed fams to improve t2d adults ' ( a ) adherence to diet and exercise recommendations and ( b ) self - efficacy and skills to engage loved ones in self - care goals in ways that facilitate behavior change . through an iterative user - centered design process followed by rigorous and multimethod usability / feasibility testing , we designed an intervention that appeals to end users , is easy to use , and is applicable to a variety of patient and family situations . pps said fams improved their diet / physical activity , communication with family members , and confidence in soliciting helpful actions / support . sps reported fams increased the amount they communicated with pps about diabetes and made it possible for them to be more helpful . comments from users indicate families find it difficult to know how to communicate about diabetes or support diabetes self - care efforts and appreciated concrete suggestions provided during coaching or in text messages . over half of the pps identified a harmful family action , but fewer ( 13% ) focused on the harmful action in skill building . patients who discussed harmful actions often preferred either asking for a helpful action to replace or offset the harmful action or asking for a helpful action from another family member instead of dealing with the family member performing the harmful action . these are productive ways to manage harmful actions , as helpful actions have been found to protect against the detrimental effects of harmful actions on patients ' a1c . for instance , only 37% of pps successfully invited a sp and a few pps did not want to discuss family issues at all . we hope to have developed appropriate and effective solutions to these problems , but the success of these changes remains unknown . we do know , however , that our iterative process dramatically reduced technical bugs and problems with content , functionality , and study processes , thereby maximizing our ability to evaluate fams . fams targets patients ' adherence to diet and exercise , but other self - care behaviors ( e.g. , medication adherence , self - monitoring of blood glucose ) are impacted by family actions [ 2 , 12 ] and affect diabetes control [ 2 , 12 ] . future work should assess patients ' expectations of others ' involvement in diabetes self - care . for instance , in other disease contexts , helpful and harmful family involvement have been shown to affect dietary adherence , self - efficacy , and depressive symptoms differently based on perceptions of the role of family in adults ' self - management . thus , future research should examine whether individuals with t2d who view their illness as an individual concern benefit from family involvement or interventions like fams . additionally , we found sps may react negatively to the idea that their actions might be less than helpful or they might nag or argue with the patient . future intervention efforts may benefit from normalizing nagging and arguing and inadvertently making self - care more challenging . this may make it less emotionally fraught for families to identify and replace these actions with more helpful ones . although mhealth interventions have improved adherence and glycemic control among adults with t2d [ 55 , 56 ] , engaging family members in these interventions remains understudied . to our knowledge , the only other mhealth intervention involving a patient - selected family member / support person for adults with t2d is mhealth + carepartner [ 57 , 58 ] . both mhealth + carepartner and fams were designed to be inclusive of patients who live alone , recruit from community clinics , and provide tailored mobile communications to an adult family member / friend . however , there are key differences between piette et al . 's approach to engaging a support person and our own . carepartners receive weekly information about the patients ' health status along with tailored advice about how to help . in contrast , fams does not provide information about patients ' progress to sps but rather aims to empower the patient to identify and communicate support needs . lessons learned from fams and mhealth + carepartner will improve future efforts to involve meaningful others in adults ' t2d self - management . we enrolled a small convenience sample with no control group and users experienced the intervention for a brief time , limiting our understanding to participants ' anecdotal accounts and short - term engagement data . down time was required for troubleshooting technical bugs , making fixes / improvements , and adding new functionality . as a result , we had to wait between each development phase and paused recruitment between rounds of usability / feasibility testing . these necessary and inevitable occurrences rarely accounted for in study planning or funding schedules nearly doubled the length of fams development and testing . down time during an evaluative trial would have been much more problematic and costly . because identifying and fixing bugs are inevitable with mhealth interventions , we stress the importance of starting rigorous multiround testing early in the design / development process rather than waiting until the intervention is developed and functioning properly to allow users to experience it . more time and energy in these phases likely pay off with faster evaluative trials with more engagement , less attrition , and fewer study limitations associated with functional problems .

family members ' helpful and harmful actions affect adherence to self - care and glycemic control among adults with type 2 diabetes ( t2d ) and low socioeconomic status . few family interventions for adults with t2d address harmful actions or use text messages to reach family members . through user - centered design and iterative usability / feasibility testing , we developed a mhealth intervention for disadvantaged adults with t2d called fams . fams delivers phone coaching to set self - care goals and improve patient participant 's ( pp ) ability to identify and address family actions that support / impede self - care . pps receive text message support and can choose to invite a support person ( sp ) to receive text messages . we recruited 19 adults with t2d from three federally qualified health centers to use fams for two weeks and complete a feedback interview . coach - reported data captured coaching success , technical data captured user engagement , and pp / sp interviews captured the fams experience . pps were predominantly african american , 83% had incomes < $ 35,000 , and 26% were married . most sps ( n = 7 ) were spouses / partners or adult children . pps reported fams increased self - care and both pps and sps reported fams improved support for and communication about diabetes . fams is usable and feasible and appears to help patients manage self - care support , although some pps may not have a sp .

1. Introduction 2. User-Centered Intervention Design 3. Iterative Feasibility and Usability Testing 4. Lessons to Improve FAMS 5. Conclusions

family members and other close loved ones participate in patients ' daily routines and are often present for self - care activities ( e.g. , food selection , meal preparation , and disease - related problem solving and coping ) [ 15 ] . for adults with type 2 diabetes ( t2d ) , the receipt of helpful actions ( i.e. , instrumental support ) is more predictive of adherence to self - care than other types of support ( e.g. according to both social control theory [ 8 , 9 ] and family systems theory [ 10 , 11 ] , family members are well - positioned to provide instrumental support for diabetes self - care and to create contexts valuing and supporting self - care adherence . , nagging , arguing , and sabotaging self - care efforts ) are less adherent to self - care [ 2 , 12 , 13 ] and have worse glycemic control . moreover , helpful and harmful actions are each strongly associated with patients ' being more or less adherent to diet and exercise recommendations , respectively . , nagging , arguing about self - care ) . according to family systems theory , harmful actions undermine patients ' efforts to initiate and sustain self - care efforts ( i.e. , undermining or sabotaging self - care efforts , such as bringing tempting unhealthy foods into patients ' homes ) [ 10 , 11 ] . families who become involved in adults ' self - care perform both helpful and harmful actions [ 2 , 12 , 1618 ] , but family interventions for adults with t2d have not adequately addressed the harmful aspects of family involvement . family interventions targeting both helpful and harmful actions may be particularly useful for racial / ethnic minorities and persons with low socioeconomic status ( ses ) with t2d . these groups have high rates of limited health literacy , more life stressors [ 2123 ] , and depression [ 24 , 25 ] , which may make them more vulnerable to the detrimental effects of others ' harmful actions on their diabetes self - care and control [ 12 , 26 ] . family interventions may also be challenging for patients with t2d who ( a ) have diverse living and family situations [ 1 , 27 , 28 ] , ( b ) live apart from the person(s ) providing the most support [ 27 , 28 ] , ( c ) have difficulty attending and/or bringing family members to in - person interventions due to competing priorities and limited resources [ 4 , 27 , 29 ] , and ( d ) are concerned family involvement will undermine their autonomy and self - efficacy . thus , patients may appreciate and benefit from one - on - one guidance on how to identify and communicate their desires and needs for specific supportive actions from family members . , cardiovascular diseases , cancer , and arthritis ) , family interventions have significant and stable effects on health over and above patient - only interventions but have been less successful in t2d [ 19 , 32 ] . therefore , we developed fams ( family - focused add - on for motivating self - care ) to help patients identify a diet or exercise goal , get desired support from family members or close loved ones , and redirect or cope with undesired or harmful actions without compromising their own health goals . fams seeks to improve patients ' adherence to diet and exercise recommendations via increased self - efficacy , increased receipt of helpful actions , and reduced receipt of harmful actions . , phone calls and text messages ) , which is ubiquitously available in the usa , even among adults with the lowest ses and racial / ethnic minority groups [ 34 , 35 ] . therefore , fams was developed from ( a ) front - end qualitative and quantitative formative research with adults with t2d and low ses [ 12 , 27 ] , alongside ( b ) early feedback from potential users ( adults with t2d and low ses ) and ( c ) members of our research team ( who used and critiqued fams during internal beta testing ) , followed by ( d ) iterative usability and feasibility testing with potential users . our objectives were to develop a family - focused intervention acceptable to patients receiving care from federally qualified health centers ( fqhcs ) , obtain feedback and data to improve the intervention , and ensure our research processes were sound prior to an evaluative trial . for mhealth interventions in diabetes [ 36 , 37 ] , user - centered design entails formative research prior to and during intervention development to understand the needs , values , and abilities of users , as well as iteratively assessing the design to improve users ' perceptions of and interactions with the technology and content . following these principles , we developed fams to harness universal text messaging ( i.e. to accommodate the diversity of family types among adults with t2d [ 1 , 27 , 28 , 38 ] , we designed fams to acknowledge that patients ' families include whomever the patient considers included , regardless of legal / biological relationships . to ensure plain language and accommodate those with literacy limitations , we ensured each sentence expressed one thought , simplified sentence structure , and used active voice . we then replaced or plainly defined multisyllabic ( three syllables ) and uncommon words and medical jargon . we worked with a digital content developer to shorten messages to 160 characters ( a common limit for text messages ) while maintaining their meaning . phone coaching seeks to improve the patient 's ability to identify family members ' actions that support or impede his / her self - care goals and his / her skills and self - efficacy to ask for needed support and manage harmful actions to meet these goals . authors lsm and kjh , who have graduate degrees in counseling , developed fams phone coaching to be deliverable by counselors / counselors - in - training or health coaches / health coaches - in - training ( i.e. among adults with t2d , health coaching has successfully improved adherence to exercise recommendations , psychological functioning , and illness - related coping . fams sends the pp one - way and two - way / interactive text message support tailored to his / her self - care goal selected during coaching and preferred time of day ( table 1 ) . patients can choose to invite a support person ( sp ) regardless of relationship type or living arrangement to receive text messages ( table 1 ) . pps were encouraged to select someone who is part of their daily life and routine and not someone with whom they have a lot of conflict . these aim to help sps be thoughtful about the support they provide and to increase communication about diabetes and the pp 's goal . before developing fams functionality , we shared fams design and content with adults with t2d through a community engaged research studio ( ces ) and made improvements based on their recommendations . the ces , conducted through the meharry - vanderbilt community engaged research core , employs direct feedback from community members ( experts ) who share similar demographic characteristics of a researcher 's desired sample to identify and address concerns ( e.g. our ces experts ( ~12 ) were diagnosed with t2d , predominantly african american ( aa ) , and with low ses - no family members were included . the ces facilitator read each text message and participants shared feedback ranging from good , like it to discussions with multiple view - points expressed . ces experts also recommended defining self - care to avoid confusion , so we replaced this phrase with a specific self - care behavior ( e.g. community experts said they would feel comfortable inviting a sp and provided feedback on text frequency ( recommended daily ) and coaching frequency ( most recommended every two weeks or monthly ) and duration ( most recommended 2030 minutes ) . fams functionality was developed and tested for usability alongside reach ( rapid education / encouragement and communications for health ) , a text messaging intervention supporting diabetes self - care . reach ( a ) sends tailored messages addressing users ' barriers to diabetes medication adherence , ( b ) sends nontailored messages addressing adherence to diet , exercise , and self - monitoring of blood glucose , ( c ) sends daily adherence assessment messages with weekly feedback and support , ( d ) sends messages with hipaa - compliant access to a1c test results , and ( e ) provides access to a helpline tethered to a clinical pharmacist . in addition to the reach elements , fams users received phone coaching and had the option to invite a support person ( sp ) to receive messages . we worked with memotext to design fams functionality and conduct three rounds of internal beta testing during which research team members served as pseudousers to identify technical bugs and improve the user experience before usability and feasibility testing . each round included a single phone coaching session with the pp followed by two weeks of text message support for the pp and ( if enrolled ) the sp . pps were adults with t2d recruited from three fqhcs in nashville , tn , via flyers , interest cards , and referrals from clinic staff . eligible pps were receiving care at one of the clinic sites and taking diabetes medications ( not caregiver administered ) . both pps and sps had to be adults ( 18 years old ) , speak and read english , have a cell phone with text messaging , and provide a social security number ( to process compensation ) . pps and sps were excluded if they had an existing diagnosis of dementia , were unable to orally communicate , or had an auditory limitation ( for interviews ) . we aimed to enroll n = 6 pps per round to satisfy the recommendation of three small studies with five participants in each round to identify all usability problems . coaches then called and invited the sp to participate , asking if they would like to receive text messages related to being a continued support for the pp and complete an interview telling the research team what they thought about fams and how to improve it . interested sps gave their consent via phone and provided a preferred time of day to receive text messages . data from pps ' enrollment survey , coaching , and enrolled sps were entered into redcap and transferred automatically to memotext via an application - programming interface for the tailoring and delivery of text messages . following each round of testing , we compiled pp- and sp - user feedback to improve fams and research processes . during the enrollment survey , pps self - reported demographic and diabetes information and completed validated survey instruments to characterize the sample ( health literacy , family involvement , and adherence to self - care behaviors [ 51 , 52 ] ) . glycemic control ( a1c ) was assessed via lab test with a blood sample drawn at enrollment . immediately after each coaching session , coaches recorded the pp 's goal and type of family action helpful or harmful identified and addressed with psychoeducation and assessed the success of each phase of the coaching protocol using a coaching assessment we developed . technical data collected by memotext captured user engagement : we assessed pps engagement as the number of text responses received out of the number of two - way texts sent . the fams user experience was assessed via semistructured interviews with the pp and , separately , the sp . , how easy the text messages were to understand , how helpful they were , and how much each fams component motivated them to reach their goal ) on a scale from 1 ( least favorable ) to 10 ( most favorable ) . interviews with pps and sps were audiorecorded with transcribed verbatim by an independent professional transcriber . nine pps invited a sp and seven sps enrolled , including four boyfriends / girlfriends / fiancs , one spouse , one ex - spouse , and one son . there were no differences ( significant or meaningful nonsignificant ) between participants who chose to invite a sp and those who did not in any variable in table 2 . ( pp , 50-year - old male , race unknown)two - thirds ( 67% ) said coaching improved communication about diabetes with family members : i talk to my husband , and i explain about the goal , and then he helped me more . ( pp , 36-year - old hispanic female)participants said they would want coaching once ( 33% ) or twice ( 33% ) per month in a longer intervention , and 65% said each session should last 1530 minutes . ( pp , 50-year - old male , race unknown ) i talk to my husband , and i explain about the goal , and then he helped me more . ( pp , 36-year - old hispanic female ) however , more than one in three participants ( 37% ) did not buy into the coaching session ( table 5 ) , and these same participants rated the coaching as less helpful in interviews . the most common reason ( n = 4 ; 21% ) was that the participant did not accept a connection between his / her goal and the actions of close loved ones . coaches reported they had an insufficient amount of time to discuss family actions before asking pps to think of how they would want such actions to change . buy into the coaching session because the skill in the coaching protocol was not a good fit for the identified family actions or his / her family relationships ( table 5 ) . ( pp , 37-year - old aa male)two pps and one sp wanted messages to be more interactive . and i will , thank you ) and sps ( e.g. ( pp , 50-year - old male , race unknown ) we made three changes to improve the goal assessment messages during testing ( table 4 ) . first , we created functional flexibility to allow pps to set a goal for 4 days . we altered feedback messages and functionality so a pp 's response of 03 implied the participant had not met their goal , 46 implied success , and 7 received especially congratulatory feedback . third , we initially developed feedback to reference progress relative to the prior week ( i.e. therefore , we changed the functionality of the feedback text to reference progress only when two weeks ' responses were provided and to default to more basic feedback if not ( e.g. six more pps wanted to have a sp receive text messages but did not provide the sp 's contact information to study staff . reasons included the following : sp was ineligible ( having no cell phone or being non - english - speaking ) , pp could not identify a sp , and sp expressed disinterest to the pp . only four pps ( 21% ) did not want to invite a sp : well , i guess i do have family members . ( pp , 50-year - old male , race unknown)my family members are really busy . ( pp , 56-year - old caucasian female ) well , i guess i do have family members . ( pp , 50-year - old male , race unknown ) my family members are really busy . ( pp , 56-year - old caucasian female ) enrolled sps reported the messages motivated them to talk with the pp about diabetes ( 9.3 1.2 , range 710 ) and improved their support of the pp ( 9.3 1.2 , range 710 ) . when asked how many messages per week they would want to receive in a six - month intervention , sps said three times per week ( n = 3 ) , daily ( n = 2 ) , or every other day ( n = 1 ) . of the six sps who completed an interview , four said fams increased their knowledge about diabetes and all reported discussing message content with pps.i think it was a great experience for me because i had never known that much about diabetes and i was able to learn a great deal about it , you know , through those texts and then talking with [ the pp ] . and ( sp , ex - wife ) i think it was a great experience for me because i had never known that much about diabetes and i was able to learn a great deal about it , you know , through those texts and then talking with [ the pp ] . ( sp , ex - wife ) in round one , a sp - pp dyad reported the sp 's text messages led to interpersonal conflict . you all make her feel like i 'm telling you bad shit about her and i 'm not that thoroughly pissed me off , and i texted back some pretty hot shit to them . with family intervention experts , we reviewed and edited all sp messages to avoid insinuating sps were performing a harmful action or experiencing conflict with the pp ( table 5 ) . , conflict with friends / family members as a risk ) and the sp ( i.e. in addition to described changes made between rounds of testing , we made several decisions and changes to fams for an upcoming evaluative trial based on these results ( table 4 ) . the first session will allocate half of the session to assessing current diet or exercise activities and collaboratively setting a smart goal , and the other half will be allocated to discussing the role of family actions / support in meeting and sustaining health goals . we hope this change will allow sufficient time for goal setting and increase the pp 's acceptance of family members ' role prior to asking pps to identify desired family changes in subsequent sessions . we added the skill building exercise cognitive behavioral coping for pps who report not having supportive family members or experiencing persistent harmful family actions . we also made changes to our process for enrolling and inviting sps to receive text messages ( table 4 ) . we designed fams to improve t2d adults ' ( a ) adherence to diet and exercise recommendations and ( b ) self - efficacy and skills to engage loved ones in self - care goals in ways that facilitate behavior change . through an iterative user - centered design process followed by rigorous and multimethod usability / feasibility testing , we designed an intervention that appeals to end users , is easy to use , and is applicable to a variety of patient and family situations . pps said fams improved their diet / physical activity , communication with family members , and confidence in soliciting helpful actions / support . sps reported fams increased the amount they communicated with pps about diabetes and made it possible for them to be more helpful . comments from users indicate families find it difficult to know how to communicate about diabetes or support diabetes self - care efforts and appreciated concrete suggestions provided during coaching or in text messages . we do know , however , that our iterative process dramatically reduced technical bugs and problems with content , functionality , and study processes , thereby maximizing our ability to evaluate fams . fams targets patients ' adherence to diet and exercise , but other self - care behaviors ( e.g. , medication adherence , self - monitoring of blood glucose ) are impacted by family actions [ 2 , 12 ] and affect diabetes control [ 2 , 12 ] . future work should assess patients ' expectations of others ' involvement in diabetes self - care . for instance , in other disease contexts , helpful and harmful family involvement have been shown to affect dietary adherence , self - efficacy , and depressive symptoms differently based on perceptions of the role of family in adults ' self - management . future intervention efforts may benefit from normalizing nagging and arguing and inadvertently making self - care more challenging . this may make it less emotionally fraught for families to identify and replace these actions with more helpful ones . although mhealth interventions have improved adherence and glycemic control among adults with t2d [ 55 , 56 ] , engaging family members in these interventions remains understudied . to our knowledge , the only other mhealth intervention involving a patient - selected family member / support person for adults with t2d is mhealth + carepartner [ 57 , 58 ] . 's approach to engaging a support person and our own . in contrast , fams does not provide information about patients ' progress to sps but rather aims to empower the patient to identify and communicate support needs . as a result , we had to wait between each development phase and paused recruitment between rounds of usability / feasibility testing . because identifying and fixing bugs are inevitable with mhealth interventions , we stress the importance of starting rigorous multiround testing early in the design / development process rather than waiting until the intervention is developed and functioning properly to allow users to experience it . more time and energy in these phases likely pay off with faster evaluative trials with more engagement , less attrition , and fewer study limitations associated with functional problems .

kras is a member of the highly homologous p21 ras family of monomeric gtpases . three isoforms ( hras , kras , and nras ) are expressed in all mammalian cells and function as molecular switches downstream of cell surface receptors , such as epidermal growth factor receptor ( egfr ) , to stimulate cell proliferation and cell survival . mutations of ras at the conserved codons 12 , 13 , or 61 result in an impaired intrinsic hydrolysis rate or binding to gtpase activating proteins ( gaps ) . despite a high degree of similarity , kras is typically mutated at codon 12 or codon 13 . while mutations at both sites are activating , due to impaired gap binding , the position of the mutation has functional and clinical relevance . metastatic colorectal cancer ( mcrc ) is one of the leading causes of cancer - related death worldwide . a third of crc tumors harbor kras mutations , 19% of these mutations are at codon 13 with almost all of the remainder at codon 12 . mutations of kras at codon 12 are more potent than codon 13 mutations at transforming cells and are associated with a more aggressive metastatic colorectal cancer phenotype . despite this , patients with codon 13 mutations display a significantly worse prognosis . furthermore , patients with codon 12 and codon 13 mutations exhibit differential responsiveness to treatment . these data suggest that each activating kras mutation generates a distinctive signaling output . early ras research supports these observations by demonstrating that variant amino acid codon mutations are not equally transforming . the mechanistic basis for this is unclear but may relate to differences in nucleotide hydrolysis rates that could translate into differential coupling with and activation of ras effectors . for example , g12d and g12v , exhibit different gtp hydrolysis rates ( g12d 40% and g12v 10% of wild - type respectively ) . alternatively , the mutations may differently affect the distribution of gtp - ras between conformational states that differ in effector recognition . various omic approaches have been previously used to identify kras signatures , typically using cell lines harboring oncogenic ras variants . a drawback with some of these studies is the variability of the genetic background between cell lines that confounds attribution of results directly to the presence of oncogenic ras . one strategy to overcome this has been the use of isogenic cells . however , almost all models employed so far have involved , either stable overexpression of oncogenic ras randomly inserted into the genome on an isogenic background or genetic ablation of a wild type or oncogenic kras allele . we exploit recently developed model cell culture systems that accurately recapitulate the genetic changes present in human crcs . specifically , we are using isogenic human sw48 crc cell - lines in which targeted homologous recombination with the endogenous kras gene has been used to knock - in a panel of kras codon 12 and 13 mutations commonly found in crc . the g12d , g12v , and g13d kras mutations present in our isogenic cell panel are the three most abundant mutations representing 75% of all cases of crc harboring a kras mutation . we have used quantitative proteomic approaches to determine ( phospho)proteomic signatures associated with each kras mutation . this combination of cell model and experimental approach represents the contemporary gold standard for precise analysis of endogenous oncogenic kras signaling . importantly , we find that each of the activating mutations that we have investigated display distinct output signatures . we identified a subset of proteins and phosphosites associated with codon 12 versus codon 13 responses . among these are the kinase proteins dclk1 and met which show the same patterns of kras - dependent overexpression across a broad panel of codon 12 mutant isogenic sw48 cells . the clones used were heterozygous knock - in ( g12v/+ ) of k - ras activating mutation kras ( cat . hd 103 - 007 0395 ) , heterozygous knock - in ( g12d/+ ) of k - ras activating mutation kras ( hd 103 - 011 00436 ) and heterozygous knock - in ( g13d/+ ) of k - ras activating mutation kras ( hd 103 - 002 0025 ) . these were referenced to homozygous kras expressing cells ( hd par-006 00276 ) , hereafter referred to as parental cells . for kras knock down studies , sw48 par and g12d cells containing doxycycline inducible shrna targeting kras were generated . the following sequences were used : shrna#a top strand : ccggcgatacagctaattcagaatcctcgaggattctgaattagctgtatcgttttt , bottom strand : aattaaaaacgatacagctaattcagaatcctcgaggattctgaattagctgtatcg . shrna#b top strand : ccggcaggctcaggacttagcaagactcgagtcttgctaagtcctgagcctgttttt , bottom strand : aattaaaaacaggctcaggacttagcaagactcgagtcttgctaagtcctgagcctg . to knock down kras , 5a medium supplemented with 10% dialyzed fbs ( dundee cell products ) . to generate light , medium and heavy stable isotope - labeled cells , arginine- and lysine - free mccoy s medium was supplemented with 200 mg / l l - proline and either l - lysine ( lys0 ) together with l - arginine ( arg0 ) , l - lysine - h4 ( lys4 ) with l - arginine - u - c6 ( arg6 ) or l - lysine - u - c6-n2 ( lys8 ) with l - arginine - u - c6-n4 ( arg10 ) at final concentrations of 28 mg / l for the arginine and 146 mg / l for the lysine until fully metabolically labeled . the extent of isotope incorporation was assessed using an r - script as described . cell lysates were prepared , quantified , subjected to sds page and in - gel tryptic digest as described previously . at least three biological replicate data sets representative of each kras versus parental sw48 were obtained ( n = 4 for kras versus parental ) . for phosphopeptide ( pser / thr / tyr ) isolation , we used filter - aided sample preparation ( fasp ) followed by fractionation using strong cation exchange ( scx ) chromatography and tio2-based phosphopeptide isolation ( based on refs ( 28 and 29 ) and described previously in refs ( 25 and 60 ) ) . in parallel , quantitative sw48 isogenic cell - line proteome analyses were carried out by resolving a 50 g aliquot of each silac mixture by sds - polyacrylamide gel electrophoresis ( sds - page ) on a 412% nupage gel ( invitrogen ) , prior to protein visualization by colloidal blue staining ( invitrogen ) . gel lanes were then cut into 48 bands each , according to protein content , in - gel digested overnight at 37 c with trypsin ( 4 ng/l working concentration ; trypsin gold , sequencing grade , promega ) to cleave c - terminal to arginine and lysine residues , dried , and redissolved in 0.05% tfa prior to lc - msms analysis of each gel slice . a total of 5 l of each sample was fractionated by nanoscale c18 high performance liquid chromatography ( hplc ) on a waters nanoacquity uplc system coupled to an ltq - orbitrapxl ( thermo fisher ) fitted with a proxeon nanoelectrospray source . peptides were loaded onto a 5 cm 180 m trap column ( beh - c18 symmetry ; waters corporation ) in 0.1% formic acid at a flow rate of 15 l / min and then resolved using a 25 cm 75 m column using a 20 min linear gradient of 3 to 62.5% acetonitrile in 0.1% formic acid at a flow rate of 400 nl / min ( column temperature of 65 c ) . the mass spectrometer acquired full ms survey scans in the orbitrap ( r = 30 000 ; m / z range 3002000 ) and performed msms on the top five multiple charged ions in the linear quadrupole ion trap ( ltq ) after fragmentation using collision - induced dissociation ( 30 ms at 35% energy ) . full scan ms ions previously selected for msms were dynamically excluded for 180 s from within a rolling exclusion list ( with n = 1 ) . phosphopeptides were also analyzed using multistage activation ( r = 60 000 , neutral loss mass list : 49.0 , 65.3 , 98.0 ) for the top six multiply charged ions , using a 60 min linear gradient of 3 to 62.5% acetonitrile in 0.1% formic acid , all other conditions as above . all spectra were acquired using xcalibur software ( version 2.0.7 ; thermo fisher scientific ) . raw ms peak list files from each experimental configuration were searched against the human ipi database ( version 3.77 ) using the andromeda search engine and processed with the maxquant software suite ( version 1.2.2.5 ) as described previously . the minimum required peptide length was set to six amino acids and two missed cleavages were allowed . cysteine carbamidomethylation ( c ) was set as a fixed modification , whereas oxidation ( m ) and s / t / y phosphorylation were considered as variable modifications . the initial precursor and fragment ion maximum mass deviations were set to 7 ppm and 0.8 da , respectively , for the search of the ipi_human_v3.77.fasta database containing 89 709 entries . the results of the database search were further processed and statistically evaluated by maxquant . proteins with at least one peptide unique to the protein sequence were considered as valid identifications . for protein quantitation , only proteins with at least three peptides ( one unique ) were selected . in addition , all experiments were also analyzed together using andromeda and maxquant in a single iteration of the pipeline . data obtained from maxquant analyses were evaluated using excel and mev ( version 4.8.1 ; www.tm4.org/mev ) . to compare the interexperimental correlation between biological replicate experiments peptides or phosphopeptides present in two or more of each experimental configuration ( 2/4 or 2/3 [ kras versus parental ] ) were log10 transformed , plotted on scatter plots and the r correlation(s ) visualized as heatmaps . peptide data were included for analysis if ratios were available for every par / mutant condition . no imputation was performed . peptides with missing ratios were excluded from the analysis . gprox analysis of log2 transformed maxquant data sets using unsupervised fuzzy c - means clustering was performed as described previously to identify coresponding genes in the proteome and phosphoproteome data sets . gene ontology analysis using david bioinformatics database was performed using the entrez gene i d identifiers of shortlisted proteins and phosphopeptides . over - represented terms within the short - lists were calculated using a background list comprising all genes identified across our experiments ( threshold count = 2 ; ease score = 1 ) . terms with a p - value < 0.1 in at least one cluster were selected , log10 transformed , hierarchically clustered and plotted as a heatmap . phosphopeptides within the data set with a phosphorylation localization probability 0.75 ( class 1 ) were analyzed for common motifs and their putative regulatory kinases using motifx and networkin v2.0 as described previously . a 25 g cell lysate was run on sds gels , and subsequently transferred to nitrocellulose membrane . membranes were then incubated with ir dye coupled secondary antibodies and detected using the odyssey system ( licor ) . within this study the following primary antibodies were used ; polyclonal anti - akap12 ( c3 , gene tex ) , monoclonal anti - met , polyclonal anti - egfr , anti - erk1/2 and anti - zo-2 ( cell signaling technologies ) , polyclonal anti - caveolin-1 ( transduction laboratories ) , monoclonal anti--actin ( abcam ) , polyclonal anti - dclk1 , and monoclonal anti--tubulin ( sigma - aldrich ) , rabbit monoclonal pan - ras ( epitomics ) , and rabbit monoclonal anti - aldh3a1 ( abcam ) . cdna was made by reverse transcription of 1 g of rna using revertaid h - minus m - mulv reverse transcriptase ( fermentas ) and oligo(dt ) primer ( promega ) . quantitative real - time pcr ( qpcr ) was performed using a real - time pcr detection system ( bio - rad ) using iq sybr green supermix . qpcr was conducted in triplicate with 1 l of cdna and 150 nmol primers . samples underwent 40 cycles of amplification at 94 c ( 30 s ) and 60 c/62 c ( 60 s ) , fluorescence was read at 60 c/62 c , and melt curves analyzed . for each sample , the ct values for dclk1 and kras were normalized to the reference gene actb and the control sample and represented as 2 . isogenic sw48 colorectal cancer cell lines harboring either wild type or a g12d , g12v , or g13d mutated kras allele were used to investigate the effects of amino - acid substitution specificity ( g12d vs g12v ) or codon - specificity ( g12d vs g13d ) on kras signaling . stable isotope labeling of amino acids in cell culture ( silac ) allows different cell populations to be selectively labeled with isotopes of arginine and lysine and analyzed by mass spectrometry in a triplexed configuration ( figure 1 ; ) . following silac labeling , cell lysates were either run directly on sds - page gels or subjected to tio2-based phosphopeptide enrichment procedures . high - resolution mass - spectrometry of gel slices or peptide fractions allowed us to compare their proteome and signaling network responses downstream of each kras mutant ( figure 1 ) . all kras mutants were compared to a parental wild - type kras control in each triplex configuration with an n of 3 or 4 biological repeats for each comparison . diagram of the experimental workflow to determine proteome and phosphopeptide status in isogenic sw48 cells . the experimental configurations adopted resulted in at least n = 3 biological replicate data sets to be obtained that were representative of each kras versus parental sw48 ( n = 4 for kras versus parental ) . in total , across all experiments , responses were measured from 2359 unique proteins in the proteome data set and 3971 unique phosphopeptides from the tio2 purifications ( 3311 phosphosites unique by sequence ; supporting information table 1 ) . a total of 65% of proteins and 35% of phosphopeptides were sampled at least twice across biological replicates ( supporting information figure 1 ) . a total of 3727 phosphosites could be assigned to a specific position within the protein with a probability of at least 0.75 ( class 1 sites ) . the 3727 class 1 phosphosites were composed of 3030 pser , 632 pthr , and 65 ptyr sites mapped to 1288 proteins . to examine experimental reproducibility and intermutation response variability , we performed cross - correlation analysis between all experimental pairs across biological replicates ( figure 2a ) . for both proteome and phosphoproteome data sets , hierarchical clustering indicates good experimental reproducibility with a least a 5-fold higher cross - correlation coefficient between biological repeats compared to any of the interisogenic cell type correlations . importantly , the consistent responses of each cell line allow us to clearly observe kras mutation - specific signaling signatures . more specifically , both hierarchical clustering and principal component analysis ( figure 2b ) indicate that although changes in the proteome and phosphoproteome outputs are similar between the g12d / g12v mutants there is a divergence between codon 12 and codon 13 mutants . closer inspection of the responses within our data sets reveals that g13d mutant cells exhibit more prevalent protein and phosphopeptide up - regulation than the g12 mutant cells ( supporting information figure 1 ) . for example , almost 50% of g13d phosphopeptides are up - regulated versus < 10% of phosphopeptides in g12 mutant cell lines ( supporting information figure 1a and 1c ) . therefore , we observe that both the type of amino acid substitution and the codon positioning of the mutation , influence the outputs of oncogenic kras , with codon position having the greatest effect . oncogenic kras variants display mutation - specific changes to proteome and phosphopeptide networks . ( a ) at both proteome and phosphoproteome levels there is consistent biological reproducibility but a high degree of variability between cell lines containing different kras mutations . isogenic sw48 cells harboring codon 12 kras mutations share greater correlation compared to cells harboring a g13d mutation . values for peptides / phosphopeptides shared between each experiment were used for r cross - correlation analysis . pearson correlation coefficient ( r ) indicates linkage strength and relationship between experimental conditions . codon 12 mutant kras cell lines share similar projections at both proteome and phosphoproteome levels . notably , there were very few proteins or phosphosites that showed significant pan - mutation responses ( supporting information table 4 ) . one protein and 29 phosphosites exhibited 1.5 fold up- or down - regulation versus wild type ras across all three g12v , g12d , and g13d cell lines . these included some of the codon - specific responders such as akap12 and dclk1 where though increases were seen in each of the mutant cell lines , there was a significant bias in favor of one or more kras variant . almost all of the genes showing pan - mutation responses are involved in mrna processing and transcriptional regulation . we clustered proteins and phosphopeptides according to their mutation - specific responses versus parental controls using proteome meta - analysis software gprox . these clusters represent groups of proteins or phosphosites that show matching profiles across each of the cell lines . six distinct phosphopeptide and proteome clusters were identified ( figure 3a , supporting information figure 2 and table 2 ) . of particular interest were those representing codon 12- versus codon 13-specific responses that are most marked in clusters 5 and 6 . ratios for proteins that exhibit a change in expression level within a kras - mutated environment were subjected to unsupervised clustering with the fuzzy c means algorithm . clusters corresponding to six different response patterns were identified . the number ( n ) of phosphopeptides in each pattern is indicated . clusters 5 and 6 contain phosphoproteins that are likely to be signatures of codon 12 and codon 13 kras mutations in colorectal cancer . ( b ) go analysis indicates that proteins associated with the cytoskeleton and cell adhesion are significantly enriched in clusters 5 and 6 . ( c ) phosphopeptides and selected proteins are highlighted within a scatter graph of g12d versus g13d responses . analysis of the proteome data revealed 115 proteins in clusters 5 and 6 for which changes in abundance are associated with kras codon 12 versus 13-specific signaling ( supporting information figure 2 ) . in this data set , gene ontology ( go ) analysis revealed that certain mitochondrial proteins involved in oxidative phosphorylation are enriched in cluster 5 , that is , these proteins are decreased in g13d relative to codon 12 mutant cells ( supporting information figure 2 ) . for example , within the proteome data set , we observe decreases in abundance of peptides from 5 of the 11 members of the cytochrome bc1 complex ( complex iii ) and succinate dehydrogenase of complex ii in g13d cells but not other components of the respiratory chain . in contrast , metabolic enzymes including those involved in gluconeogenesis are enriched in cluster 6 ( supporting information figure 2 ) ; however , the majority of enzymes within the proteome data set that are associated with glycolysis , including pyruvate kinase m2 do not significantly change between the cell lines . the most significant response was seen for the aldehyde dehydrogenase aldh3a1 that is increased in g13d cells and decreased in g12d cells . we also note the presence in clusters 5 and 6 of the membrane trafficking and organizing proteins sec23b , anxa1 , anx3 , and anx11 and the increased expression of the stage - specific colorectal cancer biomarker serpinb5 in g12d cells . a total of 274 phosphopeptides from 198 proteins representing 17% of the total number of unique sites were observed in clusters 5 and 6 of the phosphoproteome data set ( figure 3a ) . in total , 56 out of 274 phosphopeptides are associated with 27 proteins linked by go analysis to cell adhesion or cytoskeletal function in clusters 5 and 6 of the phosphoproteome data set ( figure 3b ) . among these other notable phosphopeptide representatives of clusters 5 and 6 are the hgf - receptor met thr995 and caveolin-1 ser37 sites that both exhibit > 10-fold increases in abundance in g12d versus g13d cells and the ras effector braf ser729 site that is decreased in g12d versus g13d cells . in the case of caveolin and met , a component of this change is due to the higher levels of protein expression observed in g12d and g12v cells as judged by western blotting ( figure 6 ) . phosphopeptide members of clusters 5 and 6 that originated from proteins with multiple phosphorylation sites were curated to examine the patterns of response across all detected sites within these proteins ( figure 4 and supporting information figure 3 ) . where available , proteome data are also presented ( squares ) to see the extent to which phosphopeptide responses were influenced by changes in protein abundance rather than a proportional increase in phosphorylation . in the majority of cases where comparisons could be made , proteome changes were a minor influence on phosphopeptide ratios . interestingly , most phosphosites within a protein trended in a similar direction for both cluster 5 and cluster 6 members , indicating coordinated increase or decrease of phosphorylation at multiple sites within a protein . the top 15 proteins with multiple phosphosites measured in the combined maxquant analysis and a minimum of one phosphopeptide within cluster 5 or 6 are collated together with their respective proteome values . given the central role of kinases in mediating ras responses and modulating phosphonetworks , we examined their contribution to our data sets . in total , we detected peptides from 38 kinases ( supporting information table 3 ) . a total of 35 kinase phosphopeptides out of 96 were responsive ( 1.5-fold change compared to parental control ) to the presence of at least one of the oncogenic ras mutations ( figure 5a and supporting information table 3 ) . these included core growth factor receptor - ras pathway members egfr , met , map2k2 ( mek2 ) and erk2 as well as cdc42bpb , nek9 and pak4 ( figure 5a and b ) . phosphopeptides from eight kinases were present in clusters 5 and 6 ( supporting information table 3 ) . among these this phosphosite shows specific down - regulation in g13d vs codon 12 cell lines suggesting that kras g13d is impaired in its ability to activate erk2 . to investigate the wider context of the kinases regulating the phosphosites in clusters 5 and 6 , we used networkin analysis that integrates consensus substrate motifs and contextual modeling to predict potential kinases for each phosphosite . a significant number of cluster 5 and 6 members are potential targets of kinases that regulate the cell cycle and promote proliferation ( cyclin - dependent , casein , map and mok kinases ; figure 5c ) . kinase responses and predicted kinase regulators of phosphosites associated with codon 12 versus codon 13 kras outputs . peptide ( blue ) and phosphopeptide ( red ) responses of all kinases present in our data sets are depicted ( a ) . all kinases identified in the proteome data set except dclk1 are collated with their respective phosphopeptide values ( b ) . ( c ) motifx analysis identified significantly over - represented linear phosphorylation motifs from the set of sites present in gprox phosphopeptide clusters 5 and 6 . analysis of all of the phosphosites in our data set is provided for comparison ( long list ) . average responses of the sites associated with each motif in response to the presence of kras mutations are indicated within the output column . the total number of submissions used in the motifx analysis and average response heatmap are indicated ( n ) . kinases that had constituent peptides or phosphopeptides detected in our data sets are labeled ( ) . upon inspection of our data , we have chosen to follow up several proteins based on enrichment factor , biological relevance , and availability of reagents . within our proteome data set we saw a number of proteins that were highly expressed in kras codon 12 mutant cell lines . the pre - eminent examples of this were doublecortin - like kinase-1 ( dclk1 ) and a - kinase anchor protein 12 ( akap12 ) that were up - regulated at least 8-fold in kras the distinctive pattern of expression of these proteins was confirmed by western blotting ( figure 6a ) and was recapitulated in a second independent clone of each cell line ( supporting information figure 4 ) . we also confirmed the significant up - regulation of the c - met hgf receptor and caveolin-1 in codon 12 mutant kras cells . the tight junction protein , zo-2 , and the aldehyde dehydrogenase , aldh3a1 , show the converse pattern of specific up - regulation in the g13d cell line together with significant down - regulation in codon 12 cells versus the parental cells ( figure 6a and b , supporting information figure 2c ) . increased dclk1 expression is observed across a panel of codon 12 mutant kras isogenic cell lines . ( a ) increased expression of selected hits from our silac proteome analysis were confirmed using western blotting . ( b ) western blotting of a wider panel of isogenic sw48 cells , including lines not directly analyzed by proteomics , shows that dclk1 and met follow the same patterns of codon 12 specific up - regulation whereas zo-2 is coupled to kras g13d signaling . ( c ) qpcr analysis indicates significant up - regulation of dclk1 isoform 3/4 expression in codon 12 mutant kras cells . ( d ) schematic diagram of dclk1 isoforms expressed in human and distribution of peptides observed in our data set indicates that all of the dclk1 peptides detected by mass spectrometry are in the c terminus . our observation that dclk1 is highly overexpressed in kras g12d cells is striking , given recent data that this protein is a colon cancer tumor stem cell marker . up - regulation of dclk1 is also seen in g12v cells ( figure 6a ) . to examine the extent to which this is a codon 12-specific phenomenon , we extended our analysis to a broader panel of isogenic sw48 cells expressing codon 12 variants . strikingly , we observe that dclk1 expression is significantly increased with all codon 12 mutants while codon 13 is equivalent to parental cells ( figure 6b ) . met exhibits a corresponding pattern of up - regulation across the codon 12 mutant panel potentially indicating a common mechanism promoting up - regulation . qpcr analysis indicates that the increased levels of dclk1 observed in codon 12 kras mutant cell lines are due to transcriptional up - regulation rather than via regulation of translation or protein stability ( figure 6c ) . the molecular weight of dclk1 observed in our western blotting experiments and the distribution of peptides identified by mass spectrometry indicate that this represents isoform 3 or isoform 4 of dclk1 . these consist of an active kinase domain but lack the n - terminal doublecortin - like domains required for binding to microtubules ( figure 6d ) . to investigate whether transcriptional up - regulation of dclk1 seen in g12d cells was directly kras - dependent rather than an adaptive change , we used isogenic sw48 cell lines stably expressing kras - specific shrnas that can be inducibly expressed in response to doxycycline . an almost complete loss of kras protein expression can be seen in response to either of the two independent shrnas for kras ( figure 7a ) . this is accompanied by 50% decreases in dclk1 protein expression in kras g12d cells ( figure 7a ) . qpcr - based analysis of kras and dclk1 transcripts revealed proportional reductions in kras and dclk1 expression in g12d cells in response to kras knockdown ( figure 7b ) . the induced expression of two independent shrnas specific for kras results in significant decreases in kras and dclk1 protein ( a ) and proportional decreases in dclk1 mrna ( b ) . a pan - ras antibody is used in ( a ) ; the upper band of the doublet corresponds to kras ( arrow ) . the combination of isogenic cell lines and large - scale quantitative proteomics has resulted in unprecedented depth of coverage of pathways specifically engaged by oncogenic kras variants . our first and perhaps most striking observation was that each type of activating codon mutation specifies a distinct kras signaling output . this is an important insight because to date almost all ras studies and ras - related clinical trials have treated ras mutations as being equivalent . we were interested in the mechanisms by which kras codon 12 and codon a total of 274 phosphopeptides and 115 proteins differentially responded to the presence of codon 12 versus codon 13 kras mutants . numerous proteins that we have identified have prominent links to colon cancer or properties associated with malignant cells . among these were the cell adhesion associated protein akap12 and the cell surface organizer caveolin 1 that is a tumor suppressor implicated in regulating ras signaling and kras mediated colorectal cancer cell migration . met has a well - established role in ras - mediated tumorigenesis where it is up - regulated ; furthermore , met amplification has been previously observed in colorectal tumors . although met is upstream of kras , the potential interplay between ras pathway activation feeding back to growth factor receptor signaling was recently highlighted in colon cancer cells where inhibition of braf signaling resulted in activation of egfr through loss of negative feedback . importantly , the pattern of codon - specific up - regulation of met and caveolin seen in our data is supported in a wider panel of 275 lung , pancreas , and colon cancer cell lines ( supporting information figure 5 ) . we observe a significant correlation of increased met and caveolin expression with the presence of a kras codon 12 mutation compared to nonmutated kras cells . although there are relatively few kras codon 13 mutant cells in the panel , expression of met and caveolin are not significantly different from the nonmutant subset . the most prominent example of a diametrically opposite response was seen with the aldehyde dehydrogenase aldh3a1 that is increased in g13d and decreased in codon 12 cells versus parentals . a significant correlation ( p < 0.001 ) with kras codon 13 mutation status and aldh3a1 overexpression was also seen across 275 lung , pancreas , and colon cancer cell lines ( supporting information figure 5 ) . together , these data provide important validation of the predictive utility of our data sets . our analysis revealed dclk1 to be the most amplified of all proteins in any of our kras mutant isogenic cells compared to wild type parentals , and this up - regulation is also reflected in the mrna transcript levels . this indicates a continued direct role for kras , rather than an irreversible adaptive response , or selection pressure , in regulating dclk1 expression . although dclk1 is a relatively poorly understood kinase , we note that analysis of gene coexpression across almost 1000 cell lines reveals that the microtubule stabilizing protein map1b that is a prominent phosphosite responder in the codon 12 cells is among the top 20 nearest neighbor genes with dclk1 suggesting functional cooperation between these proteins . a genome wide mutant kras synthetic lethality screen previously identified the related kinase dclk2 as a stringent hit in colorectal dld1 cells . these data suggest that dclk1 is biologically relevant to colorectal cancer cell survival . furthermore , dclk1 is also a crc tumor stem cell specific marker ; ablation of dclk1 tumor stem cells results in regression of crc polyps ; however , there was no formal linkage with kras status established in these in vivo studies . overexpression of dclk1 is seen with all variants of codon 12 kras mutant cells but not in g13d cells . however , this relationship is likely to be highly context - dependent because , in this case , interrogation of the cancer cell line encyclopaedia reveals no significant correlation between the presence of a codon 12 mutated kras allele and dclk1 levels in a panel of 275 cancer cell lines or within the subset of 61 colorectal cell lines ( supporting information figure 5 ) . our data show expression of the short forms of dclk1 containing the kinase domain but not the microtubule binding double cortin domains . although none of the previous colorectal studies have discriminated between which dclk isoforms are contributing to their results , data from studies of brain function reveal specific up - regulation of short c - terminal dclk1 transcripts in adult brain that are associated with modulating memory and cognitive abilities . our study represents the first unbiased global screen of signaling pathways downstream of endogenous oncogenic kras . our experimental approach enabled differences in outputs emanating from each kras mutant to be identified without the confounding effects of significant differences in genetic background . the majority of nodes within the immediate ras signaling network displayed differential responses at the proteome and phosphoproteome level ( figure 8) the mechanistic basis for this is currently unclear ; however , it vividly illustrates the importance of factoring precise mutation status into the designs and interpretation of experiments comparing ras function . for example , several recent studies identified genes that are synthetically lethal when depleted or inhibited in cells harboring oncogenic kras . each study used a different panel of cell lines with a variety of codon 12 or codon 13 mutations and responsiveness between cell types was inconsistent . our data predict that synthetic lethality would likely vary , depending upon which specific mutation is present , and suggest that an isogenic cell line approach will be important for identifying contingencies of drug responsiveness on mutation status . phosphosites identified in the phosphosite data set are highlighted in red , those displaying at least a 2-fold change versus parental cells in at least one of the kras mutant cell lines are highlighted in green . in summary , we have found that each of the three main kras mutations generates a distinct signaling network signature and proteome expression profile . furthermore , we have demonstrated that a key collection of genes with known functions in promoting oncogenic colorectal cancer signaling and tumorigenesis exhibit codon - specific kras dependence for their expression and/or phosphorylation . among these our analysis provides fundamental insights into basic ras biology with significant implications for the design and interpretation of large - scale studies of oncogenic ras signaling across cell panels .

oncogenic mutations of ras at codons 12 , 13 , or 61 , that render the protein constitutively active , are found in 16% of all cancer cases . among the three major ras isoforms , kras is the most frequently mutated isoform in cancer . each ras isoform and tumor type displays a distinct pattern of codon - specific mutations . in colon cancer , kras is typically mutated at codon 12 , but a significant fraction of patients have mutations at codon 13 . clinical data suggest different outcomes and responsiveness to treatment between these two groups . to investigate the differential effects upon cell status associated with kras mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic sw48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific kras mutations ( g12v , g12d , or g13d ) . each mutation generates a distinct signature , with the most variability seen between g13d and the codon 12 kras mutants . one notable example of specific up - regulation in kras codon 12 mutant sw48 cells is provided by the short form of the colon cancer stem cell marker doublecortin - like kinase 1 ( dclk1 ) that can be reversed by suppression of kras .

Introduction Materials and Methods Results Discussion Conclusions

kras is a member of the highly homologous p21 ras family of monomeric gtpases . mutations of ras at the conserved codons 12 , 13 , or 61 result in an impaired intrinsic hydrolysis rate or binding to gtpase activating proteins ( gaps ) . despite a high degree of similarity , kras is typically mutated at codon 12 or codon 13 . while mutations at both sites are activating , due to impaired gap binding , the position of the mutation has functional and clinical relevance . a third of crc tumors harbor kras mutations , 19% of these mutations are at codon 13 with almost all of the remainder at codon 12 . mutations of kras at codon 12 are more potent than codon 13 mutations at transforming cells and are associated with a more aggressive metastatic colorectal cancer phenotype . furthermore , patients with codon 12 and codon 13 mutations exhibit differential responsiveness to treatment . these data suggest that each activating kras mutation generates a distinctive signaling output . for example , g12d and g12v , exhibit different gtp hydrolysis rates ( g12d 40% and g12v 10% of wild - type respectively ) . a drawback with some of these studies is the variability of the genetic background between cell lines that confounds attribution of results directly to the presence of oncogenic ras . one strategy to overcome this has been the use of isogenic cells . specifically , we are using isogenic human sw48 crc cell - lines in which targeted homologous recombination with the endogenous kras gene has been used to knock - in a panel of kras codon 12 and 13 mutations commonly found in crc . the g12d , g12v , and g13d kras mutations present in our isogenic cell panel are the three most abundant mutations representing 75% of all cases of crc harboring a kras mutation . we identified a subset of proteins and phosphosites associated with codon 12 versus codon 13 responses . among these are the kinase proteins dclk1 and met which show the same patterns of kras - dependent overexpression across a broad panel of codon 12 mutant isogenic sw48 cells . gel lanes were then cut into 48 bands each , according to protein content , in - gel digested overnight at 37 c with trypsin ( 4 ng/l working concentration ; trypsin gold , sequencing grade , promega ) to cleave c - terminal to arginine and lysine residues , dried , and redissolved in 0.05% tfa prior to lc - msms analysis of each gel slice . raw ms peak list files from each experimental configuration were searched against the human ipi database ( version 3.77 ) using the andromeda search engine and processed with the maxquant software suite ( version 1.2.2.5 ) as described previously . gprox analysis of log2 transformed maxquant data sets using unsupervised fuzzy c - means clustering was performed as described previously to identify coresponding genes in the proteome and phosphoproteome data sets . isogenic sw48 colorectal cancer cell lines harboring either wild type or a g12d , g12v , or g13d mutated kras allele were used to investigate the effects of amino - acid substitution specificity ( g12d vs g12v ) or codon - specificity ( g12d vs g13d ) on kras signaling . diagram of the experimental workflow to determine proteome and phosphopeptide status in isogenic sw48 cells . for both proteome and phosphoproteome data sets , hierarchical clustering indicates good experimental reproducibility with a least a 5-fold higher cross - correlation coefficient between biological repeats compared to any of the interisogenic cell type correlations . more specifically , both hierarchical clustering and principal component analysis ( figure 2b ) indicate that although changes in the proteome and phosphoproteome outputs are similar between the g12d / g12v mutants there is a divergence between codon 12 and codon 13 mutants . closer inspection of the responses within our data sets reveals that g13d mutant cells exhibit more prevalent protein and phosphopeptide up - regulation than the g12 mutant cells ( supporting information figure 1 ) . for example , almost 50% of g13d phosphopeptides are up - regulated versus < 10% of phosphopeptides in g12 mutant cell lines ( supporting information figure 1a and 1c ) . therefore , we observe that both the type of amino acid substitution and the codon positioning of the mutation , influence the outputs of oncogenic kras , with codon position having the greatest effect . oncogenic kras variants display mutation - specific changes to proteome and phosphopeptide networks . ( a ) at both proteome and phosphoproteome levels there is consistent biological reproducibility but a high degree of variability between cell lines containing different kras mutations . isogenic sw48 cells harboring codon 12 kras mutations share greater correlation compared to cells harboring a g13d mutation . codon 12 mutant kras cell lines share similar projections at both proteome and phosphoproteome levels . one protein and 29 phosphosites exhibited 1.5 fold up- or down - regulation versus wild type ras across all three g12v , g12d , and g13d cell lines . these included some of the codon - specific responders such as akap12 and dclk1 where though increases were seen in each of the mutant cell lines , there was a significant bias in favor of one or more kras variant . these clusters represent groups of proteins or phosphosites that show matching profiles across each of the cell lines . clusters 5 and 6 contain phosphoproteins that are likely to be signatures of codon 12 and codon 13 kras mutations in colorectal cancer . ( b ) go analysis indicates that proteins associated with the cytoskeleton and cell adhesion are significantly enriched in clusters 5 and 6 . analysis of the proteome data revealed 115 proteins in clusters 5 and 6 for which changes in abundance are associated with kras codon 12 versus 13-specific signaling ( supporting information figure 2 ) . in this data set , gene ontology ( go ) analysis revealed that certain mitochondrial proteins involved in oxidative phosphorylation are enriched in cluster 5 , that is , these proteins are decreased in g13d relative to codon 12 mutant cells ( supporting information figure 2 ) . for example , within the proteome data set , we observe decreases in abundance of peptides from 5 of the 11 members of the cytochrome bc1 complex ( complex iii ) and succinate dehydrogenase of complex ii in g13d cells but not other components of the respiratory chain . in contrast , metabolic enzymes including those involved in gluconeogenesis are enriched in cluster 6 ( supporting information figure 2 ) ; however , the majority of enzymes within the proteome data set that are associated with glycolysis , including pyruvate kinase m2 do not significantly change between the cell lines . we also note the presence in clusters 5 and 6 of the membrane trafficking and organizing proteins sec23b , anxa1 , anx3 , and anx11 and the increased expression of the stage - specific colorectal cancer biomarker serpinb5 in g12d cells . in total , 56 out of 274 phosphopeptides are associated with 27 proteins linked by go analysis to cell adhesion or cytoskeletal function in clusters 5 and 6 of the phosphoproteome data set ( figure 3b ) . a total of 35 kinase phosphopeptides out of 96 were responsive ( 1.5-fold change compared to parental control ) to the presence of at least one of the oncogenic ras mutations ( figure 5a and supporting information table 3 ) . among these this phosphosite shows specific down - regulation in g13d vs codon 12 cell lines suggesting that kras g13d is impaired in its ability to activate erk2 . to investigate the wider context of the kinases regulating the phosphosites in clusters 5 and 6 , we used networkin analysis that integrates consensus substrate motifs and contextual modeling to predict potential kinases for each phosphosite . kinase responses and predicted kinase regulators of phosphosites associated with codon 12 versus codon 13 kras outputs . analysis of all of the phosphosites in our data set is provided for comparison ( long list ) . average responses of the sites associated with each motif in response to the presence of kras mutations are indicated within the output column . within our proteome data set we saw a number of proteins that were highly expressed in kras codon 12 mutant cell lines . the pre - eminent examples of this were doublecortin - like kinase-1 ( dclk1 ) and a - kinase anchor protein 12 ( akap12 ) that were up - regulated at least 8-fold in kras the distinctive pattern of expression of these proteins was confirmed by western blotting ( figure 6a ) and was recapitulated in a second independent clone of each cell line ( supporting information figure 4 ) . we also confirmed the significant up - regulation of the c - met hgf receptor and caveolin-1 in codon 12 mutant kras cells . the tight junction protein , zo-2 , and the aldehyde dehydrogenase , aldh3a1 , show the converse pattern of specific up - regulation in the g13d cell line together with significant down - regulation in codon 12 cells versus the parental cells ( figure 6a and b , supporting information figure 2c ) . increased dclk1 expression is observed across a panel of codon 12 mutant kras isogenic cell lines . ( b ) western blotting of a wider panel of isogenic sw48 cells , including lines not directly analyzed by proteomics , shows that dclk1 and met follow the same patterns of codon 12 specific up - regulation whereas zo-2 is coupled to kras g13d signaling . ( c ) qpcr analysis indicates significant up - regulation of dclk1 isoform 3/4 expression in codon 12 mutant kras cells . our observation that dclk1 is highly overexpressed in kras g12d cells is striking , given recent data that this protein is a colon cancer tumor stem cell marker . up - regulation of dclk1 is also seen in g12v cells ( figure 6a ) . to examine the extent to which this is a codon 12-specific phenomenon , we extended our analysis to a broader panel of isogenic sw48 cells expressing codon 12 variants . strikingly , we observe that dclk1 expression is significantly increased with all codon 12 mutants while codon 13 is equivalent to parental cells ( figure 6b ) . met exhibits a corresponding pattern of up - regulation across the codon 12 mutant panel potentially indicating a common mechanism promoting up - regulation . qpcr analysis indicates that the increased levels of dclk1 observed in codon 12 kras mutant cell lines are due to transcriptional up - regulation rather than via regulation of translation or protein stability ( figure 6c ) . these consist of an active kinase domain but lack the n - terminal doublecortin - like domains required for binding to microtubules ( figure 6d ) . to investigate whether transcriptional up - regulation of dclk1 seen in g12d cells was directly kras - dependent rather than an adaptive change , we used isogenic sw48 cell lines stably expressing kras - specific shrnas that can be inducibly expressed in response to doxycycline . an almost complete loss of kras protein expression can be seen in response to either of the two independent shrnas for kras ( figure 7a ) . qpcr - based analysis of kras and dclk1 transcripts revealed proportional reductions in kras and dclk1 expression in g12d cells in response to kras knockdown ( figure 7b ) . the induced expression of two independent shrnas specific for kras results in significant decreases in kras and dclk1 protein ( a ) and proportional decreases in dclk1 mrna ( b ) . a pan - ras antibody is used in ( a ) ; the upper band of the doublet corresponds to kras ( arrow ) . the combination of isogenic cell lines and large - scale quantitative proteomics has resulted in unprecedented depth of coverage of pathways specifically engaged by oncogenic kras variants . we were interested in the mechanisms by which kras codon 12 and codon a total of 274 phosphopeptides and 115 proteins differentially responded to the presence of codon 12 versus codon 13 kras mutants . numerous proteins that we have identified have prominent links to colon cancer or properties associated with malignant cells . among these were the cell adhesion associated protein akap12 and the cell surface organizer caveolin 1 that is a tumor suppressor implicated in regulating ras signaling and kras mediated colorectal cancer cell migration . met has a well - established role in ras - mediated tumorigenesis where it is up - regulated ; furthermore , met amplification has been previously observed in colorectal tumors . although met is upstream of kras , the potential interplay between ras pathway activation feeding back to growth factor receptor signaling was recently highlighted in colon cancer cells where inhibition of braf signaling resulted in activation of egfr through loss of negative feedback . importantly , the pattern of codon - specific up - regulation of met and caveolin seen in our data is supported in a wider panel of 275 lung , pancreas , and colon cancer cell lines ( supporting information figure 5 ) . we observe a significant correlation of increased met and caveolin expression with the presence of a kras codon 12 mutation compared to nonmutated kras cells . although there are relatively few kras codon 13 mutant cells in the panel , expression of met and caveolin are not significantly different from the nonmutant subset . the most prominent example of a diametrically opposite response was seen with the aldehyde dehydrogenase aldh3a1 that is increased in g13d and decreased in codon 12 cells versus parentals . a significant correlation ( p < 0.001 ) with kras codon 13 mutation status and aldh3a1 overexpression was also seen across 275 lung , pancreas , and colon cancer cell lines ( supporting information figure 5 ) . together , these data provide important validation of the predictive utility of our data sets . our analysis revealed dclk1 to be the most amplified of all proteins in any of our kras mutant isogenic cells compared to wild type parentals , and this up - regulation is also reflected in the mrna transcript levels . although dclk1 is a relatively poorly understood kinase , we note that analysis of gene coexpression across almost 1000 cell lines reveals that the microtubule stabilizing protein map1b that is a prominent phosphosite responder in the codon 12 cells is among the top 20 nearest neighbor genes with dclk1 suggesting functional cooperation between these proteins . these data suggest that dclk1 is biologically relevant to colorectal cancer cell survival . furthermore , dclk1 is also a crc tumor stem cell specific marker ; ablation of dclk1 tumor stem cells results in regression of crc polyps ; however , there was no formal linkage with kras status established in these in vivo studies . overexpression of dclk1 is seen with all variants of codon 12 kras mutant cells but not in g13d cells . however , this relationship is likely to be highly context - dependent because , in this case , interrogation of the cancer cell line encyclopaedia reveals no significant correlation between the presence of a codon 12 mutated kras allele and dclk1 levels in a panel of 275 cancer cell lines or within the subset of 61 colorectal cell lines ( supporting information figure 5 ) . our data show expression of the short forms of dclk1 containing the kinase domain but not the microtubule binding double cortin domains . although none of the previous colorectal studies have discriminated between which dclk isoforms are contributing to their results , data from studies of brain function reveal specific up - regulation of short c - terminal dclk1 transcripts in adult brain that are associated with modulating memory and cognitive abilities . the majority of nodes within the immediate ras signaling network displayed differential responses at the proteome and phosphoproteome level ( figure 8) the mechanistic basis for this is currently unclear ; however , it vividly illustrates the importance of factoring precise mutation status into the designs and interpretation of experiments comparing ras function . each study used a different panel of cell lines with a variety of codon 12 or codon 13 mutations and responsiveness between cell types was inconsistent . phosphosites identified in the phosphosite data set are highlighted in red , those displaying at least a 2-fold change versus parental cells in at least one of the kras mutant cell lines are highlighted in green . in summary , we have found that each of the three main kras mutations generates a distinct signaling network signature and proteome expression profile . furthermore , we have demonstrated that a key collection of genes with known functions in promoting oncogenic colorectal cancer signaling and tumorigenesis exhibit codon - specific kras dependence for their expression and/or phosphorylation .

immunization with tumor - associated antigen ( taa ) is a potential approach for cancer treatment and prevention . cancer vaccines have not been administered to prevent a tumor from occurring in healthy individuals . instead , they have been used to alleviate the suffering of patients who are already combating cancer . ga733 is an epithelial cell adhesion molecule ( epcam ) that is abundant in colorectal cancer cells . in addition , ga733 is known to mediate ca - independent homotypic cell - cell adhesion . it contains an extracellular domain with 2 epidermal growth factor-(egf- ) like repeats , followed by a cysteine - poor region , a transmembrane domain , and a short ( 26 amino acid ) cytoplasmic tail . the extracellular domain ( ecd ) of ga733 such recombinant vaccines developed over the last several decades have been expressed using many available heterologous expression systems . plants are a promising expression system that can efficiently produce recombinant proteins in large quantities without pathogenic animal contaminants . recently , the tumor - associated colorectal cancer antigen epcam ( ga733 ) was expressed in plants , and the recombinant plant - derived antigen induced a humoral immune response in balb / c mice . however , plants are not an ideal expression system for producing therapeutic proteins because of the differences in the n - glycosylation processes between plants and humans and the low expression level . plant - derived specific n - glycans contain antigenic and/or allergenic (1 , 2)-xylose and (1 , 3)-fucose , which are absent in mammalian glycans . the plant - specific glycans lack sialic acid , which may cause instability and a lower half - life . to avoid the plant - derived specific n - glycan structure , we generated an oligomannose glycan structure by retaining the recombinant protein ( ga733 and ga733-fc ) in the endoplasmic reticulum ( er ) . fusion of ga733 or ga733-fc to kdel ( the er retention motif , lys - asp - glu - leu ) helps retain the protein inside the er and at the same time enhances ga733 and ga733-fc assembly in plant cells . the antigen - antibody complex may potentially have properties similar to the parental igg , such as enhanced efficacy of vaccination by targeting the vaccine to antigen - presenting cells ( apcs ) , facilitated purification by the protein - a method , and increased half - life [ 2 , 1416 ] . furthermore , an antigen - antibody chimera was reported to provide higher expression levels , better yields , and increased stability in plant expression systems [ 2 , 17 ] . in the present study , 3 different recombinant human colorectal cancer antigen ga733 genes were expressed in a tobacco ( nicotiana tabacum ) plant expression system : ga733 fused to the immunoglobulin fc fragment ( ga733-fc ) , ga733-fc with kdel ( ga733-fck ) , and ga733 with kdel ( ga733k ) . the stability and functionality of these colorectal cancer vaccine candidates were confirmed by western blot analysis and elisa , respectively . in order to understand the fusion of fc to ga733 and its functionality , the immunogenicity of recombinant ga733-fc with oligomannose glycosylation was investigated in mice . ay189981 ) was modified by n - terminal extension with a 30-aa plant er signal peptide ( matqrranpsslhlitvfsllaavvsaevd ) from nicotiana plumbaginifolia and c - terminal extension with an er retention signal ( kdel ) . the recombinant chimeric protein ga733-fc was generated by fusing ga733 to the fc fragment of human igg1 ( val97-gly328 , genbank accession no . the lys279 ( c - terminus of ga733 ) was followed by the val97 ( n - terminus of fc fragment of human igg1 ) . the ga733-fc - encoding sequences were placed under the control of the enhanced cauliflower mosaic virus ( camv ) 35s promoter and tobacco etch viral 5-leader sequence ( tev ) . the ga733 , ga733-fc , and ga733-fck expression cassettes were subcloned into the hindiii sites of the binary plant transformation vector pbin - plus to yield pbi ga733k , pbi ga733-fck , and pbi ga733-fc , respectively ( figure 1(a ) ) . transgenic tobacco lines were selected on murashige and skoog medium ( dachfu , haarlem , netherland ) containing 100 mg / l kanamycin . transgenic plantlets were then transferred to soil in a growth chamber at a constant temperature of 23c and 70% humidity and were maintained under a 14 : 10 h light - dark cycle . transgenic and nontransgenic n. tabacum plants were grown in a greenhouse under controlled conditions . genomic dna was isolated from the fresh leaf tissue of transgenic and non - transgenic plants using a dna extraction kit ( intron biotechnology , seoul , republic of korea ) according to the manufacturer 's recommendations . pcr amplification of genomic dna was performed in order to confirm the presence of the recombinant genes using the following primer pairs : for ga733k , forward primer 5-gcg tcg aca cgg cga ctt tgc cgc tca gga a-3 , reverse primer 5-gct cta cat cag agt tca tct ttt ttt aga ccc tcg att gag-3 ; for ga733-fck , forward primer 5-gcg tcg aca cgg cga ctt ttg ccg cag ctc agg aa-3 , reverse primer 5-gct cta gat cag agt tca tct tta ccc ggg gac agg g-3 ; for ga733-fc , forward primer 5-gcg tcg aca cgg cga ctt tgc cgc agc tca gga a-3 , reverse primer 5-gct cta gat caa ccc ggg gac agg gag ag-3. pcr was performed with 38 cycles of 94c for 60 s , 55c for 60 s , and 72c for 60 s. non - transgenic plants were used as negative control , while a t - easy vector ( promega , madison , wi , usa ) containing the ga733-fck gene was used as a positive control . the expected size of the dna products for ga733k , ga733-fck , and ga733-fc was 768 , 1483 , and 1471 bp , respectively . the transcription levels of ga733k , ga733-fck , and ga733-fc mrna were quantified by performing semi - quantitative rt - pcr . total rna was extracted from transgenic and non - transgenic plants using the rneasy plant mini kit ( qiagen , valencia , ca , usa ) , according to the manufacturer 's protocol . to remove the genomic dna , 600 ng of total rna was treated using a turbo dna - free kit ( ambion , austin , tx , usa ) in a reaction volume of 20 l . the rna samples were stored at 80c until use . each rna sample was used as a template for rt reactions performed using accupower rt / pcr premix ( bioneer , daejeon , republic of korea ) . rt - pcr was performed using the following master mix : 4 l 10 rt - pcr buffer , 2 l for each primer ( 10 pmol/l ) , 2 l 10 mm dntps , and 1 l of hotstart taq dna polymerase ( bioneer ) ; the volume of the mix was adjusted to 22 l with sterilized water , and 3 l of rna was added as a template . the following primers were used in the rt - pcr reaction : for ga733k , forward primer 5-gca gct cag gaa gaa tct-3 , reverse primer 5-ctc aga gca ggt tat ttc a-3 ; for ga733-fck or ga733-fc , forward primer 5-atc tgg atc ctg gtc aaa-3 , reverse primer 5-ctc aga gca ggt tat ttc a-3 ; for actin ( genbank accession no . x69885 ) , forward primer 5-aat cca cga gac tac ata caa-3 , reverse primer 5-aga gcc tcc aat cca gac a-3. the rna was subjected to rt - pcr with the following specifications : reverse transcription at 50c for 30 min ; an initial pcr activation step at 95c for 15 min ; 38 cycles of 1 min at 94c , 1 min at 55c , and 1 min at 72c ; a final extension at 72c for 10 min . the predicted sizes of the rt - pcr products for ga733k , ga733-fck , and ga733-fc are 348 , 534 , and 534 bp , respectively . the actin gene ( 214 bp ) the rt - pcr products were analyzed on 1.0% agarose gel . in order to evaluate the stability of ga733k , ga733-fck , and ga733-fc , a leaf tissue ( 100 mg ) was homogenized in 1 pbs in the presence or absence of complete protease inhibitor cocktail ( roche diagnostics , mannheim , germany ) . the proteins in the homogenates were resolved by 12.5% sds - page and transferred to a nitrocellulose membrane ( millipore , billerica , ma , usa ) . membranes were incubated with blocking buffer ( 3% skim milk ( fluka , buchs , switzerland ) in pbs plus 0.5% ( v / v ) tween 20 ) . the blots were incubated for 1 h 30 min at rt with either mouse anti - epcam antibody ( r&d systems , minneapolis , mn , usa ) diluted in blocking buffer at 1 : 500 or mouse anti - human fc conjugated to horseradish peroxidase ( jackson immunoresearch , west grove , pa , usa ) diluted in blocking buffer at 1 : 3,000 and then incubated for 1 h and 30 min at rt with secondary antibody goat anti - mouse igg ( animal genetics , daejeon , republic of korea ) diluted in blocking buffer at 1 : 3,000 . anti - fc recognizes the fc portion of ga733-fc , while anti - human ga733 antibody detects ga733 . protein bands were visualized by exposing the membrane to an x - ray film ( fuji , tokyo , japan ) using a chemiluminescence substrate ( pierce ) . non - transgenic plants and the recombinant human epcam / trop-1 fc chimera ( r&d systems ) , of which the human epcam / trop-1 is fused with the fc fragment of human igg1 , were used as negative and positive controls , respectively . to reconfirm the expression of recombinant proteins , the leaf samples were collected from the 3 transgenic plants ( ga733k , ga733-fck , and ga733-fc ) and a non - transgenic plant and were then fixed in formalin - acetic acid - alcohol solution for 24 h. the fixed leaves were dehydrated in ethanol , cleared in xylene , and embedded in paraffin blocks . the paraffin blocks were sectioned into 7-m slices and attached to gelatin - coated slides . the tissue slides were incubated with 1% bovine serum albumin in 0.01 m pbs ( ph 7.2 ) to block nonspecific binding of the primary antibody . the primary antibodies , mouse anti - ga733 igg , and goat anti - human igg fc antibody ( animal genetics ) , were diluted 1 : 500 in pbs and applied to samples for 3 h at rt . the tissue slides were then incubated for another 3 h at rt with the secondary antibodies , alexa-488 conjugated anti - mouse igg ( invitrogen , ca , usa ) and alexa-546 conjugated anti - goat igg ( invitrogen ) , diluted 1 : 300 in pbs . in order to stain the nucleus anti - human igg and to - pro-3 fluorescence was observed using a confocal laser scanning microscope ( olympus , tokyo , japan ) at 488 nm and 633 nm , respectively . the expression and assembly of vaccine candidates in plants were further analyzed by sandwich elisa . briefly , 96-well maxisorp surface plates ( nunc , rochester , ny , usa ) were coated with 1 g / ml mouse anti - human ga733 antibody in 50 mm sodium carbonate ( ph 9.6 ) . the leaf tissues ( 100 mg ) were homogenized in 200 l of extraction buffer containing 10 mm sodium sulfate , 2% polyvinylpyrrolidone ( molecular weight , 40,000 kda ) , 3 mm sodium azide , and 0.5% tween 20 . leaf extract samples were diluted 2-fold in extraction buffer , and then serial 3-fold dilutions were made in pbs . recombinant human epcam / trop-1 fc chimera ( r&d systems , 5 g / ml ) was used as a positive control , and a rabies antibody ( bethyl , montgomery , tx , usa ) was used as a negative control . the plate was incubated with secondary antibody goat anti - human fc conjugated to horseradish peroxidase ( jackson immunoresearch ) ( 1 : 3,000 ) diluted in pbs for 2 h at rt and was detected using soluble tmb ( 3.3 , 5.5-tetramethylbenzidine ) substrate ( kpl , gaithersburg , md , usa ) . the antibody titers in 3 wells per tested sample were estimated by determining the optical density at 450 nm using an elisa reader sunrise ( tecan , mnnedorf , switzerland ) . isolation and purification procedures of the recombinant proteins were followed as described in a previous study . plant leaves were homogenized in an hr2094 aluminium blender ( philips , seoul , republic of korea ) in chilled extraction buffer ( 37.5 mm tris - hcl , 50 mm nacl , 15 mm edta , 75 mm sodium citrate , and 0.2% sodium thiosulfate ) . after centrifugation for 30 min at 15,000 g , the supernatants were further clarified through a miracloth ( calbiochem , la jolla , ca , usa ) , and solid ammonium sulfate was added 16% saturation . after 2 h incubation at 4c , the solution was centrifuged at 15,000 g for 30 min at 4c , the precipitate was discarded , and ammonium sulfate was added to the supernatant to 40% saturation . after incubation at 4c overnight , the solution was centrifuged at 15,000 g for 30 min at 4c , and the pellet was resuspended in extraction buffer at one - fifth of the original volume . soluble protein extract was applied to a protein a column ( ge healthcare , piscataway , nj , usa ) . eluates of plant - derived recombinant ga733-fc protein were dialyzed against 1 pbs buffer and brought to a final concentration of 1 mg / ml using an amicon ultra spincolumn with a 10 kda cutoff ( millipore ) . aliquots were frozen in liquid nitrogen and stored at 80c . for analysis , extracts were resolved by sds - polyacrylamide gel electrophoresis ( page ) and either stained or transferred to a nitrocellulose membrane ( millipore ) , blocked with 3% nonfat milk and murine anti - ga733 igg ( calbiochem , san diego , ca , usa ) , and followed by secondary antimurine mab conjugated to horseradish peroxidase ( sigma , st . louis , mo , usa ) diluted 1 : 10,000 to detect ga733 . non - transgenic plants and the recombinant human epcam / trop-1 fc chimera ( r&d systems ) were used as negative and positive controls , respectively . samples were first digested into glycopeptides using pepsin , as previously described . from the resulting glycopeptide mixtures , n - glycans were released using peptide n - glycosidase ( pngase ) a ( roche , basel , switzerland ) . the released n - glycans were purified using graphitized carbon resin from carbograph ( alltech , lexington , ma , usa ) . purified glycans were dried and then redissolved in a mixture of 90 l dimethyl sulfoxide ( dmso ) , 2.7 l water , and 35 l iodomethane for solid phase permethylation using a spin - column method . after the process previously described , the chloroform layer containing permethylated glycans was dried and resuspended in 4 l of a 50% methanol solution . then , it was mixed in equal volume with the matrix 2,5-dihydroxybenzoic acid prepared in 1 mm sodium acetate solution . the resulting mixtures were applied onto an maldi msp 96 polished steel chip ( bruker daltonik gmbh , bremen , germany ) and dried . matrix - assisted laser desorption / ionization - time of flight ( maldi - tof ) mass spectrometry was performed in the reflector positive ion mode using a microflex ( bruker daltonik ) . all mass spectra were acquired at a 20 kv accelerating voltage using the method recommended by the manufacturer . six - week - old female balb / c mice were purchased from da mool science ( daejeon , republic korea ) and maintained in a pathogen - free environment . all mice experiments in this study were approved by the wonkwang university animal ethics committee in accordance with the guidelines of the korean council on animal care . eight - week - old female balb / c mice ( 5 per group ) were injected 3 times with 1 , 5 , or 10 g of plant - derived ga733 or mammalian - derived ga733 ( r&d systems ) in a total volume of 100 l at 2-week intervals . the first and second immunizations were given subcutaneously ( s.c . ) with complete ( difco , detroit , mi , usa ) and incomplete freund 's adjuvant ( thermo fisher scientific , roskilde , denmark ) , respectively ; the third dose was administered intraperitoneally ( i.p . ) in saline . blood samples were collected by retroorbital bleeding before the experiment and 10 days after the second immunization ; 10 days after the third immunization , mice were bled individually via the retroorbital plexus and sacrificed . 100 l / well of sera ( 1 g / ml in pbs ) were applied to elisa plates coated with human colorectal cells from lines sw480 , sw1116 , and sw620 , and with human breast cancer cells from line mcf-7 ( 4 10 cells / well ) and incubated for 2 h at rt . 100 l / well secondary antibody hrp - conjugated goat anti - mouse igg ( animal genetics ) diluted in pbs ( 1 : 8,000 ) were applied and incubated for 2 h at rt . the signals were detected using soluble tmb ( 3.3 , 5.5-tetramethylbenzidine ) substrate ( kpl , gaithersburg , md , usa ) . the results were read by sunrise ( tecan ) at 450 nm and presented as mean sd . pcr analysis of the genomic dna was performed to confirm the presence of transgene in 10 randomly selected transgenic lines from each type of vectors among the regenerated plants ( figure 2 ) . the transgenic plants lines with ga733k , ga733-fck , and ga733-fc had the expected amplified bands at 780 , 1473 , and 1461 bp , respectively . semi - quantitative rt - pcr analysis was carried out to confirm mrna expression in randomly selected transgenic plants using specific primers ( figure 3 ) . all tested ga733k transgenic plants had the band at 384 bp , while the ga733-fck and ga733-fc transgenic plants had a band at 534 bp . the ratios of target gene to actin differed slightly among the transgenic lines , which suggested that different transgenic plants had different expression levels of transgene rna . protein extracts from ga733-fck and ga733-fc transgenic plants were analyzed by western blotting ( figure 4 ) . in order to confirm the effect of the kdel sequence on ga733-fc expression , leaf tissues were homogenized in 1 pbs buffer with or without protease inhibitor ( roche diagnostics ) . transgenic lines with ga733-fck ( t303 and t305 ) showed an approximately 65 kda protein band , similar to the positive control ; however , this 65 kda band was not detected by either the anti - human fc antibody ( figure 4 , anti - fc ) or the anti - human ga733 antibody with protease inhibitor in the transgenic lines with ga733-fc ( t103 and t108 ) ( figure 4 , anti - ga733 ) . in the t303 and t305 protein samples treated with protease inhibitor ( pi ) , an approximately 140 kda protein band was detected by the anti - fc antibody , while in the t303 protein sample without pi , the 140 kda protein band was not detected ( figure 4 , anti - fc ) . in t303 without pi , the protein bands were generally weaker than in t303 with pi . in both the t303 and t305 samples with pi , the 65 and 140 kda protein bands were detected with the ga733-specific antibody ; however , no protein bands were observed in t103 and t108 ( figure 4 , anti - ga733 ) . no band was observed in the non - transgenic plants ( data not shown ) . to confirm the expression of the ga733 and ga733-fc fused to kdel , western blot analysis was conducted ( figure 5 ) . in all the transgenic lines with ga733-fck ( t303 , t305 , and t312 ) , an approximately 65 kda band was detected by the anti - fc antibody , whereas in the transgenic lines with ga733k ( t202 , t203 , and t207 ) no protein band was detected ( figure 5 , anti - fc ) . however , in some of both the ga733k and ga733-fck transgenic lines , the 35 kda protein band was detected with the anti - ga733 antibody ( figure 5 , anti - ga733 ) . among all the tested ga733k transgenic lines treated with or without protease inhibitor , t207 seemed to have the highest expression . among all the tested ga733-fck transgenic lines , t303 had the highest expression ( figure 5 ) . in general , the density of the ga733 protein bands recognized with the anti - ga733 antibody in ga733k transgenic plants was slightly stronger than that of the ga733-fck transgenic plants , indicating that the fc did not seem to enhance the ga733 expression level ( figure 5 ) . expression of the cancer vaccine candidates ga733k , ga733-fck , and ga733-fc in transgenic plants was investigated by immunofluorescence staining ( figure 6 ) . in the analysis , ga733 , fc , and the nucleus were stained in green , red , and blue , respectively . green fluorescence activity was observed in leaf samples from ga733k and ga733-fck transgenic plants but not in ga733-fc plants ( figure 6 , anti - ga733 ) . red fluorescence was detected in leaf samples from ga733-fck plants but rarely observed in ga733-fc plants ( figure 6 , anti - fc ) . a 96-well plate was coated with mouse anti - human ga733 antibody , and soluble plant leaf protein extracts were applied . leaf extracts from 2 ga733-fck transgenic lines ( t303 and t305 ) , 2 ga733-fc transgenic lines ( t103 and t108 ) , and a ga733k transgenic line ( t207 ) were analyzed by elisa . hrp - conjugated anti - human igg fc was used as a secondary antibody to confirm ga733-fc expression . both the t303 and t305 transgenic lines ( ga733-fck ) showed significantly greater absorbance than t103 and t108 ( ga733-fc ) ( figure 7 ) . no absorbance was detected in the transgenic line with ga733k ( t207 ) , which does not have the fc fragment or with mab so57 ( antirabies antibody ) , which lacks the ga733 fragment . nt tobacco served as a negative control . a recombinant human epcam / trop-1 fc chimera ( r&d systems ) , which is the positive control , showed high absorbance . the ga733-fck protein was purified from transgenic plants using a protein a affinity column for glycan structure analysis . however , the ga733-fc protein could not be purified from ga733-fc transgenic plants since its expression was very low . ga733-fc had mostly glycan structures with mammalian - specific (1 , 6)-fucose residues , whereas ga733-fck had oligomannose glycan structures ( figure 8) . a ga733-specific serum igg antibody immune response was induced in all mice injected with purified ga733-fck or ga733-fc ( figure 9 ) . the serum of the injected mice contained iggs recognizing the human colorectal cancer cell lines sw480 , sw1116 , and sw620 , as well as the human breast cancer cell line mcf-7 . with all 3 human colorectal cancer cell lines ( sw480 , sw1116 , and sw620 ) , serum from mice injected with ga733-fck showed comparable absorbance to serum from mice injected with ga733-fc ( figure 9 ) . with all the colorectal cell lines , the absorbance rapidly decreased in the serum from mice injected with ga733-fc , whereas the absorbance level slowly decreased in the serum from mice injected with ga733-fck at dilution factor of 1 : 50 and 1 : 100 . even with the human breast cancer cell line mcf-7 , serum from mice injected with ga733-fck showed comparable absorbance to serum from mice injected with ga733-fc . here , we describe the successful expression of chimeric ga733-fc , which is a fusion of ga733 to a human igg fc fragment , in plants . in this study , 3 different transgenic plant lines expressing ga733k , ga733-fck , or ga733-fc were established in order to investigate the effect of the fc fragment and the kdel retention signal on ga733 expression and bioactivities . both pcr and rt - pcr analyses showed that all randomly tested transgenic plants with each of the transgenes ( ga733k , ga733-fck , and ga733-fc ) expressed the transgene transcript . ga733-fck transgenic plants had stronger protein expression than ga733-fc transgenic plants , which did not have any detectable protein expression . this higher expression of ga733-fck can be explained by its localization in er and consequently is protected from proteolytic activity found through the secretary pathway , in intracellular and extracellular spaces [ 21 , 22 ] . therefore , it seems that the fc fragment does not increase ga733 protein expression . in order to confirm the stability of the ga733-fc fusion protein , western blot analysis was performed , and it was shown that the protein samples homogenized with or without proteinase inhibitor had similar expression levels , which indicated that the fc fusion proteins are stable . confocal immunofluorescence analysis reconfirmed that the expression of ga733k and ga733-fck in plants was higher than the ga733-fc expression in plants . in addition , ga733-fck transgenic plants expressed ga733 fused to the human fc fragment . in the elisa analysis , the ga733-fck transgenic plants ( t303 and t305 ) showed higher absorbance than the ga733-fc transgenic plants ( t103 and t108 ) , indicating that the kdel sequence enhanced ga733-fc expression . the absorbance signal observed in the ga733-fck and ga733-fc transgenic plants was not detected using the anti - fc antibody in the ga733k transgenic plants or using mab so57 , indicating that ga733 is fused to the fc fragment and is expressed in ga733-fck transgenic plants . the higher yield of ga733-fc in lines t305 and t303 and of ga733 with kdel in lines t202 , t203 , and t207 might reflect the use of the endoplasmic reticulum retrieval motif kdel ( lys - asp - glu - leu ) , which was fused to the c - terminus of the fc fragment and/or ga733 in order to retain the recombinant protein in the endoplasmic reticulum . we observed lower expression of ga733-fc when the kdel sequence is absent in lines t103 , t108 , and t110 , which indicates that the endoplasmic reticulum is a good subcellular location for the accumulation of recombinant protein . therefore , in this study , the ga733-fc fusion protein did not provide better yield and stability than ga733 without the fc fragment fusion . nevertheless , the reason that ga733 was fused to the fc fragment was the obvious advantage of an easy one - step purification procedure using the protein a method [ 17 , 23 ] . as a source of recombinant ga733-fc antigen , soluble fc fusion proteins have become valuable reagents for immunotherapy and laboratory investigations . furthermore , the immunoadhesin antibody - like protein design resulted in soluble protein accumulation in plant tissues . ga733-fc could not be purified from transgenic plants since the expression was very low . however , the soluble ga733-fck protein was successfully purified using a protein - a affinity column for glycan structure analysis and in vivo animal immunizations . in the glycosylation analysis , ga733-fc had mostly glycan structures with mammalian - specific (1 , 6)-fucose residues , whereas ga733-fck had high - mannose glycan structures . these results indicate that the kdel er retention signal efficiently retained the glycoproteins , yielding proteins with high mannose . in balb / c mice , the immune response to it seems likely that this differential immunoreactivity is due to the different glycosylation patterns in ga733-fck and ga733-fc . the reactivity of anti - ga733-fck sera with all 3 tested human colorectal cancer cell lines ( sw480 , sw1116 , and sw620 ) appeared to be slightly higher than that of ga733-fc sera at sera dilutions of 1 : 50 and 1 : 100 ( figure 9 ) . the reactivity of both anti - ga733-fck and anti - ga733-fc sera was much weaker with the human breast cancer cell line mcf-7 than with the human colorectal cancer cell lines ( figure 9 ) . however , a similar trend in differential immunoreactivity between ga733-fck and ga733-fc was also observed at sera dilutions of 1 : 50 and 1 : 100 with the human breast cancer cell line mcf-7 . the antigen - antibody complex can enhance the efficiency and effectiveness of vaccination by targeting the vaccine to apcs . it has not been reported that the common fc - receptors on mouse apc do interact efficiently with human igg1 fc however , in our unpublished data , the human fc of a full size human monoclonal antibody has interaction with the mouse fc receptor ( cd64 ) . thus , it is speculated that the human fc fragment should be specific for cd64 located on the surface of dendritic cells , which is a known entry portal to the antigen - processing pathway in mice . the fc fragment is specific for cd64 located on the surface of dendritic cells , which is a known entry portal to the antigen - processing pathway . in model systems , the antigen delivered by antibody has been shown to be processed and presented by dendritic cells at least 100- to 1000-fold more efficiently than nontargeted antigen . in addition , the oligomannose - type n - glycan structure would be expected to cause an enhanced immune response through the mannose receptor ( mr ) on macrophages and dendritic cells recognizing the oligomannose of ga733-fck . according to a previous study , the er - type glycans on glycoprotein which are also known as oligomannose glycan - type can render the glycoprotein more immunogenic , producing igg against the oligomannose glycosylated protein itself . in this study , the data clearly demonstrate expression of the colorectal cancer vaccine candidate ga733 and the antigen - antibody complex protein ga733-fc in tobacco plant expression systems . fusion of the fc fragment of human igg to the c - terminus of ga733 and the er retention kdel in ga733-fck generating oligomannose glycosylated proteins is an ideal strategy to easily purify recombinant ga733 vaccine candidate proteins and to enhance accumulation of the recombinant proteins with oligomannose for comparable immunogenicity to the non - kdel - tagged mammalian - derived proteins in a plant expression system .

the tumor - associated antigen ga733 is a cell - surface glycoprotein highly expressed in colorectal carcinomas . in this study , 3 recombinant genes were constructed as follows : ga733 tagged to the er retention sequence kdel ( ga733k ) , ga733 fused to the immunoglobulin fc fragment ( ga733-fc ) , and ga733-fc fused to the er retention sequence ( ga733-fck ) . agrobacterium - mediated transformation was used to generate transgenic plants expressing recombinant genes . the presence of transgenes was confirmed by genomic pcr . western blot , confocal immunofluorescence , and sandwich elisa showed the expression of recombinant proteins . the stability , flexibility , and bioactivity of recombinant proteins were analyzed and demonstrated through n - glycosylation analysis , animal trials , and sera elisa . our results suggest that the kdel retained proteins in er with oligomannose glycan structure and enhanced protein accumulation level . the sera of mice immunized with ga733-fck purified from plants contained immunoglobulins which were at least as efficient as the mammalian - derived ga733-fc at recognizing human colorectal cancer cell lines . thus , a plant system can be used to express the kdel fusion protein with oligomannose glycosylation , and this protein induces an immune response which is comparable to non - kdel - tagged , mammalian - derived proteins .

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

immunization with tumor - associated antigen ( taa ) is a potential approach for cancer treatment and prevention . ga733 is an epithelial cell adhesion molecule ( epcam ) that is abundant in colorectal cancer cells . in addition , ga733 is known to mediate ca - independent homotypic cell - cell adhesion . it contains an extracellular domain with 2 epidermal growth factor-(egf- ) like repeats , followed by a cysteine - poor region , a transmembrane domain , and a short ( 26 amino acid ) cytoplasmic tail . plants are a promising expression system that can efficiently produce recombinant proteins in large quantities without pathogenic animal contaminants . recently , the tumor - associated colorectal cancer antigen epcam ( ga733 ) was expressed in plants , and the recombinant plant - derived antigen induced a humoral immune response in balb / c mice . however , plants are not an ideal expression system for producing therapeutic proteins because of the differences in the n - glycosylation processes between plants and humans and the low expression level . plant - derived specific n - glycans contain antigenic and/or allergenic (1 , 2)-xylose and (1 , 3)-fucose , which are absent in mammalian glycans . to avoid the plant - derived specific n - glycan structure , we generated an oligomannose glycan structure by retaining the recombinant protein ( ga733 and ga733-fc ) in the endoplasmic reticulum ( er ) . fusion of ga733 or ga733-fc to kdel ( the er retention motif , lys - asp - glu - leu ) helps retain the protein inside the er and at the same time enhances ga733 and ga733-fc assembly in plant cells . the antigen - antibody complex may potentially have properties similar to the parental igg , such as enhanced efficacy of vaccination by targeting the vaccine to antigen - presenting cells ( apcs ) , facilitated purification by the protein - a method , and increased half - life [ 2 , 1416 ] . in the present study , 3 different recombinant human colorectal cancer antigen ga733 genes were expressed in a tobacco ( nicotiana tabacum ) plant expression system : ga733 fused to the immunoglobulin fc fragment ( ga733-fc ) , ga733-fc with kdel ( ga733-fck ) , and ga733 with kdel ( ga733k ) . the stability and functionality of these colorectal cancer vaccine candidates were confirmed by western blot analysis and elisa , respectively . in order to understand the fusion of fc to ga733 and its functionality , the immunogenicity of recombinant ga733-fc with oligomannose glycosylation was investigated in mice . ay189981 ) was modified by n - terminal extension with a 30-aa plant er signal peptide ( matqrranpsslhlitvfsllaavvsaevd ) from nicotiana plumbaginifolia and c - terminal extension with an er retention signal ( kdel ) . the recombinant chimeric protein ga733-fc was generated by fusing ga733 to the fc fragment of human igg1 ( val97-gly328 , genbank accession no . the lys279 ( c - terminus of ga733 ) was followed by the val97 ( n - terminus of fc fragment of human igg1 ) . genomic dna was isolated from the fresh leaf tissue of transgenic and non - transgenic plants using a dna extraction kit ( intron biotechnology , seoul , republic of korea ) according to the manufacturer 's recommendations . pcr amplification of genomic dna was performed in order to confirm the presence of the recombinant genes using the following primer pairs : for ga733k , forward primer 5-gcg tcg aca cgg cga ctt tgc cgc tca gga a-3 , reverse primer 5-gct cta cat cag agt tca tct ttt ttt aga ccc tcg att gag-3 ; for ga733-fck , forward primer 5-gcg tcg aca cgg cga ctt ttg ccg cag ctc agg aa-3 , reverse primer 5-gct cta gat cag agt tca tct tta ccc ggg gac agg g-3 ; for ga733-fc , forward primer 5-gcg tcg aca cgg cga ctt tgc cgc agc tca gga a-3 , reverse primer 5-gct cta gat caa ccc ggg gac agg gag ag-3. pcr was performed with 38 cycles of 94c for 60 s , 55c for 60 s , and 72c for 60 s. non - transgenic plants were used as negative control , while a t - easy vector ( promega , madison , wi , usa ) containing the ga733-fck gene was used as a positive control . the transcription levels of ga733k , ga733-fck , and ga733-fc mrna were quantified by performing semi - quantitative rt - pcr . total rna was extracted from transgenic and non - transgenic plants using the rneasy plant mini kit ( qiagen , valencia , ca , usa ) , according to the manufacturer 's protocol . rt - pcr was performed using the following master mix : 4 l 10 rt - pcr buffer , 2 l for each primer ( 10 pmol/l ) , 2 l 10 mm dntps , and 1 l of hotstart taq dna polymerase ( bioneer ) ; the volume of the mix was adjusted to 22 l with sterilized water , and 3 l of rna was added as a template . the predicted sizes of the rt - pcr products for ga733k , ga733-fck , and ga733-fc are 348 , 534 , and 534 bp , respectively . in order to evaluate the stability of ga733k , ga733-fck , and ga733-fc , a leaf tissue ( 100 mg ) was homogenized in 1 pbs in the presence or absence of complete protease inhibitor cocktail ( roche diagnostics , mannheim , germany ) . non - transgenic plants and the recombinant human epcam / trop-1 fc chimera ( r&d systems ) , of which the human epcam / trop-1 is fused with the fc fragment of human igg1 , were used as negative and positive controls , respectively . to reconfirm the expression of recombinant proteins , the leaf samples were collected from the 3 transgenic plants ( ga733k , ga733-fck , and ga733-fc ) and a non - transgenic plant and were then fixed in formalin - acetic acid - alcohol solution for 24 h. the fixed leaves were dehydrated in ethanol , cleared in xylene , and embedded in paraffin blocks . the primary antibodies , mouse anti - ga733 igg , and goat anti - human igg fc antibody ( animal genetics ) , were diluted 1 : 500 in pbs and applied to samples for 3 h at rt . the expression and assembly of vaccine candidates in plants were further analyzed by sandwich elisa . the leaf tissues ( 100 mg ) were homogenized in 200 l of extraction buffer containing 10 mm sodium sulfate , 2% polyvinylpyrrolidone ( molecular weight , 40,000 kda ) , 3 mm sodium azide , and 0.5% tween 20 . isolation and purification procedures of the recombinant proteins were followed as described in a previous study . after centrifugation for 30 min at 15,000 g , the supernatants were further clarified through a miracloth ( calbiochem , la jolla , ca , usa ) , and solid ammonium sulfate was added 16% saturation . after 2 h incubation at 4c , the solution was centrifuged at 15,000 g for 30 min at 4c , the precipitate was discarded , and ammonium sulfate was added to the supernatant to 40% saturation . for analysis , extracts were resolved by sds - polyacrylamide gel electrophoresis ( page ) and either stained or transferred to a nitrocellulose membrane ( millipore ) , blocked with 3% nonfat milk and murine anti - ga733 igg ( calbiochem , san diego , ca , usa ) , and followed by secondary antimurine mab conjugated to horseradish peroxidase ( sigma , st . non - transgenic plants and the recombinant human epcam / trop-1 fc chimera ( r&d systems ) were used as negative and positive controls , respectively . purified glycans were dried and then redissolved in a mixture of 90 l dimethyl sulfoxide ( dmso ) , 2.7 l water , and 35 l iodomethane for solid phase permethylation using a spin - column method . all mice experiments in this study were approved by the wonkwang university animal ethics committee in accordance with the guidelines of the korean council on animal care . eight - week - old female balb / c mice ( 5 per group ) were injected 3 times with 1 , 5 , or 10 g of plant - derived ga733 or mammalian - derived ga733 ( r&d systems ) in a total volume of 100 l at 2-week intervals . 100 l / well of sera ( 1 g / ml in pbs ) were applied to elisa plates coated with human colorectal cells from lines sw480 , sw1116 , and sw620 , and with human breast cancer cells from line mcf-7 ( 4 10 cells / well ) and incubated for 2 h at rt . pcr analysis of the genomic dna was performed to confirm the presence of transgene in 10 randomly selected transgenic lines from each type of vectors among the regenerated plants ( figure 2 ) . the transgenic plants lines with ga733k , ga733-fck , and ga733-fc had the expected amplified bands at 780 , 1473 , and 1461 bp , respectively . protein extracts from ga733-fck and ga733-fc transgenic plants were analyzed by western blotting ( figure 4 ) . to confirm the expression of the ga733 and ga733-fc fused to kdel , western blot analysis was conducted ( figure 5 ) . in all the transgenic lines with ga733-fck ( t303 , t305 , and t312 ) , an approximately 65 kda band was detected by the anti - fc antibody , whereas in the transgenic lines with ga733k ( t202 , t203 , and t207 ) no protein band was detected ( figure 5 , anti - fc ) . in general , the density of the ga733 protein bands recognized with the anti - ga733 antibody in ga733k transgenic plants was slightly stronger than that of the ga733-fck transgenic plants , indicating that the fc did not seem to enhance the ga733 expression level ( figure 5 ) . expression of the cancer vaccine candidates ga733k , ga733-fck , and ga733-fc in transgenic plants was investigated by immunofluorescence staining ( figure 6 ) . in the analysis , ga733 , fc , and the nucleus were stained in green , red , and blue , respectively . leaf extracts from 2 ga733-fck transgenic lines ( t303 and t305 ) , 2 ga733-fc transgenic lines ( t103 and t108 ) , and a ga733k transgenic line ( t207 ) were analyzed by elisa . both the t303 and t305 transgenic lines ( ga733-fck ) showed significantly greater absorbance than t103 and t108 ( ga733-fc ) ( figure 7 ) . no absorbance was detected in the transgenic line with ga733k ( t207 ) , which does not have the fc fragment or with mab so57 ( antirabies antibody ) , which lacks the ga733 fragment . a recombinant human epcam / trop-1 fc chimera ( r&d systems ) , which is the positive control , showed high absorbance . the ga733-fck protein was purified from transgenic plants using a protein a affinity column for glycan structure analysis . however , the ga733-fc protein could not be purified from ga733-fc transgenic plants since its expression was very low . ga733-fc had mostly glycan structures with mammalian - specific (1 , 6)-fucose residues , whereas ga733-fck had oligomannose glycan structures ( figure 8) . the serum of the injected mice contained iggs recognizing the human colorectal cancer cell lines sw480 , sw1116 , and sw620 , as well as the human breast cancer cell line mcf-7 . with all 3 human colorectal cancer cell lines ( sw480 , sw1116 , and sw620 ) , serum from mice injected with ga733-fck showed comparable absorbance to serum from mice injected with ga733-fc ( figure 9 ) . with all the colorectal cell lines , the absorbance rapidly decreased in the serum from mice injected with ga733-fc , whereas the absorbance level slowly decreased in the serum from mice injected with ga733-fck at dilution factor of 1 : 50 and 1 : 100 . here , we describe the successful expression of chimeric ga733-fc , which is a fusion of ga733 to a human igg fc fragment , in plants . in this study , 3 different transgenic plant lines expressing ga733k , ga733-fck , or ga733-fc were established in order to investigate the effect of the fc fragment and the kdel retention signal on ga733 expression and bioactivities . both pcr and rt - pcr analyses showed that all randomly tested transgenic plants with each of the transgenes ( ga733k , ga733-fck , and ga733-fc ) expressed the transgene transcript . this higher expression of ga733-fck can be explained by its localization in er and consequently is protected from proteolytic activity found through the secretary pathway , in intracellular and extracellular spaces [ 21 , 22 ] . therefore , it seems that the fc fragment does not increase ga733 protein expression . in order to confirm the stability of the ga733-fc fusion protein , western blot analysis was performed , and it was shown that the protein samples homogenized with or without proteinase inhibitor had similar expression levels , which indicated that the fc fusion proteins are stable . confocal immunofluorescence analysis reconfirmed that the expression of ga733k and ga733-fck in plants was higher than the ga733-fc expression in plants . in addition , ga733-fck transgenic plants expressed ga733 fused to the human fc fragment . in the elisa analysis , the ga733-fck transgenic plants ( t303 and t305 ) showed higher absorbance than the ga733-fc transgenic plants ( t103 and t108 ) , indicating that the kdel sequence enhanced ga733-fc expression . the absorbance signal observed in the ga733-fck and ga733-fc transgenic plants was not detected using the anti - fc antibody in the ga733k transgenic plants or using mab so57 , indicating that ga733 is fused to the fc fragment and is expressed in ga733-fck transgenic plants . the higher yield of ga733-fc in lines t305 and t303 and of ga733 with kdel in lines t202 , t203 , and t207 might reflect the use of the endoplasmic reticulum retrieval motif kdel ( lys - asp - glu - leu ) , which was fused to the c - terminus of the fc fragment and/or ga733 in order to retain the recombinant protein in the endoplasmic reticulum . we observed lower expression of ga733-fc when the kdel sequence is absent in lines t103 , t108 , and t110 , which indicates that the endoplasmic reticulum is a good subcellular location for the accumulation of recombinant protein . therefore , in this study , the ga733-fc fusion protein did not provide better yield and stability than ga733 without the fc fragment fusion . nevertheless , the reason that ga733 was fused to the fc fragment was the obvious advantage of an easy one - step purification procedure using the protein a method [ 17 , 23 ] . ga733-fc could not be purified from transgenic plants since the expression was very low . in the glycosylation analysis , ga733-fc had mostly glycan structures with mammalian - specific (1 , 6)-fucose residues , whereas ga733-fck had high - mannose glycan structures . these results indicate that the kdel er retention signal efficiently retained the glycoproteins , yielding proteins with high mannose . in balb / c mice , the immune response to it seems likely that this differential immunoreactivity is due to the different glycosylation patterns in ga733-fck and ga733-fc . the reactivity of anti - ga733-fck sera with all 3 tested human colorectal cancer cell lines ( sw480 , sw1116 , and sw620 ) appeared to be slightly higher than that of ga733-fc sera at sera dilutions of 1 : 50 and 1 : 100 ( figure 9 ) . the reactivity of both anti - ga733-fck and anti - ga733-fc sera was much weaker with the human breast cancer cell line mcf-7 than with the human colorectal cancer cell lines ( figure 9 ) . however , a similar trend in differential immunoreactivity between ga733-fck and ga733-fc was also observed at sera dilutions of 1 : 50 and 1 : 100 with the human breast cancer cell line mcf-7 . thus , it is speculated that the human fc fragment should be specific for cd64 located on the surface of dendritic cells , which is a known entry portal to the antigen - processing pathway in mice . the fc fragment is specific for cd64 located on the surface of dendritic cells , which is a known entry portal to the antigen - processing pathway . in addition , the oligomannose - type n - glycan structure would be expected to cause an enhanced immune response through the mannose receptor ( mr ) on macrophages and dendritic cells recognizing the oligomannose of ga733-fck . according to a previous study , the er - type glycans on glycoprotein which are also known as oligomannose glycan - type can render the glycoprotein more immunogenic , producing igg against the oligomannose glycosylated protein itself . in this study , the data clearly demonstrate expression of the colorectal cancer vaccine candidate ga733 and the antigen - antibody complex protein ga733-fc in tobacco plant expression systems . fusion of the fc fragment of human igg to the c - terminus of ga733 and the er retention kdel in ga733-fck generating oligomannose glycosylated proteins is an ideal strategy to easily purify recombinant ga733 vaccine candidate proteins and to enhance accumulation of the recombinant proteins with oligomannose for comparable immunogenicity to the non - kdel - tagged mammalian - derived proteins in a plant expression system .

human ifn-2 ( il-28a ) is a relatively new cytokine , in which the genomic structure resembles that of the il-10 family , but the protein structure is more closely related to type i ifn than to interleukin- ( il- ) 10 [ 1 , 2 ] . for example , it can induce antiviral activity in cell lines , though the potency is weaker than other ifns , and has the potential antitumor effect against human lung cancer cells . it has also been discovered that ifn-2 is capable of exacerbating t - cell - mediated autoimmune diseases such as uveitis . treatment with ifn-2 completely halts and reverses the development of collagen - induced arthritis , dramatically reduces the numbers of proinflammatory il-17-producing th17 and t cells in the joints and inguinal lymph nodes , and restricts recruitment of il-1b - expressing neutrophils . however , ifn-2 seems not effective in inducing tr1 cells and can not induce proliferation of regulatory t cells from cord blood cd4(+ ) t cells . recently , it was found that the expression level of ifn-2 mrna was significantly increased during naturally occurring respiratory viral infections in children with asthma and that ifn-2 modulates lung dendritic cells ( dc ) function to promote th1 immune skewing and suppresses allergic airway disease . these suggest that ifn-2 is not only involved in autoimmune diseases but also associated with allergic airway disorders . we therefore investigated the potential involvement of ifn-2 in allergic airway diseases in the present study . to our surprise , information on the ifn-2 expressing cells is very limited . it was found that ifn-2 expressed in tracheobronchial tissue cells from the patients with copd . dc express moderate quantity of ifn-2 when using lipopolysaccharide ( lps ) as the maturation stimulus , and vitiligo patient skin and/or peripheral blood mononuclear cells express ifn-2 mrna . in order to understand the role of ifn-2 , we examined the cell origins of ifn-2 in the present study . the aim of the study is to investigate the expression of ifn-2 in peripheral blood of allergic airway disorders , its correlation with cytokines and tryptase , and its potential cell location . we found that the levels of ifn-2 were elevated in the plasma of ar and ar + as and that several cell types express ifn-2 . trypsin , leupeptin , collagenase ( type i ) , hyaluronidase ( type i ) , rabbit anti - human ifn-2 antibody , and bovine serum albumin ( bsa , fraction v ) were purchased from sigma aldrich ( st . louis , mo , usa ) . the sequences of the active and reverse peptides were par-2 , trans - cinnamoyl - leu - ile - gly - arg - leu - orn - amide ( tc - ligrlo - nh2 ) and trans - cinnamoyl - orn - leu - arg - gly - ile - leu - amide ( tc - olrgil - nh2 ) , sligkv - nh2 , and vkgils - nh2 ; par-2 antagonist peptide phe - ser - leu - leu - arg - asn - nh2 ( fsllrn - nh2 ) was synthesized in cl bio - scientific inc . dulbecco 's modified eagle 's medium ( dmem ) and fetal calf serum ( fcs ) were obtained from hyclone ( logan , ut , usa ) . human ifn-2 , il-4 , il-10 , and il-12 elisa kits were purchased from r&d systems ( minneapolis , mn ) . foxp3 fix / perm buffer set , rbc lysis buffer ( 10x ) , fitc - anti - human cd123 , percp - anti - human cd16 , percp - anti - human hla - dr , percp / cy5.5-anti - human cd25 , percp / cy5.5-anti - human il-17a , pe / cy7-anti - human cd8 , pe / cy7-anti - human cd14 , and pe / cy7 conjugated rat anti - human il-4 antibodies were purchased from biolegend ( san diego , ca , usa ) . fixation / permeabilization solution kit , fitc - anti - human cd4 , apc - anti - human cd19 , apc - anti - human ifn- , alexa fluor 647-anti - human foxp3 , and pe conjugated rat anti - mouse igm antibodies were purchased from bd pharmingen ( san jose , ca , usa ) . fitc or pe conjugated goat anti - rabbit igg antibody was purchased from santa cruz biotec ( santa cruz , ca , usa ) . biotinylated rabbit anti - human ifn-2 was purchased from bioss ( beijing , china ) . dab + substrate chromogen system and extravidin - peroxidase conjugate were purchased from chemicon international inc . recombinant human lung -tryptase was obtained from promega ( madison , wi , usa ) . exscript rt reagent kit and sybr premix ex taq ( perfect real time ) were obtained from takara biotechnology co. , ltd . oligonucleotide primers for real - time pcr were synthesized by invitrogen biotechnology co. ( shanghai , china ) . most of the general chemicals such as salts and buffer components were of analytical grade . a total of 33 allergic rhinitis ( ar ) , 26 asthma , 12 combined rhinitis with asthma ( ar + as ) , and 20 healthy control subjects ( hc ) were recruited in the study . the diagnosing criterion of asthma was conformed to the global initiative for asthma , and diagnosis for allergic rhinitis was based on allergic rhinitis and its impact on asthma ( aria ) . all patients were asked to stop antiallergy medication for at least 2 weeks prior to attending the study ( those who could not stop antiallergy drugs were excluded ) . the informed consent from each volunteer according to the declaration of helsinki and agreement with the ethical committee of the first affiliated hospital of liaoning medical university and general hospital of shenyang military area command were obtained . the general characteristics of the patients and control subjects were summarized in table 1 . peripheral venous blood sample ( 10 ml ) was collected from each patient or hc and was immediately processed to collect cells and plasma for analysis . specimens of human tissues for immunohistochemistry and flow cytometry analysis were collected from the department of pathology , the first affiliated hospital of liaoning medical university . the protocol for ethical use of human tissue in research was according to the declaration of helsinki ( 2000 ) and approved by the committees of the first affiliated hospital of liaoning medical university . to detect ifn-2 expression on leukocytes excluding t cells , the following antibodies were added to different testing tubes : ( 1 ) to detect ifn-2 expression in basophils : fitc - anti - human cd123 and percp - anti - human hla - dr ; ( 2 ) to detect ifn-2 expression in cd16 + polynucleated cells , cd16 polynucleated cells , and cd14 + cells and cd19 + cells : percp - anti - human cd16 , pe / cy7-anti - human cd14 , and apc - anti - human cd19 before 200 l of whole blood being added at room temperature for 15 min in dark . following ligation of red blood cells , white blood cells were fixed and permeabilized by using cytofix / cytoperm fixation / permeabilization kit according to the manufacturer 's instructions . following washing with bd washing buffer , the cell pellets were resuspended and rabbit anti - human ifn-2 followed by pe or fitc conjugated goat anti - rabbit igg antibodies were added at 4c for 30 min . finally , cells were resuspended in fluorescence - activated cell sorting- ( facs- ) flow solution and analyzed with facsverse flow cytometer ( bd biosciences , san jose , ca ) . data were analyzed with cellquest software ( bd immunocytometry systems ) . for detection of ifn-2 expression in t cells , peripheral blood mononucleated cells ( pbmc ) were isolated by using lymphoprep according to the manufacturer 's instruction . the following antibodies were then added to different testing tubes : ( 1 ) fitc - anti - human cd4 , percp / cy5.5-anti - human cd25 , pe / cy7-anti - human cd8 , and rabbit anti - human ifn-2 followed by alexa fluor 647-anti - human foxp3 and pe conjugated goat anti - rabbit igg antibodies to detect cd8 + t cells and regulatory t cells ( treg ) ; ( 2 ) fitc - anti - human cd4 , apc - anti - human ifn- , pe / cy7 conjugated rat anti - human il-4 , percp / cy5.5-anti - human il-17a , and rabbit anti - human ifn-2 followed by pe conjugated goat anti - rabbit igg antibodies to detect th1 , th2 , and th17 cells . cells were then incubated with each labeled monoclonal antibody including ( 1 ) pe / cy7 conjugated mouse anti - human tryptase , anti - human chymase antibody cc4 ( igm subtype ) , pe conjugated rat anti - mouse igm , rabbit anti - human ifn-2 , and fitc conjugated goat anti - rabbit igg antibodies to detect mast cells ; ( 2 ) pe / cy7-anti - human cd14 , apc - anti - human cd19 , rabbit anti - human ifn-2 , and fitc conjugated goat anti - rabbit igg antibodies to detect macrophages and b cells at 4c for 30 min in dark . after washing , the cell pellets were resuspended in facs - flow solution and analyzed with facsverse flow cytometer . tissues were fixed in carnoy 's fixative , dehydrated , and embedded in paraffin wax . sections ( 4 m ) were dewaxed , rehydrated , and incubated for 10 min with 0.5% h2o2 in methanol followed by 0.1% sodium azide for 10 min in order to inhibit endogenous peroxidase activity . pbs containing 5% bsa was added for 1 h and the same solution was employed as the diluent for the antibodies added subsequently . sequential sections of tonsil , lung , or nasal polyps were incubated with biotinylated rabbit anti - human ifn-2 for 2 h. after washing with pbst , extravidin - peroxidase conjugate was applied to sections for 1 h. staining was developed over 4 min by using dab chromogen system before being counterstained with mayer 's haematoxylin and mounted in aquamount . for each section , the number of positively stained cells was counted in at least 30 fields ( the area of each field equals 0.19 mm ) . the human lung carcinoma cell line a549 ( morphology : epithelial ) was obtained from the american type culture collection ( manassas , va , usa ) . cells were grown in dulbecco 's modified eagle 's medium ( dmem ) , supplemented with 10% ( v / v ) fetal calf serum ( fcs ) , 100 u / ml penicillin , and 100 g / ml streptomycin . cells were cultured in 75 cm tissue culture flasks ( falcon ) at 37c in a 5% ( v / v ) co2 , water - saturated atmosphere . for challenge experiments , cells were detached from culture flasks using trypsin , seeded into 12-well cell culture plates , and grown to about 80% confluence . the cells were then cultured with the serum - free basal medium for an additional 16 h before challenge . cells were exposed to tryptase ( 2 g / ml , 1 g / ml = 7.4 nm ) with or without its inhibitor leupeptin ( 3 g / ml ) , 100 m of sligkv - nh2 with or without par-2 antagonist fsllrn - nh2 ( 400 m ) and its reverse peptide vkgils - nh2 , and 100 m of tc - ligrlo - nh2 with or without par-2 antagonist fsllrn - nh2 ( 400 m ) and its reverse peptide tc - olrgil - nh2 , respectively . cells ( 1.5 10 per well ) were collected at 2 h or 6 h , centrifuged at 4c , and stored at 80c until use . the expression of ifn-2 mrna in a549 cells was determined by qpcr following the manufacture 's protocol . briefly , after synthesizing cdna from total rna by using superscript first strand synthesis system for rt - pcr and oligo - dt primers , real - time pcr was performed by using sybr premix ex taq kit on the abi prism 7700 sequence detection system ( perkin elmer applied systems , foster city , ca , usa ) . sequence - specific standard curves were generated using 10-fold serial dilutions of plasmid dna , and the values for the initial concentrations of unknown samples were calculated by using the software ( version 1.7 ) provided with the abi 7700 system . the primers for ifn-2 were forward : 5-caccctgcaccatatcctct-3 , reverse : 5-ggagggtcagacacacaggt-3 and for -actin were forward : 5-agagctacgagctgcctgac-3 , reverse : 5-agcactgtgttggcgtacag-3. levels of tryptase , il-4 , il-10 , il-12 , and ifn-2 in the plasma of ar , asthma , ar + as , and hc were measured by using elisa kits according to the manufacturer 's instructions . data were expressed as mean sem . where analysis of variance indicated significant differences between groups with anova , student 's t - test was applied . data for allergic patients are presented as scatter plot . where kruskal - wallis analysis indicated significant differences between groups , for the preplanned comparisons of interest , the paired mann - whitney u test was employed . in order to evaluate the potential role of ifn-2 in allergic airway disorders , the most direct evidence is to examine the changes of its levels in clinical specimen . we therefore examined the levels of ifn-2 in the plasma and its cellular location in blood of the patients with ar and asthma . the results showed that the levels of ifn-2 were elevated by 17.9% and 14.2% in the plasma of ar and combined rhinitis with asthma ( ar + as ) , but not of asthma ( figure 1(a ) ) . the plasma levels of tryptase were increased by 34.7% and 38.3% in the patients with ar and asthma , but not ar + as ( figure 1(b ) ) . the plasma levels of il-4 were increased by 21.1% in the patients with asthma but decreased by 55.3% and 26.3% in ar and ar + as ( figure 1(c ) ) . the plasma levels of il-10 ( figure 1(d ) ) and il-12 ( figure 1(e ) ) were decreased by 29.8% and 100% in the patients with ar , by 54.3% and 100% in the patients with asthma , and by 100% and 100% in the patients with ar + as , respectively . there were positive correlation between ifn-2 and tryptase and negative correlation between ifn-2 and il-10 in the plasma of ar . similarly , plasma ifn-2 positively correlates with tryptase , and il-10 positively correlates with il-12 in asthma ( table 2 ) . in order to identify the potential sources of ifn-2 the results showed that ifn-2 was predominately expressed in the cd16 + ( representing neutrophils ) ( figure 2(a)(f ) ) and cd14 + cells ( representing monocytes ) ( figure 2(a)(e ) ) and weakly expressed in cd19 + ( representing b cells ) ( figure 2(a)(a ) ) , cd8 + cells ( representing cytotoxic t cells ) ( figure 2(a)(b ) ) , and basophils ( figure 2(a)(g ) ) . cd4 + t cells ( figure 2(a)(d ) ) and cd16 polynucleated cells ( representing eosinophils ) ( figure 2(a)(c ) ) seemed not to express ifn-2 in hc ( figure 2(b ) ) . however ifn-2 expression was upregulated by 43.5% and 49.1% in ar , by 125% and 42.3% in asthma , and by 99% and 72.8% in ar + as in cytotoxic t cells and eosinophils but downregulated by 57% and 76.3% in ar , by 86.4% and 81.6% in asthma , and by 58.1% and 37.2% in ar + as in monocytes and neutrophils , respectively ( figure 2(b ) ) . in order to further investigate the potential source of ifn-2 , we examined the expression of ifn-2 in cells of various tissue origins by using immunohistochemical staining technique . the results showed that ifn-2 clearly expresses in glandular epithelial cells and some large cells ( most likely mast cells or macrophages ) in tonsillar tissue ( figure 3(b ) ) and in some large cells in lung tissue ( figure 3(d ) ) and nasal polyps ( figure 3(f ) ) as compared with the negative control tissues ( figures 3(a ) , 3(c ) , and 3(f ) ) . to confirm the immunohistochemical staining observations , we examined ifn-2 expression in dispersed human tonsil and lung mast cells , b cells , and macrophages by flow cytometry analysis . the results showed that approximately 2.1% , 4.5% , and 7.0% dispersed tonsil cells are ifn-2 + mct mast cells , mctc mast cells , and macrophages . however , 2.5% , 3.3% , 0.44% , and 0.14% dispersed cells are ifn-2 + mct mast cells , mctc mast cells , macrophages , and b cells ( figure 4 ) . positive correlation of ifn-2 with tryptase implicated that the increased level of ifn-2 in the plasma of patients with ar and ar + as may be elicited by mast cell tryptase . to confirm this anticipation , we examined the effect of tryptase and agonist peptides of par-2 on ifn-2 mrna expression in a549 cells . it was found that the expression of ifn-2 mrna over baseline control was increased by approximately 1.4- and 0.5-fold when the cells were incubated with tryptase at 2 g / ml for 2 and 6 h ( figure 5 ) . similarly , sligkv - nh2 and tc - ligrlo - nh2 induced approximately 1.4- and 0.9-fold increase in expression of ifn-2 mrna over baseline control , respectively , when they were incubated with a549 cells for 2 h ( figure 5 ) . at 6 h following incubation with sligkv - nh2 and tc - ligrlo - nh2 , the expression of ifn-2 mrna was enhanced by approximately 0.6- and 1.0-fold , respectively ( figure 5 ) . the reverse peptides vkgils - nh2 and tc - olrgil - nh2 showed little effect on the expression of ifn-2 mrna in a549 cells following 2 and 6 h incubation periods ( figure 5 ) . since fsllrn - nh2 and leupeptin were able to inhibit tryptase induced upregulation of expression of ifn-2 mrna and fsllrn - nh2 suppressed sligkv - nh2 and tc - ligrlo - nh2 induced upregulation of ifn-2 mrna expression ( figure 5 ) , the action of tryptase is likely to be mediated by par-2 and requires its enzymatic activity . we have demonstrated for the first time that the levels of ifn-2 are elevated in plasma of the patients with ar and ar + as , but not with asthma , which provides the first hard evidence for proving that ifn-2 may participate in adoptive immune response such as allergic airway reactions . the recent reports that the expression level of ifn-2 mrna was significantly increased during naturally occurring respiratory viral infections in children with asthma and that ifn-2 was capable of exacerbating a t - cell - mediated autoimmune disease may support our observation . it is difficult to evaluate the role of ifn-2 in allergic airway inflammation at this stage as we do not know if the increased serum level of ifn-2 is a causative or resulting factor in the pathogenesis of the allergic airway disorders . our observation that elevated ifn-2 levels were positively correlated to tryptase level in the plasma of ar suggests that these two compounds are likely released from the same source . since tryptase is a relatively selective marker of mast cell degranulation and the most abundant secretory product from mast cells , it is likely that ifn-2 is also released from mast cells upon degranulation . indeed , we have demonstrated in the present study that large numbers of tonsil and lung mct and mctc subtypes of mast cells express ifn-2 , confirming that mast cells are the major source of ifn-2 . our previous report that ifn-1 ( il-29 ) highly expressed in mast cells may support our current observation . however , unlike tryptase acting as a potent proinflammatory mediator which is capable of provoking microvascular leakage in the skin of guinea pigs , stimulating the release of histamine from dispersed human tonsil mast cells , and inducing accumulation of eosinophils and neutrophil in the peritoneum of mice , ifn-2 appears to act as a suppressor of allergic airway diseases . for example , ifn-2 can modulate lung dc function to promote th1 immune skewing and suppress allergic airway disease . since the information on the role of ifn-2 in allergy is very limited , the study that treatment with ifn-2 completely halts and reverses the development of collagen - induced arthritis , dramatically reduces numbers of proinflammatory il-17-producing th17 and t cells in the joints and inguinal lymph nodes , and restricts recruitment of il-1b - expressing neutrophils may support the anticipation that ifn-2 may play an inhibitory role in allergic airway diseases . since a large population of macrophages express ifn-2 , it is likely one of major sources of ifn-2 , considering huge numbers of macrophages in lung and tonsil . epithelial cells could be another source of ifn-2 as tonsil glandular epithelial cells express ifn-2 , and a549 cells express ifn-2 mrna . our observation that tryptase induced upregulation of expression of ifn-2 mrna in a549 cells is mediated by par-2 and requires tryptase enzymatic activity implicates that tryptase may provoke ifn-2 production in lung epithelial cells through activation of par-2 , and released ifn-2 could contribute to the elevated plasma level of ifn-2 in allergic airway disorders . is known of the relationship between pars and ifn-s , our previous report that the actions of thrombin on a549 cells are most likely carried out through hydrolytic cleavage of n - terminal of par-1 may help to understand our observation above . we have also observed the declined plasma levels of il-10 and il-12 in the allergic patients . since the correlation between il-12 and il-10 levels in serum has been reported in the patients with atopic dermatitis , and diminished il-12 levels were previously found in the serum of allergic patients , our observation may further suggest that reduced il-10 and il-12 production may contribute to the pathogenesis of the airway allergic disorders . the negative correlation between ifn-2 and il-10 in the plasma of ar suggested they are not likely to be released from same sources , which means that if mast cells are major source of ifn-2 , they should not be the major source for il-10 in ar . in order to identify the potential source of increased ifn-2 our data showed that ifn-2 expression was downregulated in ar , in asthma , and in ar + as in monocytes and neutrophils . since neutrophils and monocytes are predominant ifn-2-expressing cells in blood of hc , the decreased expression of ifn-2 in these 2 cell types could contribute to diminished level of ifn-2 in the plasma of asthma , even though ifn-2 expression appeared to be upregulated in blood cytotoxic t cells and eosinophils in asthma as cytotoxic t cells only weakly express and eosinophils do not express ifn-2 in hc . downregulation of expression of ifn-2 in peripheral blood monocytes and neutrophils of ar and ar + as seemed to conflict with the observation of increased level of ifn-2 in the plasma of ar and ar + as , which suggests that there must be some other sources to generate large amount of ifn-2 apart from blood leukocytes . moreover since helper t cells including regulatory t cells do not express ifn-2 , they are one of the major sources of il-10 , which may at least partially explain the negative correlation between ifn-2 and il-10 in the plasma of ar . in conclusion , the elevated levels of ifn-2 in the plasma of ar and ar + as and positive correlations of plasma ifn-2 with tryptase in ar and asthma indicate that ifn-2 is likely to contribute to the pathogenesis of allergic airway disorders . mast cells , macrophages , and epithelial cells in human tonsil and lung tissues express ifn-2 , and upregulated ifn-2 expression was observed in cd8 + t cells and eosinophils of allergic airway disorders indicate that they are the potential sources of ifn-2 .

this study investigated the expression levels of interferon- ( ifn- ) 2 in peripheral blood and tissues . the results showed that the levels of ifn-2 were elevated by 17.9% and 14.2% in the plasma of allergic rhinitis ( ar ) and combined rhinitis with asthma ( ar + as ) , which was positively correlated with the level of tryptase but negatively correlated with the level of il-10 . ifn-2 was predominately expressed in the cd16 + cells and cd14 + cells in healthy control subjects ( hc ) but upregulated only in cd8 + cells of ar and in eosinophils of asthma . it was observed that approximately 6.6% and 7.0% dispersed tonsil cells and 5.8% and 0.44% dispersed lung cells are ifn-2 + mast cells and macrophages . moreover , tryptase and agonist peptides of par-2 induced enhanced ifn-2 mrna expression in a549 cells . in conclusion , the elevated levels of ifn-2 in the plasma of ar and ar + as indicate that ifn-2 is likely to contribute to the pathogenesis of allergic airway disorders . the potential origins of the elevated plasma ifn-2 include mast cells , macrophages , and epithelial cells in tissues , neutrophils , monocytes , cd8 + t cells , and eosinophils in peripheral blood . development of ifn-2 related therapy may help to treat or prevent allergic airway disorders .

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

it has also been discovered that ifn-2 is capable of exacerbating t - cell - mediated autoimmune diseases such as uveitis . treatment with ifn-2 completely halts and reverses the development of collagen - induced arthritis , dramatically reduces the numbers of proinflammatory il-17-producing th17 and t cells in the joints and inguinal lymph nodes , and restricts recruitment of il-1b - expressing neutrophils . recently , it was found that the expression level of ifn-2 mrna was significantly increased during naturally occurring respiratory viral infections in children with asthma and that ifn-2 modulates lung dendritic cells ( dc ) function to promote th1 immune skewing and suppresses allergic airway disease . these suggest that ifn-2 is not only involved in autoimmune diseases but also associated with allergic airway disorders . we therefore investigated the potential involvement of ifn-2 in allergic airway diseases in the present study . it was found that ifn-2 expressed in tracheobronchial tissue cells from the patients with copd . dc express moderate quantity of ifn-2 when using lipopolysaccharide ( lps ) as the maturation stimulus , and vitiligo patient skin and/or peripheral blood mononuclear cells express ifn-2 mrna . in order to understand the role of ifn-2 , we examined the cell origins of ifn-2 in the present study . the aim of the study is to investigate the expression of ifn-2 in peripheral blood of allergic airway disorders , its correlation with cytokines and tryptase , and its potential cell location . we found that the levels of ifn-2 were elevated in the plasma of ar and ar + as and that several cell types express ifn-2 . the sequences of the active and reverse peptides were par-2 , trans - cinnamoyl - leu - ile - gly - arg - leu - orn - amide ( tc - ligrlo - nh2 ) and trans - cinnamoyl - orn - leu - arg - gly - ile - leu - amide ( tc - olrgil - nh2 ) , sligkv - nh2 , and vkgils - nh2 ; par-2 antagonist peptide phe - ser - leu - leu - arg - asn - nh2 ( fsllrn - nh2 ) was synthesized in cl bio - scientific inc . foxp3 fix / perm buffer set , rbc lysis buffer ( 10x ) , fitc - anti - human cd123 , percp - anti - human cd16 , percp - anti - human hla - dr , percp / cy5.5-anti - human cd25 , percp / cy5.5-anti - human il-17a , pe / cy7-anti - human cd8 , pe / cy7-anti - human cd14 , and pe / cy7 conjugated rat anti - human il-4 antibodies were purchased from biolegend ( san diego , ca , usa ) . a total of 33 allergic rhinitis ( ar ) , 26 asthma , 12 combined rhinitis with asthma ( ar + as ) , and 20 healthy control subjects ( hc ) were recruited in the study . the diagnosing criterion of asthma was conformed to the global initiative for asthma , and diagnosis for allergic rhinitis was based on allergic rhinitis and its impact on asthma ( aria ) . the informed consent from each volunteer according to the declaration of helsinki and agreement with the ethical committee of the first affiliated hospital of liaoning medical university and general hospital of shenyang military area command were obtained . the protocol for ethical use of human tissue in research was according to the declaration of helsinki ( 2000 ) and approved by the committees of the first affiliated hospital of liaoning medical university . to detect ifn-2 expression on leukocytes excluding t cells , the following antibodies were added to different testing tubes : ( 1 ) to detect ifn-2 expression in basophils : fitc - anti - human cd123 and percp - anti - human hla - dr ; ( 2 ) to detect ifn-2 expression in cd16 + polynucleated cells , cd16 polynucleated cells , and cd14 + cells and cd19 + cells : percp - anti - human cd16 , pe / cy7-anti - human cd14 , and apc - anti - human cd19 before 200 l of whole blood being added at room temperature for 15 min in dark . for detection of ifn-2 expression in t cells , peripheral blood mononucleated cells ( pbmc ) were isolated by using lymphoprep according to the manufacturer 's instruction . the following antibodies were then added to different testing tubes : ( 1 ) fitc - anti - human cd4 , percp / cy5.5-anti - human cd25 , pe / cy7-anti - human cd8 , and rabbit anti - human ifn-2 followed by alexa fluor 647-anti - human foxp3 and pe conjugated goat anti - rabbit igg antibodies to detect cd8 + t cells and regulatory t cells ( treg ) ; ( 2 ) fitc - anti - human cd4 , apc - anti - human ifn- , pe / cy7 conjugated rat anti - human il-4 , percp / cy5.5-anti - human il-17a , and rabbit anti - human ifn-2 followed by pe conjugated goat anti - rabbit igg antibodies to detect th1 , th2 , and th17 cells . cells were then incubated with each labeled monoclonal antibody including ( 1 ) pe / cy7 conjugated mouse anti - human tryptase , anti - human chymase antibody cc4 ( igm subtype ) , pe conjugated rat anti - mouse igm , rabbit anti - human ifn-2 , and fitc conjugated goat anti - rabbit igg antibodies to detect mast cells ; ( 2 ) pe / cy7-anti - human cd14 , apc - anti - human cd19 , rabbit anti - human ifn-2 , and fitc conjugated goat anti - rabbit igg antibodies to detect macrophages and b cells at 4c for 30 min in dark . cells were grown in dulbecco 's modified eagle 's medium ( dmem ) , supplemented with 10% ( v / v ) fetal calf serum ( fcs ) , 100 u / ml penicillin , and 100 g / ml streptomycin . cells were exposed to tryptase ( 2 g / ml , 1 g / ml = 7.4 nm ) with or without its inhibitor leupeptin ( 3 g / ml ) , 100 m of sligkv - nh2 with or without par-2 antagonist fsllrn - nh2 ( 400 m ) and its reverse peptide vkgils - nh2 , and 100 m of tc - ligrlo - nh2 with or without par-2 antagonist fsllrn - nh2 ( 400 m ) and its reverse peptide tc - olrgil - nh2 , respectively . the expression of ifn-2 mrna in a549 cells was determined by qpcr following the manufacture 's protocol . levels of tryptase , il-4 , il-10 , il-12 , and ifn-2 in the plasma of ar , asthma , ar + as , and hc were measured by using elisa kits according to the manufacturer 's instructions . in order to evaluate the potential role of ifn-2 in allergic airway disorders , the most direct evidence is to examine the changes of its levels in clinical specimen . we therefore examined the levels of ifn-2 in the plasma and its cellular location in blood of the patients with ar and asthma . the results showed that the levels of ifn-2 were elevated by 17.9% and 14.2% in the plasma of ar and combined rhinitis with asthma ( ar + as ) , but not of asthma ( figure 1(a ) ) . the plasma levels of tryptase were increased by 34.7% and 38.3% in the patients with ar and asthma , but not ar + as ( figure 1(b ) ) . the plasma levels of il-4 were increased by 21.1% in the patients with asthma but decreased by 55.3% and 26.3% in ar and ar + as ( figure 1(c ) ) . the plasma levels of il-10 ( figure 1(d ) ) and il-12 ( figure 1(e ) ) were decreased by 29.8% and 100% in the patients with ar , by 54.3% and 100% in the patients with asthma , and by 100% and 100% in the patients with ar + as , respectively . there were positive correlation between ifn-2 and tryptase and negative correlation between ifn-2 and il-10 in the plasma of ar . similarly , plasma ifn-2 positively correlates with tryptase , and il-10 positively correlates with il-12 in asthma ( table 2 ) . in order to identify the potential sources of ifn-2 the results showed that ifn-2 was predominately expressed in the cd16 + ( representing neutrophils ) ( figure 2(a)(f ) ) and cd14 + cells ( representing monocytes ) ( figure 2(a)(e ) ) and weakly expressed in cd19 + ( representing b cells ) ( figure 2(a)(a ) ) , cd8 + cells ( representing cytotoxic t cells ) ( figure 2(a)(b ) ) , and basophils ( figure 2(a)(g ) ) . cd4 + t cells ( figure 2(a)(d ) ) and cd16 polynucleated cells ( representing eosinophils ) ( figure 2(a)(c ) ) seemed not to express ifn-2 in hc ( figure 2(b ) ) . however ifn-2 expression was upregulated by 43.5% and 49.1% in ar , by 125% and 42.3% in asthma , and by 99% and 72.8% in ar + as in cytotoxic t cells and eosinophils but downregulated by 57% and 76.3% in ar , by 86.4% and 81.6% in asthma , and by 58.1% and 37.2% in ar + as in monocytes and neutrophils , respectively ( figure 2(b ) ) . in order to further investigate the potential source of ifn-2 , we examined the expression of ifn-2 in cells of various tissue origins by using immunohistochemical staining technique . the results showed that ifn-2 clearly expresses in glandular epithelial cells and some large cells ( most likely mast cells or macrophages ) in tonsillar tissue ( figure 3(b ) ) and in some large cells in lung tissue ( figure 3(d ) ) and nasal polyps ( figure 3(f ) ) as compared with the negative control tissues ( figures 3(a ) , 3(c ) , and 3(f ) ) . to confirm the immunohistochemical staining observations , we examined ifn-2 expression in dispersed human tonsil and lung mast cells , b cells , and macrophages by flow cytometry analysis . the results showed that approximately 2.1% , 4.5% , and 7.0% dispersed tonsil cells are ifn-2 + mct mast cells , mctc mast cells , and macrophages . however , 2.5% , 3.3% , 0.44% , and 0.14% dispersed cells are ifn-2 + mct mast cells , mctc mast cells , macrophages , and b cells ( figure 4 ) . positive correlation of ifn-2 with tryptase implicated that the increased level of ifn-2 in the plasma of patients with ar and ar + as may be elicited by mast cell tryptase . to confirm this anticipation , we examined the effect of tryptase and agonist peptides of par-2 on ifn-2 mrna expression in a549 cells . it was found that the expression of ifn-2 mrna over baseline control was increased by approximately 1.4- and 0.5-fold when the cells were incubated with tryptase at 2 g / ml for 2 and 6 h ( figure 5 ) . similarly , sligkv - nh2 and tc - ligrlo - nh2 induced approximately 1.4- and 0.9-fold increase in expression of ifn-2 mrna over baseline control , respectively , when they were incubated with a549 cells for 2 h ( figure 5 ) . at 6 h following incubation with sligkv - nh2 and tc - ligrlo - nh2 , the expression of ifn-2 mrna was enhanced by approximately 0.6- and 1.0-fold , respectively ( figure 5 ) . the reverse peptides vkgils - nh2 and tc - olrgil - nh2 showed little effect on the expression of ifn-2 mrna in a549 cells following 2 and 6 h incubation periods ( figure 5 ) . since fsllrn - nh2 and leupeptin were able to inhibit tryptase induced upregulation of expression of ifn-2 mrna and fsllrn - nh2 suppressed sligkv - nh2 and tc - ligrlo - nh2 induced upregulation of ifn-2 mrna expression ( figure 5 ) , the action of tryptase is likely to be mediated by par-2 and requires its enzymatic activity . we have demonstrated for the first time that the levels of ifn-2 are elevated in plasma of the patients with ar and ar + as , but not with asthma , which provides the first hard evidence for proving that ifn-2 may participate in adoptive immune response such as allergic airway reactions . the recent reports that the expression level of ifn-2 mrna was significantly increased during naturally occurring respiratory viral infections in children with asthma and that ifn-2 was capable of exacerbating a t - cell - mediated autoimmune disease may support our observation . it is difficult to evaluate the role of ifn-2 in allergic airway inflammation at this stage as we do not know if the increased serum level of ifn-2 is a causative or resulting factor in the pathogenesis of the allergic airway disorders . our observation that elevated ifn-2 levels were positively correlated to tryptase level in the plasma of ar suggests that these two compounds are likely released from the same source . since tryptase is a relatively selective marker of mast cell degranulation and the most abundant secretory product from mast cells , it is likely that ifn-2 is also released from mast cells upon degranulation . indeed , we have demonstrated in the present study that large numbers of tonsil and lung mct and mctc subtypes of mast cells express ifn-2 , confirming that mast cells are the major source of ifn-2 . however , unlike tryptase acting as a potent proinflammatory mediator which is capable of provoking microvascular leakage in the skin of guinea pigs , stimulating the release of histamine from dispersed human tonsil mast cells , and inducing accumulation of eosinophils and neutrophil in the peritoneum of mice , ifn-2 appears to act as a suppressor of allergic airway diseases . since the information on the role of ifn-2 in allergy is very limited , the study that treatment with ifn-2 completely halts and reverses the development of collagen - induced arthritis , dramatically reduces numbers of proinflammatory il-17-producing th17 and t cells in the joints and inguinal lymph nodes , and restricts recruitment of il-1b - expressing neutrophils may support the anticipation that ifn-2 may play an inhibitory role in allergic airway diseases . epithelial cells could be another source of ifn-2 as tonsil glandular epithelial cells express ifn-2 , and a549 cells express ifn-2 mrna . our observation that tryptase induced upregulation of expression of ifn-2 mrna in a549 cells is mediated by par-2 and requires tryptase enzymatic activity implicates that tryptase may provoke ifn-2 production in lung epithelial cells through activation of par-2 , and released ifn-2 could contribute to the elevated plasma level of ifn-2 in allergic airway disorders . is known of the relationship between pars and ifn-s , our previous report that the actions of thrombin on a549 cells are most likely carried out through hydrolytic cleavage of n - terminal of par-1 may help to understand our observation above . we have also observed the declined plasma levels of il-10 and il-12 in the allergic patients . since the correlation between il-12 and il-10 levels in serum has been reported in the patients with atopic dermatitis , and diminished il-12 levels were previously found in the serum of allergic patients , our observation may further suggest that reduced il-10 and il-12 production may contribute to the pathogenesis of the airway allergic disorders . the negative correlation between ifn-2 and il-10 in the plasma of ar suggested they are not likely to be released from same sources , which means that if mast cells are major source of ifn-2 , they should not be the major source for il-10 in ar . in order to identify the potential source of increased ifn-2 our data showed that ifn-2 expression was downregulated in ar , in asthma , and in ar + as in monocytes and neutrophils . since neutrophils and monocytes are predominant ifn-2-expressing cells in blood of hc , the decreased expression of ifn-2 in these 2 cell types could contribute to diminished level of ifn-2 in the plasma of asthma , even though ifn-2 expression appeared to be upregulated in blood cytotoxic t cells and eosinophils in asthma as cytotoxic t cells only weakly express and eosinophils do not express ifn-2 in hc . downregulation of expression of ifn-2 in peripheral blood monocytes and neutrophils of ar and ar + as seemed to conflict with the observation of increased level of ifn-2 in the plasma of ar and ar + as , which suggests that there must be some other sources to generate large amount of ifn-2 apart from blood leukocytes . moreover since helper t cells including regulatory t cells do not express ifn-2 , they are one of the major sources of il-10 , which may at least partially explain the negative correlation between ifn-2 and il-10 in the plasma of ar . in conclusion , the elevated levels of ifn-2 in the plasma of ar and ar + as and positive correlations of plasma ifn-2 with tryptase in ar and asthma indicate that ifn-2 is likely to contribute to the pathogenesis of allergic airway disorders . mast cells , macrophages , and epithelial cells in human tonsil and lung tissues express ifn-2 , and upregulated ifn-2 expression was observed in cd8 + t cells and eosinophils of allergic airway disorders indicate that they are the potential sources of ifn-2 .

a common and serious complication of type 1 diabetes mellitus ( t1 dm ) is diabetic autonomic neuropathy [ 1 , 2 ] . cardiovascular autonomic neuropathy ( can ) is a subset of diabetic autonomic neuropathy characterized by impaired autonomic control of the cardiovascular ( cv ) system . for instance , can has been reported to increase the mortality of diabetic patients by a factor of 3.45 . clinically , the most common methods for assessing can are heart rate variability ( hrv ) analysis and baroreflex sensitivity ( brs ) [ 3 , 5 , 6 ] . in t1 dm , aspects of the baroreflex arc can be impaired , such that both baroreceptor activity and excitability are blunted [ 8 , 9 ] and the aortic depressor nerves undergo axonal atrophy . as well , autonomic efferents , primarily of the parasympathetic nervous system ( psns ) , have decreased activity , reduced responsiveness , and decreased neurochemical activity in the heart [ 10 , 11 ] . impairment of central nervous system regions has also been reported as the limiting factor of brs [ 12 , 13 ] . reduced heart rate variability ( hrv ) is often the earliest symptom of can . whether measured by time domain analysis or by frequency domain analysis and whether in clinical or experimental t1 dm , hrv is consistently reported to be reduced in t1 dm [ 5 , 1417 ] . exercise has been demonstrated to be an effective means of improving deficits in hrv and brs in both clinical and experimental t1 dm [ 1821 ] . such improvements have been attributed to improved insulin sensitivity , increased endogenous antioxidant and anti - inflammatory mediators , and improved autonomic control of the cv system [ 2224 ] . despite similar reductions in hrv and brs , there are marked differences in early - stage changes to other cv parameters between clinical and experimental t1 dm . specifically , in clinical t1 dm , increases in heart rate ( hr ) and blood pressure ( bp ) are commonly reported in early autonomic neuropathy [ 1 , 3 , 14 , 24 , 2628 ] . in contrast , experimental stz - induced t1 dm is regularly associated with decreased bp and hr , beginning shortly after diabetes induction [ 15 , 16 , 2931 ] . due to these opposing initial changes in bp and hr , exercise training is often observed to produce contrasting outcomes on cv parameters in experimental and clinical t1 dm , namely , increased bp and hr in experimental t1 dm and decreased bp and hr in clinical t1 dm [ 19 , 25 , 3234 ] . as a result , both the increase and decrease of these cv factors are concurrently cited as exercise - mediated improvements to can with little consideration of the fact that the changes are opposed between these two contexts of t1 dm . this is important because if animal models do not accurately reproduce t1 dm pathology , then the outcomes of experimental studies may not translate to the treatment of human can , as the mechanisms underlying the pathology and exercise modifications may differ . another important difference between experimental and clinical t1 dm is the common omission of insulin treatment in experimental diabetes leading to severe hyperglycemia ranging from roughly 17 to 25 mm blood glucose concentrations ( [ bg ] ) [ 15 , 16 , 29 , 30 ] . as the severity and duration of hyperglycemia have been shown to influence the degree of diabetic neuropathy , acute and steep elevations of [ bg ] in stz - induced t1 dm may not only cause early onset neuropathy to the psns but also cause acute neuropathy of the sympathetic nervous system ( sns ) and directly affect the sinoatrial ( sa ) node . these changes may mediate the observed reduction in bp and hr that arise acutely in experimental t1 dm and in late - stage clinical t1 dm [ 2 , 3538 ] . indeed , intensive insulin therapy has been shown to restore bp and hr to non - t1 dm levels in stz - induced t1 dm rats [ 25 , 39 ] . yet , despite the use of insulin therapy in clinical t1 dm , it is often the case that chronic , moderate hyperglycemia is maintained as a result of difficulties in regulating [ bg ] in response to dynamic influences on glycemic control , such as food intake and exercise [ 40 , 41 ] . this is often resultant of a tendency to err on the side of moderate hyperglycemia in order to circumvent the acute discomfort and danger associated with hypoglycemic episodes , which occur more frequently with diabetic neuropathy due to the impairment of the glucagon response [ 40 , 42 , 43 ] . to address this , our laboratory established a model of t1 dm using a multiple low - dose stz - treatment and insulin therapy to replicate the moderate hyperglycemia observed in clinical t1 dm . in our previous studies that employed this model , we observed impairments in glucose tolerance , vascular responsiveness , cardiac function , and bone health , which were improved with high intensity aerobic exercise training [ 4447 ] . the purpose of the current study was to investigate whether our model of multiple low - dose stz - induced t1 dm with insulin therapy would induce deficits in cardiovascular autonomic function more representative of clinical t1 dm , and if high intensity aerobic training could prevent those deficits . we hypothesised that ( 1 ) our model of stz - induced t1 dm would elicit indices of can , including a blunted brs ( bradycardia and tachycardia response ) , lowered hrv and intrinsic heart rate , increased vascular sympathetic tone , and increased mean arterial pressure , and ( 2 ) high intensity aerobic exercise training would prevent or ameliorate the indications of can . the protocols used in this investigation were approved by the university of western ontario council on animal care and conformed to the guidelines of the canadian council on animal care . eight - week - old male sprague - dawley rats were obtained from charles river laboratories canada ( saint - constant , quebec ) . the rats were housed in pairs and maintained on a 12-hour dark / light cycle at a constant temperature ( 20 1c ) and relative humidity ( 50% ) . sixty - four rats were randomly assigned to one of four groups as follows : ( 1 ) sedentary control ( c , n = 16 ) ; ( 2 ) exercised control ( cx , n = 16 ) ; ( 3 ) sedentary t1 dm ( d , n = 16 ) ; ( 4 ) exercised t1 dm ( dx , n = 16 ) . all functional and blood endpoint measures were acquired 24 hours after the final exercise bout . upon arrival rats subsequently , t1 dm was induced over five consecutive days by multiple intraperitoneal ( ip ) injections of 20 mg / kg streptozotocin ( stz , sigma - aldrich ) dissolved in a citrate buffer ( 0.1 m , ph 4.5 ) . diabetes was confirmed by blood glucose measurements greater than or equal to 18 mm on two consecutive days . if necessary , subsequent 20 mg / kg stz injections were administered until diabetes was confirmed . following the confirmation of diabetes , insulin pellets ( 1 pellet ; 2 u insulin / day ; linplant , linshin canada , inc . , insulin pellet doses were then monitored for 1 week and adjusted ( 0.5 pellets ) in order to obtain daily nonfasting blood glucose concentrations in the moderate hyperglycemic range of 917 mm . insulin dose was determined by multiplying the total quantity of pellet implanted ( 0.5 pellet increments ) by the amount of insulin released per pellet ( 2 units of insulin / day / pellet ) divided by the body weight ( kg ) of the rat . blood was obtained from the saphenous vein by venous puncture with a 30-gauge needle and measured via freestyle lite blood glucose monitoring system ( abbott diabetes care inc . , intravenous glucose tolerance tests ( ivgtt ) were performed on all animals prior to t1 dm induction ( pre - t1 dm ) and at the end of week 10 of the exercise training period . rats were fasted for approximately 8 to 12 hours prior to the assay and did not perform exercise on the day of their ivgtt . a sterile - filtered dextrose solution ( 50% dextrose , 50% ddh2o ) was injected ( 1 g / kg ) into the lateral tail vein of the conscious rat . following dextrose infusion , blood glucose was measured at 5 minutes , at 10 minutes , and then at 10-minute intervals thereafter until blood glucose levels plateaued . prior to the initiation of the exercise training program , rats were familiarized with the exercise equipment on two consecutive days . the familiarization consisted of two 15-minute sessions of running at progressive treadmill speeds up to 30 meters per minute ( m / min ) . the treadmill was a custom - built apparatus fabricated by the physical plant at university of western ontario and has been used in many previous studies [ 4447 ] . the exercise training program consisted of 1 hour of motor - driven treadmill running per day at 27 m / min with a 6-degree incline , 5 days per week , for 10 weeks . the exercise intensity was determined based on earlier research that investigated oxygen uptake in rats at various treadmill speeds . the chosen intensity was found to represent approximately 7585% vo2max [ 48 , 49 ] . to achieve a surgical plane of anesthesia , rats were placed in an induction chamber circulating 4% isoflurane ( 96% o2 ) . once motor reflexes were undetectable , rats were transferred to a nosecone delivering 3% isoflurane ( 97% o2 ) and placed on a hot water pad ( 37c ) . rats were cannulated with saline - infused polyethylene ( pe90 ) catheters in the right jugular vein and carotid artery and each catheter was attached to a three - way stopcock . the jugular vein catheter was used for drug infusions and the carotid artery catheter was connected in series with a pressure transducer ( px272 , edwards life sciences , irvine , california , usa ) for arterial blood pressure measurements . at the end of the preparative surgery , rats were injected ip with a 25 mg / kg cocktail of urethane ( 16 mg / ml ) and -chloralose ( 100 mg / ml ) , an anesthetic cocktail that has been shown to have the least inhibition of baseline cv control and autonomic function . a total of 10 ml of urethane/-chloralose was made , 5 ml of which was diluted to 50% with ddh2o and was used as needed to maintain anesthesia throughout data collection . isoflurane anesthesia was gradually removed , whereby urethane/-chloralose was the primary anesthesia used during data collection . heart rate ( hr ) , systolic blood pressure ( sbp ) , and mean arterial pressure ( map ) were determined from the blood pressure pulse waveform and were collected while the rats were under urethane/-chloralose anesthesia in the supine position . data were obtained using a powerlab data acquisition system , digitized , and recorded at 1000 hz using the bundled labchart 7 pro software ( adinstruments , colorado springs , co , usa ) . prior to drug infusions , 5 minutes of spontaneous electrocardiogram data was sampled at 1000 hz and analyzed with labchart hrv analysis software ( adinstruments ) . time domain analysis of the standard deviation between normal peak pulses of the pressure pulse waveform ( sdnn ) was quantified as a measure of the total variability of the hr . frequency domain analysis of the high frequency ( hf ) band of the fast fourier transform ( fft ) of the data was assessed as an index of parasympathetically mediated hrv . baroreflex sensitivity ( brs ) was assessed using the modified oxford technique [ 51 , 52 ] . the brs was quantified using the slope of the linear regression line representing the linear portion of the sigmoidal heart rate - systolic blood pressure relationship ( hr { bpm}/sbp { mmhg } ) after rapid bolus injections ( ~5 s ) of phenylephrine ( pe , 12 g / kg , 10 g / ml ) and sodium nitroprusside ( snp , 60 g / kg , 110 g / ml ) dissolved in ddh2o . ( 2007 ) . for each drug , the catheter was first filled with a 0.2 ml volume to ensure accuracy of the drug dose . after a stable baseline was obtained , a bolus injection of snp was rapidly infused and the reflex sns mediated tachycardia response was measured . the analysis began at the onset of sbp decrease after snp infusion and ended when sbp reached its nadir . this was followed by a saline flush to washout any remaining snp in the catheter . after a stable baseline was reestablished , this same procedure was then followed using pe to measure psns mediated reflex bradycardia , except that analysis began at the onset of sbp increase and ended when sbp reached its zenith . responses to pe and snp were plotted separately and only regression lines ( slopes ) with correlation coefficients ( r ) 0.70 and p < 0.05 were accepted [ 53 , 54 ] . to measure the sympathetic contribution to baseline vascular resistance , map was assessed before and after a bolus injection of the -adrenergic receptor blocker , prazosin ( 85 g / kg , 500 g / ml ) . following this protocol to ensure physiological testing did not confound serum neuropeptide y concentration [ npy ] measurement , a subset of animals from each group did not undergo surgery for heart rate variability , baroreflex sensitivity , mean arterial pressure , or vascular sympathetic tone measurements . rather , at the end of the 10-week exercise training period these animals were anesthetized via intraperitoneal injection of sodium pentobarbital ( 65 mg / kg ) and blood serum samples for [ npy ] measurement were collected upon euthanasia . serum [ npy ] was measured using an npy elisa kit ( uscn life sciences inc . ) according to the manufacturer 's instructions . following the euthanasia of animals for blood [ npy ] measurement , hearts were extracted and immediately arrested by placing them in ice cold krebs - henseleit buffer ( khb ) . hearts were cannulated for unpaced retrograde aortic constant flow perfusion ( 15 ml / min ) of coronary arteries with khb ( containing 120 mm nacl , 4.63 m kcl , 1.17 mm kh2po4 , 1.25 mm cacl2 , 1.2 mm mgcl2 , 20 mm nahco3 , and 8 mm glucose gassed with 95% o2 and 5% co2 ) that was maintained at 37c . body weight and blood glucose concentrations were compared using a two - way repeated measures anova , while endpoint measures were compared by two - way anova , with the exception of endpoint insulin dose , which was compared using a one - tailed t - test . when significance was found , pairwise comparisons were made using the fisher lsd post hoc test . data are represented as mean standard error , with a significance level set at p < 0.05 . all groups increased in body weight over the course of the study ( p < 0.05 , figure 1(a ) ) . at the end of the study , the body weights of the t1 dm groups ( d and dx ) were lower than non - t1 dm groups ( c and cx ) , and exercised groups ( cx and dx ) weighed less than their nonexercised counterparts ( c and d ; p < 0.05 ) . following the confirmation of diabetes , weekly [ bg ] was mostly maintained in the targeted range of 917 mm ; however , the [ bg ] did move outside of this range periodically . the [ bg ] in the t1 dm groups were elevated in comparison to the non - t1 dm groups ( p < 0.05 ; figure 1(b ) ) . within the non - t1 dm and t1 dm groups , there was no difference in [ bg ] between nonexercised and exercised groups ( c versus cx and d versus dx ; p > 0.05 ) . the glucose clearance rate ( kg ) of the diabetic groups ( d and dx ) decreased from pre - t1 dm to week 10 of training ( p < 0.05 ) , whereas kg of the cx group increased ( p < 0.05 ; figure 2(a ) ) . both diabetic groups had significantly lower kg values than both the control groups ( c and cx ) at week 10 ( p < 0.05 ; figure 2(a ) ) . the amount of insulin supplementation that the dx group received was significantly less than the amount the d group received at week 10 ( p < 0.05 ; figure 2(b ) ) . for resting hr and map , there was not a significant difference between groups at week 10 ( figures 3(a ) and 3(b ) , resp . ) . however , for the intrinsic heart rate ( ihr ) , there was main effect of both exercise and t1 dm , where t1 dm decreased the ihr , while exercise increased ihr ( p < 0.05 , figure 3(c ) ) . further , within the t1 dm groups ( d and dx ) , exercise increased ihr , while within the nonexercised groups ( c and d ) t1 dm decreased ihr ( p < 0.05 ) . total hrv at week 10 , as measured by the standard deviation of the normal pulse wave peaks ( sdnn ) , was not significantly different between groups ( figure 4(a ) ) . however , there was a main effect of exercise on the hf contribution to hrv , where exercise increased hf hrv ( p < 0.05 , figure 4(b ) ) . particularly , within the t1 dm groups ( d and dx ) , exercise increased hf hrv ( p < 0.05 ) . in response to snp infusion , there was not a significant difference between groups in the tachycardia brs response ( figure 5(a ) ) . however , a significant interaction between t1 dm and exercise was observed for brs during the bradycardia response to phenylephrine ( p < 0.05 , figure 5(b ) ) . more specifically , within the t1 dm groups ( d and dx ) exercise prevented the reduction in brs that was observed in the d group ( p < 0.05 ) . an interaction between t1 dm and exercise was observed for the prazosin - induced change in map ( p < 0.05 , figure 6(a ) ) . within the nonexercised groups ( c and d ) , t1 dm resulted in an increased change in map ( p < 0.05 ) . within the t1 dm groups ( d and dx ) exercise prevented the increased change in map observed in the d group ( p < 0.05 ) . there was also a main effect of t1 dm on [ npy ] ( p < 0.05 , figure 6(b ) ) . within the nonexercised groups ( c and d ) and exercised groups ( cx and dx ) , serum [ npy ] this study demonstrated that a multiple low - dose stz model with moderate hyperglycemia , maintained using insulin therapy , produced deficits in cardiovascular autonomic function without inducing the resting bradycardia or hypotension typical of other stz models . this study also showed that high intensity aerobic exercise training can prevent deficits of cardiovascular autonomic function caused by t1 dm . furthermore , because [ bg ] was held within a moderate hyperglycemic range , the observed exercise - mediated improvements to indications of can were independent of changes in [ bg ] and , instead , may primarily have been the result of improvements to other aspects of glucoregulation and/or the preservation of autonomic nervous system function . although we found time domain analysis of total hrv , as measured by the sdnn , did not demonstrate differences between groups , frequency domain analysis exposed a reduction in the hf power in the d group compared with the dx group . since the hf power corresponds to the level of vagally mediated parasympathetic hrv , these results demonstrate not only the detrimental effects of t1 dm on autonomic cardiac control but also the benefits of exercise training toward ameliorating those effects . these findings are similar to those of other experiments of both experimental [ 16 , 21 ] and clinical diabetes [ 18 , 56 , 57 ] . ( 2009 ) reported that stz - induced t1 dm reduced the hf component of hrv , which was improved by exercise . also , they found that the vagal tonus of the control exercised rats did not differ from sedentary controls . likewise , chen et al . ( 2008 ) reported that children with t1 dm who performed a high level of physical activity did not differ from controls in hrv ; however , children with t1 dm who had low level of physical activity had significantly reduced hrv compared to both active children with t1 dm and non - t1 dm children . thus , the current study provides support that exercise can be an effective means to improve hrv in t1 dm . both tachycardia and bradycardia responses were studied in the context of brs analysis in order to explore the control features related to unloading or loading of the baroreceptors , respectively . some discrepancy exists between different experimental models of t1 dm and their impact on brs measures . investigations using the hyperglycemic non - obese diabetic ( nod ) t1 dm mouse model have shown elevations in brs measures rather than attenuated responses . in contrast , tachycardic - snp and bradycardic - pe responses have been shown to be lower in stz - induced t1 dm hyperglycemic rats in comparison to non - t1 dm controls but were improved with exercise training . in the current study , the slope of the hypotensive tachycardia response was not significantly different between any of the groups suggesting that responses to baroreceptor unloading are not affected by t1 dm or exercise . however , t1 dm reduced the bradycardia response to baroreceptor loading , which was nullified by concurrent exercise training . these findings are in line with previous reports demonstrating a bradycardia change in pe - brs without an accompanying change in snp - brs , which was improved following aerobic exercise . discrepancies in brs responses in t1 dm models seem to be closely associated with both the duration and the severity of diabetes . a recent study examining the time - course of brs changes in response to stz - induced hyperglycemia reported that alteration of the snp - brs was not evident until 12 weeks of diabetes , while a change in pe - brs was evident as early as 4 weeks after induction . interestingly , the animals in the aforementioned study were moderately hyperglycemic ( 1618 mm ) , suggesting that the severity of the hyperglycemia may play a role in the progression of this neuropathy . vinik and ziegler ( 2007 ) reported that poor glycemic control and duration of diabetes play a central role in progression of cardiovascular autonomic neuropathy . yet , it is not clear what role insulin therapy may play in the neuropathy . insulin supplementation to stz - induced t1 dm rats can modify the changes in brs sensitivity evident at 48 weeks of t1 dm . indeed , in clinical t1 dm patients , intensive therapy is well documented to slow the progression and delay the appearance of abnormal autonomic function . however , the current study provides evidence that the ability of exercise to ameliorate cardiovascular autonomic dysfunction may be independent of its ability to reduce [ bg ] , which challenges the direct relationship between [ bg ] and can suggested by previous studies [ 6769 ] . the ivgtt performed at the conclusion of the 10-week exercise period demonstrated an increased glucose clearance rate ( kg ) and therefore glucose tolerance , in the cx group compared to the preexercise training period . however , in both the sedentary and exercise diabetic groups there was an equal decline in kg to nearly the same rate . this decrease was significantly different from pre - t1 dm values and the week 10 values of the c and cx groups . while this would normally indicate that both of the diabetic groups developed equally impaired glucose tolerance , it was also the case that the dx group required approximately half of the dosage of exogenous insulin compared to the d group to maintain their [ bg ] in the 917 mm range . with double the insulin dose , it is likely that the total serum insulin over a given time during ivgtt would have been greater in the d than dx group , and with their kg being equal , that would indicate that there was a greater insulin sensitivity in the diabetic exercise group [ 70 , 71 ] . together , these ivgtt results demonstrate that exercise training improved glucose tolerance and insulin sensitivity . furthermore , since the [ bg ] of the diabetic groups in this study was held in a constant range , any abovementioned exercise - induced improvements to cv autonomic function would not have been mediated through a reduction in systemic [ bg ] but may have been the result of improvements in insulin sensitivity and glucose utilization [ 72 , 73 ] . this should be borne in mind when considering the effects of diabetes and exercise on indices of cv autonomic function , such as hrv and brs . an alternative mechanism by which exercise can influence brs was reported by bernardi et al . they demonstrated that a reduced parasympathetic brs in patients with t1 dm was improved by both oxygen supplementation and deep breathing to the same degree , which indicated the increased respiration and oxygen delivery resultant of exercise could have been mediating increases in brs . this led the authors to suggest that hypoxia in t1 dm functionally restrains parasympathetic activity . however , reduced brs could also be attributed to defects in the baroreceptors , baroreceptor afferent nerves , cns structures , or efferent fibres of the baroreflex circuit [ 7 , 8 , 61 ] . in the present study , the finding that the tachycardia response of the baroreflex was unimpaired by t1 dm , while the bradycardia response was , suggests that the afferent arm and central regulators of the baroreflex were not dysfunctional and that the observed decrement of baroreflex bradycardia may have been caused partly by alterations in efferent parasympathetic outflow [ 8 , 29 ] . another interesting outcome of the current study was the alteration of sympathetic vasomotor control in the d group , which was also modified by concurrent exercise training . in this study , prazosin treatment resulted in a drop in map that was approximately twofold greater in the d group compared to the c and dx groups , which is indicative of a much greater sympathetic contribution to the maintenance of baseline vascular resistance [ 75 , 76 ] . similarly , martinez - nieves and dunbar ( 1999 ) reported that male t1 dm rats had a greater decrease in map after a bolus injection of prazosin compared to their control cohorts . however , they postulated that an elevated prazosin response could be the result of increased 1-adrenergic receptor sensitivity . yet , in this study , the finding that treatment with pe , an 1-adrenergic receptor agonist , did not result in a greater peak sbp , nor a greater percent increase in sbp from baseline in the t1 dm group ( data not shown ) , argues against a receptor - based sensitivity mechanism and , rather , suggests that efferent sympathetic outflow may have been elevated in the d group . however , we can not determine the mechanism that resulted in prazosin showing a preferential decrease in map in the d group versus dx or c based on the data in this study . yet , in line with the current results , such elevations in resting sympathetic activity would make activation of the brs response to snp - induced hypotension more difficult . the conclusion above regarding sympathetic hyperactivity in the d group is supported by measurements of neuropeptide y [ npy ] obtained in this study . [ npy ] is coreleased with norepinephrine from perivascular and cardiac sympathetic nerve terminals during sympathetic activation [ 78 , 79 ] . in clinical t1 dm , a diabetes - related decrease in [ npy ] is attributed to impaired sympathetic function , whereas increased [ npy ] is attributed to sympathetic overactivity [ 7981 ] . in the current study , serum [ npy ] was greater in both of the t1 dm groups in comparison to their control groups . thus , despite the ability of exercise to preserve reflex cardiac function in t1 dm , hyperglycemia itself appears to have impacted basal vascular adrenergic activity in both t1 dm groups . as both t1 dm groups were maintained at equally elevated [ bg ] , there may have been a correspondingly similar stimulation of peripheral sympathetic activation and npy release [ 79 , 82 , 83 ] . despite improvements by exercise training to deficits of cardiovascular autonomic function , no observable statistical differences in either map or hr were evident between any of the groups . indeed , it has been shown that alterations in autonomic function occur before or without alterations in map and hr and are uncorrelated to changes in sympathetic tone . the observed changes in basal sympathetic activity may assist in the maintenance of blood pressure , ventricular function , and cardiac output during the early stage of diabetes , which is supported by our findings that inhibition of sympathetic activity results in a greater decrease in map in diabetic rats than normal rats . in that respect , we previously reported that although t1 dm animals demonstrated significant alterations in myocardial dimensions and structure , measurements of cardiac performance ( ejection fraction , fractional shortening , and cardiac output measurements ) were unchanged . to evaluate the heart rate of these animals without neural influence , we measured the beat rate of denervated hearts using the isolated langendorff technique . we found that the ihr of the d group was lower than both c and dx groups , which would support the notion that decreased ihr masked the effects of sympathetic overactivity in the current study . further , it supports evidence that stz - induced diabetes may have a direct effect on heart rate by modifying the heart itself [ 86 , 87 ] . interestingly , in some studies , insulin therapy was only able to partially reverse bradycardia and it was shown that stz - treatment itself could lengthen the action potential duration in the sa node , slowing the hr . however , if hyperglycemia or stz directly affected cardiac muscle or the sa node and caused a decreased ihr in the d group , it is also the case that exercise training rescued or prevented the deficit , as the ihr of the dx group was not different from the cx group . thus , previous experimental t1 dm studies that reported that stz - induced bradycardia and hypotension were caused by can , and that exercise - induced normalization of hr and bp was evidence of improvements in autonomic function , may really have been observing changes in intrinsic cardiac function which were independent of autonomic control . such changes could instead have been due to depressed sarcoplasmic reticulum function or impaired calcium handling [ 87 , 89 , 90 ] . therefore , the direct effects of stz on the heart and ihr require further examination and should be taken into consideration in future studies that investigate the autonomic regulation of cv function in stz - induced t1 dm models . an important consideration regarding the design of the current study was the use of anesthetized rats . in order to accurately reflect cardiovascular parameters in such a state , we selected an anaesthetic regime that provides the lowest level of influence on baseline and reflexive cv control attainable in rodent models . a light plane anesthesia 0.51.2 g / kg has been shown to maintain the integrity of the cardiovascular system , where higher doses of urethane ( above 1.5 g / kg ) can produce hypotension and bradycardia , as well as high rates of mortality [ 91 , 92 ] . in the current study we used a minimal dose of 25 mg / kg , which was reported in previous studies by our laboratory to have little influence on neurovascular blood flow measures [ 9395 ] . that being said , it can not be determined to what extent , if at all , the autonomic nervous system was augmented by the urethane - chloralose treatment in comparison to conscious animals . further work examining a comparison of our anesthesia regime with freely moving conscious animals ( using telemetry devices ) will better address this matter . in this study , t1 dm induced indications of parasympathetic withdrawal , sympathetic overactivity , and , despite a decreased ihr , no change in resting map or hr . however , concurrent exercise training with t1 dm maintained the sensitivity of the parasympathetically mediated baroreflex bradycardia , prevented an increase in vascular sympathetic tone , maintained a higher bodyweight , and prevented a decrease in ihr . the ability of exercise training to preserve parasympathetic function in this model of t1 dm indicates that the exercise - mediated improvements to parasympathetic function are independent of alterations in [ bg ] . however , the finding that [ npy ] remained elevated suggests that hyperglycemia has a direct impact on adrenergic activity . taken together , our t1 dm model of progressive stz induction and insulin treatment induced autonomic impairments similar to those observed in clinical t1 dm and demonstrates the novelty of this model for investigating the effectiveness of high intensity aerobic exercise training as a means to prevent the progression of can in t1 dm . thus , although not examined in this study , the mechanisms that underlie the physiological changes caused by t1 dm and exercise can be the focus of future investigations using this model .

indices of cardiovascular autonomic neuropathy ( can ) in experimental models of type 1 diabetes mellitus ( t1 dm ) are often contrary to clinical data . here , we investigated whether a relatable insulin - treated model of t1 dm would induce deficits in cardiovascular ( cv ) autonomic function more reflective of clinical results and if exercise training could prevent those deficits . sixty - four rats were divided into four groups : sedentary control ( c ) , sedentary t1 dm ( d ) , control exercise ( cx ) , or t1 dm exercise ( dx ) . diabetes was induced via multiple low - dose injections of streptozotocin and blood glucose was maintained at moderate hyperglycemia ( 917 mm ) through insulin supplementation . exercise training consisted of daily treadmill running for 10 weeks . compared to c , d had blunted baroreflex sensitivity , increased vascular sympathetic tone , increased serum neuropeptide y ( npy ) , and decreased intrinsic heart rate . in contrast , dx differed from d in all measures of can ( except npy ) , including heart rate variability . these findings demonstrate that this t1 dm model elicits deficits and exercise - mediated improvements to cv autonomic function which are reflective of clinical t1 dm .

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

a common and serious complication of type 1 diabetes mellitus ( t1 dm ) is diabetic autonomic neuropathy [ 1 , 2 ] . cardiovascular autonomic neuropathy ( can ) is a subset of diabetic autonomic neuropathy characterized by impaired autonomic control of the cardiovascular ( cv ) system . clinically , the most common methods for assessing can are heart rate variability ( hrv ) analysis and baroreflex sensitivity ( brs ) [ 3 , 5 , 6 ] . in t1 dm , aspects of the baroreflex arc can be impaired , such that both baroreceptor activity and excitability are blunted [ 8 , 9 ] and the aortic depressor nerves undergo axonal atrophy . as well , autonomic efferents , primarily of the parasympathetic nervous system ( psns ) , have decreased activity , reduced responsiveness , and decreased neurochemical activity in the heart [ 10 , 11 ] . reduced heart rate variability ( hrv ) is often the earliest symptom of can . exercise has been demonstrated to be an effective means of improving deficits in hrv and brs in both clinical and experimental t1 dm [ 1821 ] . such improvements have been attributed to improved insulin sensitivity , increased endogenous antioxidant and anti - inflammatory mediators , and improved autonomic control of the cv system [ 2224 ] . despite similar reductions in hrv and brs , there are marked differences in early - stage changes to other cv parameters between clinical and experimental t1 dm . specifically , in clinical t1 dm , increases in heart rate ( hr ) and blood pressure ( bp ) are commonly reported in early autonomic neuropathy [ 1 , 3 , 14 , 24 , 2628 ] . in contrast , experimental stz - induced t1 dm is regularly associated with decreased bp and hr , beginning shortly after diabetes induction [ 15 , 16 , 2931 ] . due to these opposing initial changes in bp and hr , exercise training is often observed to produce contrasting outcomes on cv parameters in experimental and clinical t1 dm , namely , increased bp and hr in experimental t1 dm and decreased bp and hr in clinical t1 dm [ 19 , 25 , 3234 ] . as a result , both the increase and decrease of these cv factors are concurrently cited as exercise - mediated improvements to can with little consideration of the fact that the changes are opposed between these two contexts of t1 dm . this is important because if animal models do not accurately reproduce t1 dm pathology , then the outcomes of experimental studies may not translate to the treatment of human can , as the mechanisms underlying the pathology and exercise modifications may differ . another important difference between experimental and clinical t1 dm is the common omission of insulin treatment in experimental diabetes leading to severe hyperglycemia ranging from roughly 17 to 25 mm blood glucose concentrations ( [ bg ] ) [ 15 , 16 , 29 , 30 ] . these changes may mediate the observed reduction in bp and hr that arise acutely in experimental t1 dm and in late - stage clinical t1 dm [ 2 , 3538 ] . indeed , intensive insulin therapy has been shown to restore bp and hr to non - t1 dm levels in stz - induced t1 dm rats [ 25 , 39 ] . yet , despite the use of insulin therapy in clinical t1 dm , it is often the case that chronic , moderate hyperglycemia is maintained as a result of difficulties in regulating [ bg ] in response to dynamic influences on glycemic control , such as food intake and exercise [ 40 , 41 ] . to address this , our laboratory established a model of t1 dm using a multiple low - dose stz - treatment and insulin therapy to replicate the moderate hyperglycemia observed in clinical t1 dm . in our previous studies that employed this model , we observed impairments in glucose tolerance , vascular responsiveness , cardiac function , and bone health , which were improved with high intensity aerobic exercise training [ 4447 ] . the purpose of the current study was to investigate whether our model of multiple low - dose stz - induced t1 dm with insulin therapy would induce deficits in cardiovascular autonomic function more representative of clinical t1 dm , and if high intensity aerobic training could prevent those deficits . we hypothesised that ( 1 ) our model of stz - induced t1 dm would elicit indices of can , including a blunted brs ( bradycardia and tachycardia response ) , lowered hrv and intrinsic heart rate , increased vascular sympathetic tone , and increased mean arterial pressure , and ( 2 ) high intensity aerobic exercise training would prevent or ameliorate the indications of can . sixty - four rats were randomly assigned to one of four groups as follows : ( 1 ) sedentary control ( c , n = 16 ) ; ( 2 ) exercised control ( cx , n = 16 ) ; ( 3 ) sedentary t1 dm ( d , n = 16 ) ; ( 4 ) exercised t1 dm ( dx , n = 16 ) . upon arrival rats subsequently , t1 dm was induced over five consecutive days by multiple intraperitoneal ( ip ) injections of 20 mg / kg streptozotocin ( stz , sigma - aldrich ) dissolved in a citrate buffer ( 0.1 m , ph 4.5 ) . diabetes was confirmed by blood glucose measurements greater than or equal to 18 mm on two consecutive days . , insulin pellet doses were then monitored for 1 week and adjusted ( 0.5 pellets ) in order to obtain daily nonfasting blood glucose concentrations in the moderate hyperglycemic range of 917 mm . , intravenous glucose tolerance tests ( ivgtt ) were performed on all animals prior to t1 dm induction ( pre - t1 dm ) and at the end of week 10 of the exercise training period . following dextrose infusion , blood glucose was measured at 5 minutes , at 10 minutes , and then at 10-minute intervals thereafter until blood glucose levels plateaued . prior to the initiation of the exercise training program , rats were familiarized with the exercise equipment on two consecutive days . the exercise training program consisted of 1 hour of motor - driven treadmill running per day at 27 m / min with a 6-degree incline , 5 days per week , for 10 weeks . at the end of the preparative surgery , rats were injected ip with a 25 mg / kg cocktail of urethane ( 16 mg / ml ) and -chloralose ( 100 mg / ml ) , an anesthetic cocktail that has been shown to have the least inhibition of baseline cv control and autonomic function . heart rate ( hr ) , systolic blood pressure ( sbp ) , and mean arterial pressure ( map ) were determined from the blood pressure pulse waveform and were collected while the rats were under urethane/-chloralose anesthesia in the supine position . data were obtained using a powerlab data acquisition system , digitized , and recorded at 1000 hz using the bundled labchart 7 pro software ( adinstruments , colorado springs , co , usa ) . following this protocol to ensure physiological testing did not confound serum neuropeptide y concentration [ npy ] measurement , a subset of animals from each group did not undergo surgery for heart rate variability , baroreflex sensitivity , mean arterial pressure , or vascular sympathetic tone measurements . rather , at the end of the 10-week exercise training period these animals were anesthetized via intraperitoneal injection of sodium pentobarbital ( 65 mg / kg ) and blood serum samples for [ npy ] measurement were collected upon euthanasia . hearts were cannulated for unpaced retrograde aortic constant flow perfusion ( 15 ml / min ) of coronary arteries with khb ( containing 120 mm nacl , 4.63 m kcl , 1.17 mm kh2po4 , 1.25 mm cacl2 , 1.2 mm mgcl2 , 20 mm nahco3 , and 8 mm glucose gassed with 95% o2 and 5% co2 ) that was maintained at 37c . body weight and blood glucose concentrations were compared using a two - way repeated measures anova , while endpoint measures were compared by two - way anova , with the exception of endpoint insulin dose , which was compared using a one - tailed t - test . at the end of the study , the body weights of the t1 dm groups ( d and dx ) were lower than non - t1 dm groups ( c and cx ) , and exercised groups ( cx and dx ) weighed less than their nonexercised counterparts ( c and d ; p < 0.05 ) . within the non - t1 dm and t1 dm groups , there was no difference in [ bg ] between nonexercised and exercised groups ( c versus cx and d versus dx ; p > 0.05 ) . the glucose clearance rate ( kg ) of the diabetic groups ( d and dx ) decreased from pre - t1 dm to week 10 of training ( p < 0.05 ) , whereas kg of the cx group increased ( p < 0.05 ; figure 2(a ) ) . both diabetic groups had significantly lower kg values than both the control groups ( c and cx ) at week 10 ( p < 0.05 ; figure 2(a ) ) . for resting hr and map , there was not a significant difference between groups at week 10 ( figures 3(a ) and 3(b ) , resp . ) however , for the intrinsic heart rate ( ihr ) , there was main effect of both exercise and t1 dm , where t1 dm decreased the ihr , while exercise increased ihr ( p < 0.05 , figure 3(c ) ) . further , within the t1 dm groups ( d and dx ) , exercise increased ihr , while within the nonexercised groups ( c and d ) t1 dm decreased ihr ( p < 0.05 ) . total hrv at week 10 , as measured by the standard deviation of the normal pulse wave peaks ( sdnn ) , was not significantly different between groups ( figure 4(a ) ) . particularly , within the t1 dm groups ( d and dx ) , exercise increased hf hrv ( p < 0.05 ) . however , a significant interaction between t1 dm and exercise was observed for brs during the bradycardia response to phenylephrine ( p < 0.05 , figure 5(b ) ) . more specifically , within the t1 dm groups ( d and dx ) exercise prevented the reduction in brs that was observed in the d group ( p < 0.05 ) . an interaction between t1 dm and exercise was observed for the prazosin - induced change in map ( p < 0.05 , figure 6(a ) ) . within the nonexercised groups ( c and d ) , t1 dm resulted in an increased change in map ( p < 0.05 ) . within the t1 dm groups ( d and dx ) exercise prevented the increased change in map observed in the d group ( p < 0.05 ) . there was also a main effect of t1 dm on [ npy ] ( p < 0.05 , figure 6(b ) ) . within the nonexercised groups ( c and d ) and exercised groups ( cx and dx ) , serum [ npy ] this study demonstrated that a multiple low - dose stz model with moderate hyperglycemia , maintained using insulin therapy , produced deficits in cardiovascular autonomic function without inducing the resting bradycardia or hypotension typical of other stz models . this study also showed that high intensity aerobic exercise training can prevent deficits of cardiovascular autonomic function caused by t1 dm . furthermore , because [ bg ] was held within a moderate hyperglycemic range , the observed exercise - mediated improvements to indications of can were independent of changes in [ bg ] and , instead , may primarily have been the result of improvements to other aspects of glucoregulation and/or the preservation of autonomic nervous system function . since the hf power corresponds to the level of vagally mediated parasympathetic hrv , these results demonstrate not only the detrimental effects of t1 dm on autonomic cardiac control but also the benefits of exercise training toward ameliorating those effects . these findings are similar to those of other experiments of both experimental [ 16 , 21 ] and clinical diabetes [ 18 , 56 , 57 ] . ( 2008 ) reported that children with t1 dm who performed a high level of physical activity did not differ from controls in hrv ; however , children with t1 dm who had low level of physical activity had significantly reduced hrv compared to both active children with t1 dm and non - t1 dm children . thus , the current study provides support that exercise can be an effective means to improve hrv in t1 dm . some discrepancy exists between different experimental models of t1 dm and their impact on brs measures . in contrast , tachycardic - snp and bradycardic - pe responses have been shown to be lower in stz - induced t1 dm hyperglycemic rats in comparison to non - t1 dm controls but were improved with exercise training . however , t1 dm reduced the bradycardia response to baroreceptor loading , which was nullified by concurrent exercise training . interestingly , the animals in the aforementioned study were moderately hyperglycemic ( 1618 mm ) , suggesting that the severity of the hyperglycemia may play a role in the progression of this neuropathy . vinik and ziegler ( 2007 ) reported that poor glycemic control and duration of diabetes play a central role in progression of cardiovascular autonomic neuropathy . insulin supplementation to stz - induced t1 dm rats can modify the changes in brs sensitivity evident at 48 weeks of t1 dm . indeed , in clinical t1 dm patients , intensive therapy is well documented to slow the progression and delay the appearance of abnormal autonomic function . the ivgtt performed at the conclusion of the 10-week exercise period demonstrated an increased glucose clearance rate ( kg ) and therefore glucose tolerance , in the cx group compared to the preexercise training period . however , in both the sedentary and exercise diabetic groups there was an equal decline in kg to nearly the same rate . this decrease was significantly different from pre - t1 dm values and the week 10 values of the c and cx groups . while this would normally indicate that both of the diabetic groups developed equally impaired glucose tolerance , it was also the case that the dx group required approximately half of the dosage of exogenous insulin compared to the d group to maintain their [ bg ] in the 917 mm range . with double the insulin dose , it is likely that the total serum insulin over a given time during ivgtt would have been greater in the d than dx group , and with their kg being equal , that would indicate that there was a greater insulin sensitivity in the diabetic exercise group [ 70 , 71 ] . together , these ivgtt results demonstrate that exercise training improved glucose tolerance and insulin sensitivity . furthermore , since the [ bg ] of the diabetic groups in this study was held in a constant range , any abovementioned exercise - induced improvements to cv autonomic function would not have been mediated through a reduction in systemic [ bg ] but may have been the result of improvements in insulin sensitivity and glucose utilization [ 72 , 73 ] . this should be borne in mind when considering the effects of diabetes and exercise on indices of cv autonomic function , such as hrv and brs . similarly , martinez - nieves and dunbar ( 1999 ) reported that male t1 dm rats had a greater decrease in map after a bolus injection of prazosin compared to their control cohorts . yet , in this study , the finding that treatment with pe , an 1-adrenergic receptor agonist , did not result in a greater peak sbp , nor a greater percent increase in sbp from baseline in the t1 dm group ( data not shown ) , argues against a receptor - based sensitivity mechanism and , rather , suggests that efferent sympathetic outflow may have been elevated in the d group . the conclusion above regarding sympathetic hyperactivity in the d group is supported by measurements of neuropeptide y [ npy ] obtained in this study . in clinical t1 dm , a diabetes - related decrease in [ npy ] is attributed to impaired sympathetic function , whereas increased [ npy ] is attributed to sympathetic overactivity [ 7981 ] . in the current study , serum [ npy ] was greater in both of the t1 dm groups in comparison to their control groups . as both t1 dm groups were maintained at equally elevated [ bg ] , there may have been a correspondingly similar stimulation of peripheral sympathetic activation and npy release [ 79 , 82 , 83 ] . despite improvements by exercise training to deficits of cardiovascular autonomic function , no observable statistical differences in either map or hr were evident between any of the groups . indeed , it has been shown that alterations in autonomic function occur before or without alterations in map and hr and are uncorrelated to changes in sympathetic tone . in that respect , we previously reported that although t1 dm animals demonstrated significant alterations in myocardial dimensions and structure , measurements of cardiac performance ( ejection fraction , fractional shortening , and cardiac output measurements ) were unchanged . to evaluate the heart rate of these animals without neural influence , we measured the beat rate of denervated hearts using the isolated langendorff technique . further , it supports evidence that stz - induced diabetes may have a direct effect on heart rate by modifying the heart itself [ 86 , 87 ] . thus , previous experimental t1 dm studies that reported that stz - induced bradycardia and hypotension were caused by can , and that exercise - induced normalization of hr and bp was evidence of improvements in autonomic function , may really have been observing changes in intrinsic cardiac function which were independent of autonomic control . therefore , the direct effects of stz on the heart and ihr require further examination and should be taken into consideration in future studies that investigate the autonomic regulation of cv function in stz - induced t1 dm models . in this study , t1 dm induced indications of parasympathetic withdrawal , sympathetic overactivity , and , despite a decreased ihr , no change in resting map or hr . however , concurrent exercise training with t1 dm maintained the sensitivity of the parasympathetically mediated baroreflex bradycardia , prevented an increase in vascular sympathetic tone , maintained a higher bodyweight , and prevented a decrease in ihr . the ability of exercise training to preserve parasympathetic function in this model of t1 dm indicates that the exercise - mediated improvements to parasympathetic function are independent of alterations in [ bg ] . taken together , our t1 dm model of progressive stz induction and insulin treatment induced autonomic impairments similar to those observed in clinical t1 dm and demonstrates the novelty of this model for investigating the effectiveness of high intensity aerobic exercise training as a means to prevent the progression of can in t1 dm . thus , although not examined in this study , the mechanisms that underlie the physiological changes caused by t1 dm and exercise can be the focus of future investigations using this model .

c57bl/6j mice were purchased from jackson laboratory ( bar harbor , me , usa ) and housed under a 12:12-hour light : dark cycle with access to food and water ad libitum . for intraperitoneal injections , a 25-gauge needle was inserted , and a 100-l volume was injected ( 250 g cr2-fh in pbs or pbs only ) . cr2-fh expressing cho cells were generously provided by s tomlinson , and protein was generated and purified as published previously . all experiments were approved by the medical university of south carolina institutional animal care and use committee and performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research . eight - week - old c57bl/6j male mice were divided into three groups ( n = 6 per group ) . the control group was kept in a filtered air environment , and the two experimental groups were subjected to cigarette smoke . cigarette smoke exposure was carried out ( 5 hours per day , 5 days per week ) by burning 3r4f reference cigarettes ( university of kentucky , louisville , ky , usa ) using a smoking machine ( model te-10 ; teague enterprises , woodland , ca , usa ) for 6 months as published previously . carboxyhemoglobin levels in the mice after 2 , 12 , and 24 weeks of cigarette exposure as determined in venous blood by dual beam spectrophotometry were between 8% and 12% immediately after exposure , which is consistent with values reported in the literature for modeling the effects of chronic cse . the average concentration of total suspended particulates present in the chamber was 130 mg / m and was monitored twice daily . following 6 months of cse , smoke - exposed animals were randomized into two groups ( 1 , control pbs ; 2 , cr2-fh treated ) , and mice were followed for 12 weeks post smoking cessation . during this visual acuity and contrast sensitivity of mice were measured by observing their optomotor responses to moving sine - wave gratings ( optomotry ; cerebral mechanics , inc . , mice reflexively respond to rotating vertical gratings by moving their head in the direction of grating rotation . to observe these movements , mice were placed individually on the central elevated pedestal surrounded by a square array of computer monitors that display stimulus gratings . mice were monitored via an overhead closed - circuit tv camera that allowed the observer to view only the central platform and not the rotating grating . mice were allowed to adjust to the chamber for 2 minutes with the monitors displaying a 50% gray uniform field prior to testing , and monitors returned to a homogenous gray between trials . all tests were conducted under photopic conditions with a mean luminance of 52 cd m. visual acuity was measured by finding the spatial frequency threshold of each animal at a constant speed ( 12/s ) and contrast ( 100% ) with a staircase procedure that systematically increased the spatial frequency of the grating until the animal no longer exhibited detectable responses . contrast sensitivity was determined by taking the reciprocal of the contrast threshold at a fixed spatial frequency ( 0.131 cyc / deg ) and speed ( 12/s ) . it has previously been determined that this spatial frequency falls within the range of maximal contrast sensitivity for 9-month - old c57bl/6j mice ( data not shown ) . contrast of the pattern was decreased systematically in a staircase manner until the animal stopped responding . the eyes were enucleated , and a slit was cut into the cornea to allow for rapid influx of fixative . eyes were fixed overnight in 2.5% glutaraldehyde , 1% formaldehyde , 3% sucrose , and 1 mm mgso4 in 0.1 m cacodylate buffer , ph 7.4 . the eyes were then dissected , and small central portions were osmicated for 60 minutes in 0.5% oso4 in 0.1 m cacodylate buffer , processed in maleate buffer for en bloc staining with uranyl acetate , dehydrated in graded ethanols , and processed for resin embedding as published previously . serial sections were cut at 90 nm on a leica ultramicrotome ( buffalo grove , il , usa ) onto carbon - coated formvar films supported by nickel slot - grids . electron microscopy images were captured using a jeol jem 1400 ( peabody , ma , usa ) transmission electron microscope using serialem ( peabody , ma , usa ) software to automate image capture overnight with 1200 to 1500 images captured per section , yielding datasets that were then processed with the ncr toolset to generate image mosaics with corrections for image aberrations induced by electron microscopy . images were evaluated using adobe photoshop ( adobe systems , san jose , ca , usa ) and imagej ( http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) software as published previously . for each animal , two rpe cells were outlined using the apical processes and the basal lamina ( thickness ) as well as the basolateral walls ( length ) as borders , and features were examined . a masking layer for all mitochondria present within an rpe cell was created to calculate average mitochondrial number and size . to determine mitochondrial position , the centroid coordinates for each mitochondrion were calculated as a percentage of the corresponding rpe length and thickness , respectively . coordinates were assigned to basolateral , basal , central , or apical compartments based on normalized x - y coordinates . a masking layer was created over segments of brm , using choroidal intercapillary pillars and the basement membrane of the rpe and choriocapillaris ( cc ) as boundaries . the area of the masking layer was divided by the length of each segment to determine an average brm thickness for each segment . we then calculated a weighted thickness average , based on the percentage length of each segment versus the entire 200-m section being analyzed . finally , we determined the average brm thickness by summing weighted averages for each section . the number of outer collagenous layer ( ocl ) deposits was calculated along a 200-m section of brm . an ocl deposit was defined as the presence of any discrete focal nodule of homogenous material of intermediate electron density between the ocl of brm and the basement membrane of the cc . outer nuclear layer ( onl ) thickness was determined by averaging measurements taken at five arbitrary points along a 200-m section of retinal tissue , using the outer plexiform layer ( upper ) and outer limiting membrane ( lower ) as boundaries . fenestrations were counted along a 100-m section of brm / cc and quantified as the number of fenestrations per mm . mller cell percent area was determined by subtracting the area occupied by the rod / cone somas in a 600-m area of the onl and dividing by the total area analyzed , using the same masking technique as described for brm thickness analysis . this area was also used to assess the number of nuclei within the onl . from the second eye of each animal , rpe / choroid / sclera ( henceforth referred to as rpe / choroid ) and retina fractions were isolated and stored at 80c until they were used . quantitative rt - pcr ( qrt - pcr ) analyses were performed as previously described in detail . in short , real - time pcr analyses were performed in triplicate in a geneamp 5700 sequence detection system ( applied biosystems , foster city , ca , usa ) using standard cycling conditions . quantitative values were obtained by the cycle number , normalizing genes of interest to -actin , and determining fold difference between room air and cse within treatments . quantitative rt - pcr primer sequences for data consisting of multiple groups and repeated measures , linear regression analyses of the individual animals followed by tukey 's multiple comparisons test ( p < 0.05 ) were used ; single comparisons were analyzed using student 's t - test assuming equal variance ( p < 0.05 ; statview ; sas institute , inc . , cary , nc , usa ) . fold changes in qrt - pcr experiments were analyzed by z - test ( treatment versus never smokers ; p < 0.05 ) and t - test using the holm - sidak method ( cse mice , pbs versus cr2-fh , with = 5.0% ; p < 0.01 ) . c57bl/6j mice were purchased from jackson laboratory ( bar harbor , me , usa ) and housed under a 12:12-hour light : dark cycle with access to food and water ad libitum . for intraperitoneal injections , a 25-gauge needle was inserted , and a 100-l volume was injected ( 250 g cr2-fh in pbs or pbs only ) . cr2-fh expressing cho cells were generously provided by s tomlinson , and protein was generated and purified as published previously . all experiments were approved by the medical university of south carolina institutional animal care and use committee and performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research . eight - week - old c57bl/6j male mice were divided into three groups ( n = 6 per group ) . the control group was kept in a filtered air environment , and the two experimental groups were subjected to cigarette smoke . cigarette smoke exposure was carried out ( 5 hours per day , 5 days per week ) by burning 3r4f reference cigarettes ( university of kentucky , louisville , ky , usa ) using a smoking machine ( model te-10 ; teague enterprises , woodland , ca , usa ) for 6 months as published previously . carboxyhemoglobin levels in the mice after 2 , 12 , and 24 weeks of cigarette exposure as determined in venous blood by dual beam spectrophotometry were between 8% and 12% immediately after exposure , which is consistent with values reported in the literature for modeling the effects of chronic cse . the average concentration of total suspended particulates present in the chamber was 130 mg / m and was monitored twice daily . following 6 months of cse , smoke - exposed animals were randomized into two groups ( 1 , control pbs ; 2 , cr2-fh treated ) , and mice were followed for 12 weeks post smoking cessation . during this visual acuity and contrast sensitivity of mice were measured by observing their optomotor responses to moving sine - wave gratings ( optomotry ; cerebral mechanics , inc . , mice reflexively respond to rotating vertical gratings by moving their head in the direction of grating rotation . to observe these movements , mice were placed individually on the central elevated pedestal surrounded by a square array of computer monitors that display stimulus gratings . mice were monitored via an overhead closed - circuit tv camera that allowed the observer to view only the central platform and not the rotating grating . mice were allowed to adjust to the chamber for 2 minutes with the monitors displaying a 50% gray uniform field prior to testing , and monitors returned to a homogenous gray between trials . all tests were conducted under photopic conditions with a mean luminance of 52 cd m. visual acuity was measured by finding the spatial frequency threshold of each animal at a constant speed ( 12/s ) and contrast ( 100% ) with a staircase procedure that systematically increased the spatial frequency of the grating until the animal no longer exhibited detectable responses . contrast sensitivity was determined by taking the reciprocal of the contrast threshold at a fixed spatial frequency ( 0.131 cyc / deg ) and speed ( 12/s ) . it has previously been determined that this spatial frequency falls within the range of maximal contrast sensitivity for 9-month - old c57bl/6j mice ( data not shown ) . contrast of the pattern was decreased systematically in a staircase manner until the animal stopped responding . the eyes were enucleated , and a slit was cut into the cornea to allow for rapid influx of fixative . eyes were fixed overnight in 2.5% glutaraldehyde , 1% formaldehyde , 3% sucrose , and 1 mm mgso4 in 0.1 m cacodylate buffer , ph 7.4 . the eyes were then dissected , and small central portions were osmicated for 60 minutes in 0.5% oso4 in 0.1 m cacodylate buffer , processed in maleate buffer for en bloc staining with uranyl acetate , dehydrated in graded ethanols , and processed for resin embedding as published previously . serial sections were cut at 90 nm on a leica ultramicrotome ( buffalo grove , il , usa ) onto carbon - coated formvar films supported by nickel slot - grids . electron microscopy images were captured using a jeol jem 1400 ( peabody , ma , usa ) transmission electron microscope using serialem ( peabody , ma , usa ) software to automate image capture overnight with 1200 to 1500 images captured per section , yielding datasets that were then processed with the ncr toolset to generate image mosaics with corrections for image aberrations induced by electron microscopy . images were evaluated using adobe photoshop ( adobe systems , san jose , ca , usa ) and imagej ( http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) software as published previously . for each animal , two rpe cells were outlined using the apical processes and the basal lamina ( thickness ) as well as the basolateral walls ( length ) as borders , and features were examined . a masking layer for all mitochondria present within an rpe cell was created to calculate average mitochondrial number and size . to determine mitochondrial position , the centroid coordinates for each mitochondrion were calculated as a percentage of the corresponding rpe length and thickness , respectively . coordinates were assigned to basolateral , basal , central , or apical compartments based on normalized x - y coordinates . a masking layer was created over segments of brm , using choroidal intercapillary pillars and the basement membrane of the rpe and choriocapillaris ( cc ) as boundaries . the area of the masking layer was divided by the length of each segment to determine an average brm thickness for each segment . we then calculated a weighted thickness average , based on the percentage length of each segment versus the entire 200-m section being analyzed . finally , we determined the average brm thickness by summing weighted averages for each section . the number of outer collagenous layer ( ocl ) deposits was calculated along a 200-m section of brm . an ocl deposit was defined as the presence of any discrete focal nodule of homogenous material of intermediate electron density between the ocl of brm and the basement membrane of the cc . outer nuclear layer ( onl ) thickness was determined by averaging measurements taken at five arbitrary points along a 200-m section of retinal tissue , using the outer plexiform layer ( upper ) and outer limiting membrane ( lower ) as boundaries . fenestrations were counted along a 100-m section of brm / cc and quantified as the number of fenestrations per mm . mller cell percent area was determined by subtracting the area occupied by the rod / cone somas in a 600-m area of the onl and dividing by the total area analyzed , using the same masking technique as described for brm thickness analysis . from the second eye of each animal , rpe / choroid / sclera ( henceforth referred to as rpe / choroid ) and retina fractions were isolated and stored at 80c until they were used . quantitative rt - pcr ( qrt - pcr ) analyses were performed as previously described in detail . in short , real - time pcr analyses were performed in triplicate in a geneamp 5700 sequence detection system ( applied biosystems , foster city , ca , usa ) using standard cycling conditions . quantitative values were obtained by the cycle number , normalizing genes of interest to -actin , and determining fold difference between room air and cse within treatments . for data consisting of multiple groups and repeated measures , linear regression analyses of the individual animals followed by tukey 's multiple comparisons test ( p < 0.05 ) were used ; single comparisons were analyzed using student 's t - test assuming equal variance ( p < 0.05 ; statview ; sas institute , inc . , fold changes in qrt - pcr experiments were analyzed by z - test ( treatment versus never smokers ; p < 0.05 ) and t - test using the holm - sidak method ( cse mice , pbs versus cr2-fh , with = 5.0% ; p < 0.01 ) . there is ample evidence that morphologic and cellular alterations in dry amd lead to vision loss or impairment as measured by changes in visual acuity and contrast sensitivity . in particular , structural changes to brm , including thickening and lipid deposition , can lead to impaired exchange of waste and nutrients between the rpe and choroid as well as a reduction in the generation and delivery of 11-cis - retinal , the chromophore essential for proper visual function , to photoreceptor outer segments . however , it is unclear to what degree these changes are reversible . here , we generated amd - associated morphologic alterations and loss of contrast sensitivity in mice exposed to long - term smoke inhalation as reported previously . after 6 months of cse , animals were randomly split into two groups and assessed for visual function using optokinetic responses ( reported as pretreatment data in fig . 1 ) . as reported previously , spatial acuity in c57bl/6j mice was not affected by cse , resulting in acuity measures that did not differ from those of age - matched room air controls ( never smokers ; fig . however , contrast sensitivity was significantly reduced in cse mice , which exhibited a significant decrease in contrast sensitivity compared to never smokers ( group 1 , to be treated with pbs : 4.57 0.97 ; or group 2 , to be treated with cr2-fh : 5.78 0.52 ; versus never smokers : 10.14 0.41 ; p < 0.01 ; fig . 1b , pretreatment ) . optomotor responses were analyzed in c57bl/6j mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with nothing , pbs , or alternative complement pathway inhibitor , cr2-fh . ( a ) visual acuity was measured by identifying the spatial frequency threshold at a constant speed ( 12/s ) and contrast ( 100% ) . ( b ) contrast sensitivity was measured by taking the reciprocal of the contrast threshold at a fixed spatial frequency ( 0.131 cyc / deg ) and speed ( 12/s ) . we previously determined that this spatial frequency falls within the range of maximal contrast sensitivity for 9-month - old c57bl/6j mice ( data not shown ) . after cse , mice showed a significant reduction in contrast sensitivity compared to controls , which was still maintained after 3 months of cse cessation . however , smoke - exposed mice treated with cr2-fh showed a contrast sensitivity that was similar to room air controls and significantly higher than in pbs - treated mice following cse . data are expressed as mean sem ( n = 46 per condition ; * p < 0.05 ) . after confirming that cse animals exhibited loss of contrast sensitivity , mice were returned to room air and treated for 3 consecutive months with either cr2-fh given three times per week or pbs , using intraperitoneal injections . cr2-fh has both a targeting domain ( scrs 14 of cr2 ) that binds c3 fragments deposited at sites of complement activation and a complement inhibitory domain ( scrs 15 of fh ) . after 3 months of smoking cessation and treatment , animals were retested for visual function ( reported as posttreatment data in fig . as expected , spatial acuity in cse mice treated with either pbs or cr2-fh did not differ from mice exposed to room air ( fig . 1a ; p = 0.44 ) . on the other hand , cse mice treated with pbs still exhibited a robust decrease in contrast sensitivity ( smoke + pbs ; 4.89 1.14 ) compared to never smokers ( control ; 9.97 0.53 ; p < 0.001 ; fig . importantly , contrast sensitivity threshold in cse mice treated with cr2-fh recovered to levels similar to those of age - matched never smokers ( smoke + cr2-fh : 9.97 0.53 versus control never smokers : 9.14 0.39 ; p = 0.29 ) . linear regression analysis for pre- and posttreatment contrast sensitivity confirmed the marked improvement in cr2-fh treated animals ( p < 0.0001 ) . changes in gene expression following smoke exposure for six gene categories have been shown previously . to recap , after 6 months of cse , expression of genes whose products are involved in photoreceptor cell function , complement inhibition , and inhibition of angiogenic and lysosomal function was downregulated , whereas increases were observed for genes whose products are involved in complement activation , angiogenesis , oxidative stress , and mitochondrial fission and fusion . here , we asked how cessation of smoking alone , or cessation of smoking coupled with cr2-fh therapy , affects expression of the same set of genes ( fig . analysis of marker gene expression in smoke - exposed mice following cessation of cigarette smoke exposure ( cse ) and treatment with pbs or alternative complement pathway inhibitor , cr2-fh , using quantitative rt - pcr on cdna generated from retina and rpe / choroid / sclera fraction . please note that with the exception of rho , opn1sw , and opn1mw , which were examined in the retina fraction , all other genes were examined in the rpe / choroid / sclera fraction . quantitative values were obtained by cycle number ( ct value ) , determining the difference between the mean experimental and control ( actb ) ct values for pbs- and cr2-fh treated mice following cse versus room air exposed mice ( fold difference ) . candidates were examined from a number of categories including photoreceptor cell function ( rho , opn1sw , opn1mw , rpe65 ) , complement activation ( c3 , hc , cfb , cfd , cfh , cd55 , cd59a ) , control of angiogenesis ( vegfa , serpinf1 ) , oxidative stress ( hif1a , cp ) , autophagy ( lyz1 , lamp2 , klc3 ) , mitochondrial function ( mfn1 , mt - co1 , dnm1l , ndufb8 , pfkfb1 , hmox1 ) , and immune response ( il-17 and ror ) . significant changes were identified in several categories for cr2-fh treated mice , in particular , suggesting decreased complement activation and enhanced energy production . statistics represent comparisons between pbs- and cr2-fh - treated animals following cse versus never smokers and differences between cse mice treated with pbs versus cr2-fh . data are expressed as mean sem ( n = 3 per condition ; * p < 0.05 analyzing fold changes between treatment groups versus never smokers ; # p < 0.01 analyzing fold change differences between cse mice treated with pbs versus cr2-fh ) . three months after smoking cessation , pbs - treated animals had normal levels of gene expression for photoreceptor function , pro- and antiangiogenic factors , oxidative stress , and autophagy . misregulated genes , on the other hand , involved those mediating mitochondrial fission and fusion , as well as complement activation . interestingly , a significant increase in factors regulating the complement ap pathway ( fb and fd [ factor d ] ) was observed . the effects of cr2-fh treatment compared to pbs treatment following smoking cessation involved two major categories : energy metabolism and complement activation . cr2-fh was also found to reduce mitochondrial stress , as genes affecting mitochondrial fission and fusion were normalized and an increase in the protective response gene heme oxygenase 1 was observed . loss in contrast sensitivity following smoke exposure has previously been shown to be associated with specific morphologic alterations in rpe , brm , and mller cells . here , we asked whether these same features play a role in the recovery of visual function in cse mice treated with cr2-fh . we analyzed electron micrographs obtained from cr2-fh and pbs cohorts and age - matched never smokers , analyzing outer retina structure ( fig . 4 ) . cr2-fh rescues ultrastructural changes in the outer retina in mice following cigarette exposure . electron micrographs of the outer retina obtained from age - matched mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with pbs ( b ) ( smoke + pbs ) , cr2-fh ( c ) ( smoke + alternative complement pathway inhibitor , cr2-fh ) , or 3 additional months of room air ( a ) ( control , never smokers ) were compared . while all animals had the same number of photoreceptors per row ( 9 ) , the nuclei were more densely packed in the smoke + pbs when compared to the control and cr2-fh treated animals . ipl , inner plexiform layer ; inl , inner nuclear layer ; opl , outer plexiform layer ; onl , outer nuclear layer ; is , inner segments ; os , outer segments . cr2-fh rescues ultrastructural changes in rpe / brm / cc complex in mice following cigarette exposure . electron micrographs of the retinal pigment epithelium / bruch 's membrane / choriocapillaris complex ( rpe / brm / cc ) obtained from age - matched mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with pbs ( smoke + pbs ) , cr2-fh ( smoke + alternative complement pathway inhibitor , cr2-fh ) , or 3 additional months of room air ( control , never smokers ) were compared , providing an rpe / brm / cc overview ( a c ) , or focusing on mitochondria ( d f ) and brm ( g i ) . in an animal raised in room air ( control , never smoker ) , brm exhibits an organized pentalaminar structure consisting of rpe basement membrane , inner collagenous layer , middle elastic layer , outer collagenous layer , and choriocapillaris basement membrane , and the choriocapillaris endothelium has fenestrations ( arrowheads ) along the entire membrane ( a , g ) . ( b , h ) the rpe / brm / cc in a mouse following cse ( smoke + pbs ) exhibits pathologic changes . bruch 's membrane is disorganized , no longer showing a pentalaminar structure , and large deposits ( asterisks ) are present in the outer collagen layer ( ocl ) . notably , there is significant fenestration loss and/or endothelial cell thickening adjacent to ocl deposits . ( c , i ) the rpe / brm / cc appears normal in mice treated with cr2-fh following cse ( smoke + cr2-fh ) . the morphologic features of mitochondria shown in mice exposed to smoke mitochondria have degraded outer membranes and disorganized cristae ( e ) , but normal appearance in never smokers ( d ) and mice treated with cr2-fh following cse ( f ) . bruch 's membrane is a pentalaminar structure consisting of the basement membrane of the rpe , the inner collagenous layer , the middle elastic layer , the ocl , and the basement membrane of the cc . the pentalaminar structure of brm was disrupted as evidenced by an inconsistent middle elastic layer ( figs . areas of brm disorganization were especially prevalent near large deposits located in the ocl ( asterisks ) that are absent in never smokers . in contrast , never smokers exhibit a high fenestration count with few to no ocl deposits . f ) were significantly altered in cse animals , characterized by poorly defined outer membranes and disorganized cristae . finally , mitochondria , which in a healthy rpe cell are distributed in a basal to apical gradient but lose this arrangement in unhealthy cells such as those exposed to cse , were found to localize in a pattern that was not different between never smokers and cse mice in any of the four localization bins analyzed ( fig . 5 ) . taken together , these results are consistent with previous findings from our lab obtained from animals examined immediately following the completion of the 6-month cse period , with the exception of the lack of mitochondrial mislocalization . bruch 's membrane appeared intact , exhibiting the full pentalaminar structure , without any apparent thickening . few deposits were found in brm , and fenestrations were evenly distributed along the cc . likewise , the mitochondrial phenotype also appeared to be healthy , as evidenced by a clear outer membrane and organized cristae . 3 ) by determining their centroid coordinates as a percentage of the corresponding retinal pigment epithelium ( rpe ) length and thickness , respectively . each centroid was subsequently assigned to one of four bins ( basolateral , basal , central , or apical ) and expressed as percent of total mitochondria ( mito ) . mitochondria are localized predominantly to the basolateral and basal compartments of the rpe cells with fewer localized in the central and apical portions in never smokers . while we previously reported that cigarette smoke exposure ( cse ) affects the mitochondrial distribution in c57bl/6j mice after 6 months , with mitochondria exhibiting an apical shift from the basal to central compartment , mitochondrial distribution is back to normal 3 months after the cessation of cse ( smoke + pbs ) , and is not further affected by complement inhibition ( smoke + cr2-fh ) . electron microscopy results were quantified ( table 2 based on data such as those in figs . 3 , 4 ) focusing on morphologic correlates of energy metabolism in the rpe ( mitochondria ) , nutrient and waste transport ( brm thickness , ocl deposit count , and fenestrations in the cc ) , and photoreceptor function ( onl thickness , photoreceptor cell counts , and size of mller cells ) . mitochondria were found to be significantly larger in cse mice compared to never smokers ( p < 0.05 ) and made up a larger percentage of total rpe cell area ( p < 0.05 ) . the total number of mitochondria was unchanged between all three groups as was their localization . a thickening of brm in cse mice was observed compared to never smokers ; however , this did not reach statistical significance . cigarette smoke exposure triggers the formation of ocl deposits in brm , which are maintained in cse mice treated with vehicle ( p < 0.01 ) but removed in those treated with cr2-fh . fenestrations counted along 100-m brm / cc sections revealed , on average , a 53% decline of the fenestrations in cse mice ( p = 0.002 ) , a deficit that was reduced to 17% in mice treated with cr2-fh , which is not statistically different from never smokers ( p = 0.06 ) . in addition , we measured the thickness of the onl , photoreceptor cell counts , and the area of the onl occupied by mller cells . smoke - exposed wt mice had a significantly thinner onl when compared to room air controls ( p < 0.05 ) , which is due to mller cell hypotrophy ( p < 0.01 ) as opposed to a loss in photoreceptors . in contrast , treatment with cr2-fh restored the thickness of the onl to normal levels ( p < 0.05 ) even though there was still evidence of mller cell hypotrophy ( p < 0.01 ) . quantification of morphologic structures from mice treated with alternative complement pathway inhibitor cr2-fh following cigarette smoke exposure ( cse ) there is ample evidence that morphologic and cellular alterations in dry amd lead to vision loss or impairment as measured by changes in visual acuity and contrast sensitivity . in particular , structural changes to brm , including thickening and lipid deposition , can lead to impaired exchange of waste and nutrients between the rpe and choroid as well as a reduction in the generation and delivery of 11-cis - retinal , the chromophore essential for proper visual function , to photoreceptor outer segments . however , it is unclear to what degree these changes are reversible . here , we generated amd - associated morphologic alterations and loss of contrast sensitivity in mice exposed to long - term smoke inhalation as reported previously . after 6 months of cse , animals were randomly split into two groups and assessed for visual function using optokinetic responses ( reported as pretreatment data in fig . 1 ) . as reported previously , spatial acuity in c57bl/6j mice was not affected by cse , resulting in acuity measures that did not differ from those of age - matched room air controls ( never smokers ; fig . however , contrast sensitivity was significantly reduced in cse mice , which exhibited a significant decrease in contrast sensitivity compared to never smokers ( group 1 , to be treated with pbs : 4.57 0.97 ; or group 2 , to be treated with cr2-fh : 5.78 0.52 ; versus never smokers : 10.14 0.41 ; p < 0.01 ; fig . 1b , pretreatment ) . optomotor responses were analyzed in c57bl/6j mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with nothing , pbs , or alternative complement pathway inhibitor , cr2-fh . ( a ) visual acuity was measured by identifying the spatial frequency threshold at a constant speed ( 12/s ) and contrast ( 100% ) . ( b ) contrast sensitivity was measured by taking the reciprocal of the contrast threshold at a fixed spatial frequency ( 0.131 cyc / deg ) and speed ( 12/s ) . we previously determined that this spatial frequency falls within the range of maximal contrast sensitivity for 9-month - old c57bl/6j mice ( data not shown ) . after cse , mice showed a significant reduction in contrast sensitivity compared to controls , which was still maintained after 3 months of cse cessation . however , smoke - exposed mice treated with cr2-fh showed a contrast sensitivity that was similar to room air controls and significantly higher than in pbs - treated mice following cse . data are expressed as mean sem ( n = 46 per condition ; * p < 0.05 ) . after confirming that cse animals exhibited loss of contrast sensitivity , mice were returned to room air and treated for 3 consecutive months with either cr2-fh given three times per week or pbs , using intraperitoneal injections . cr2-fh has both a targeting domain ( scrs 14 of cr2 ) that binds c3 fragments deposited at sites of complement activation and a complement inhibitory domain ( scrs 15 of fh ) . after 3 months of smoking cessation and treatment , animals were retested for visual function ( reported as posttreatment data in fig . as expected , spatial acuity in cse mice treated with either pbs or cr2-fh did not differ from mice exposed to room air ( fig . 1a ; p = 0.44 ) . on the other hand , cse mice treated with pbs still exhibited a robust decrease in contrast sensitivity ( smoke + pbs ; 4.89 1.14 ) compared to never smokers ( control ; 9.97 0.53 ; p < 0.001 ; fig . importantly , contrast sensitivity threshold in cse mice treated with cr2-fh recovered to levels similar to those of age - matched never smokers ( smoke + cr2-fh : 9.97 0.53 versus control never smokers : 9.14 0.39 ; p = 0.29 ) . linear regression analysis for pre- and posttreatment contrast sensitivity confirmed the marked improvement in cr2-fh treated animals ( p < 0.0001 ) . changes in gene expression following smoke exposure for six gene categories have been shown previously . to recap , after 6 months of cse , expression of genes whose products are involved in photoreceptor cell function , complement inhibition , and inhibition of angiogenic and lysosomal function was downregulated , whereas increases were observed for genes whose products are involved in complement activation , angiogenesis , oxidative stress , and mitochondrial fission and fusion . , we asked how cessation of smoking alone , or cessation of smoking coupled with cr2-fh therapy , affects expression of the same set of genes ( fig . analysis of marker gene expression in smoke - exposed mice following cessation of cigarette smoke exposure ( cse ) and treatment with pbs or alternative complement pathway inhibitor , cr2-fh , using quantitative rt - pcr on cdna generated from retina and rpe / choroid / sclera fraction . please note that with the exception of rho , opn1sw , and opn1mw , which were examined in the retina fraction , all other genes were examined in the rpe / choroid / sclera fraction . quantitative values were obtained by cycle number ( ct value ) , determining the difference between the mean experimental and control ( actb ) ct values for pbs- and cr2-fh treated mice following cse versus room air exposed mice ( fold difference ) . candidates were examined from a number of categories including photoreceptor cell function ( rho , opn1sw , opn1mw , rpe65 ) , complement activation ( c3 , hc , cfb , cfd , cfh , cd55 , cd59a ) , control of angiogenesis ( vegfa , serpinf1 ) , oxidative stress ( hif1a , cp ) , autophagy ( lyz1 , lamp2 , klc3 ) , mitochondrial function ( mfn1 , mt - co1 , dnm1l , ndufb8 , pfkfb1 , hmox1 ) , and immune response ( il-17 and ror ) . significant changes were identified in several categories for cr2-fh treated mice , in particular , suggesting decreased complement activation and enhanced energy production . statistics represent comparisons between pbs- and cr2-fh - treated animals following cse versus never smokers and differences between cse mice treated with pbs versus cr2-fh . data are expressed as mean sem ( n = 3 per condition ; * p < 0.05 analyzing fold changes between treatment groups versus never smokers ; # p < 0.01 analyzing fold change differences between cse mice treated with pbs versus cr2-fh ) . three months after smoking cessation , pbs - treated animals had normal levels of gene expression for photoreceptor function , pro- and antiangiogenic factors , oxidative stress , and autophagy . misregulated genes , on the other hand , involved those mediating mitochondrial fission and fusion , as well as complement activation . interestingly , a significant increase in factors regulating the complement ap pathway ( fb and fd [ factor d ] ) was observed . the effects of cr2-fh treatment compared to pbs treatment following smoking cessation involved two major categories : energy metabolism and complement activation . cr2-fh was also found to reduce mitochondrial stress , as genes affecting mitochondrial fission and fusion were normalized and an increase in the protective response gene heme oxygenase 1 was observed . loss in contrast sensitivity following smoke exposure has previously been shown to be associated with specific morphologic alterations in rpe , brm , and mller cells . here , we asked whether these same features play a role in the recovery of visual function in cse mice treated with cr2-fh . we analyzed electron micrographs obtained from cr2-fh and pbs cohorts and age - matched never smokers , analyzing outer retina structure ( fig . electron micrographs of the outer retina obtained from age - matched mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with pbs ( b ) ( smoke + pbs ) , cr2-fh ( c ) ( smoke + alternative complement pathway inhibitor , cr2-fh ) , or 3 additional months of room air ( a ) ( control , never smokers ) were compared . while all animals had the same number of photoreceptors per row ( 9 ) , the nuclei were more densely packed in the smoke + pbs when compared to the control and cr2-fh treated animals . ipl , inner plexiform layer ; inl , inner nuclear layer ; opl , outer plexiform layer ; onl , outer nuclear layer ; is , inner segments ; os , outer segments . cr2-fh rescues ultrastructural changes in rpe / brm / cc complex in mice following cigarette exposure . electron micrographs of the retinal pigment epithelium / bruch 's membrane / choriocapillaris complex ( rpe / brm / cc ) obtained from age - matched mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with pbs ( smoke + pbs ) , cr2-fh ( smoke + alternative complement pathway inhibitor , cr2-fh ) , or 3 additional months of room air ( control , never smokers ) were compared , providing an rpe / brm / cc overview ( a c ) , or focusing on mitochondria ( d f ) and brm ( g i ) . in an animal raised in room air ( control , never smoker ) , brm exhibits an organized pentalaminar structure consisting of rpe basement membrane , inner collagenous layer , middle elastic layer , outer collagenous layer , and choriocapillaris basement membrane , and the choriocapillaris endothelium has fenestrations ( arrowheads ) along the entire membrane ( a , g ) . ( b , h ) the rpe / brm / cc in a mouse following cse ( smoke + pbs ) exhibits pathologic changes . bruch 's membrane is disorganized , no longer showing a pentalaminar structure , and large deposits ( asterisks ) are present in the outer collagen layer ( ocl ) . notably , there is significant fenestration loss and/or endothelial cell thickening adjacent to ocl deposits . ( c , i ) the rpe / brm / cc appears normal in mice treated with cr2-fh following cse ( smoke + cr2-fh ) . the morphologic features of mitochondria shown in mice exposed to smoke mitochondria have degraded outer membranes and disorganized cristae ( e ) , but normal appearance in never smokers ( d ) and mice treated with cr2-fh following cse ( f ) . bruch 's membrane is a pentalaminar structure consisting of the basement membrane of the rpe , the inner collagenous layer , the middle elastic layer , the ocl , and the basement membrane of the cc . the pentalaminar structure of brm was disrupted as evidenced by an inconsistent middle elastic layer ( figs . areas of brm disorganization were especially prevalent near large deposits located in the ocl ( asterisks ) that are absent in never smokers . in contrast , never smokers exhibit a high fenestration count with few to no ocl deposits . f ) were significantly altered in cse animals , characterized by poorly defined outer membranes and disorganized cristae . finally , mitochondria , which in a healthy rpe cell are distributed in a basal to apical gradient but lose this arrangement in unhealthy cells such as those exposed to cse , were found to localize in a pattern that was not different between never smokers and cse mice in any of the four localization bins analyzed ( fig . these results are consistent with previous findings from our lab obtained from animals examined immediately following the completion of the 6-month cse period , with the exception of the lack of mitochondrial mislocalization . bruch 's membrane appeared intact , exhibiting the full pentalaminar structure , without any apparent thickening . few deposits were found in brm , and fenestrations were evenly distributed along the cc . likewise , the mitochondrial phenotype also appeared to be healthy , as evidenced by a clear outer membrane and organized cristae . 3 ) by determining their centroid coordinates as a percentage of the corresponding retinal pigment epithelium ( rpe ) length and thickness , respectively . each centroid was subsequently assigned to one of four bins ( basolateral , basal , central , or apical ) and expressed as percent of total mitochondria ( mito ) . mitochondria are localized predominantly to the basolateral and basal compartments of the rpe cells with fewer localized in the central and apical portions in never smokers . while we previously reported that cigarette smoke exposure ( cse ) affects the mitochondrial distribution in c57bl/6j mice after 6 months , with mitochondria exhibiting an apical shift from the basal to central compartment , mitochondrial distribution is back to normal 3 months after the cessation of cse ( smoke + pbs ) , and is not further affected by complement inhibition ( smoke + cr2-fh ) . electron microscopy results were quantified ( table 2 based on data such as those in figs . 3 , 4 ) focusing on morphologic correlates of energy metabolism in the rpe ( mitochondria ) , nutrient and waste transport ( brm thickness , ocl deposit count , and fenestrations in the cc ) , and photoreceptor function ( onl thickness , photoreceptor cell counts , and size of mller cells ) . mitochondria were found to be significantly larger in cse mice compared to never smokers ( p < 0.05 ) and made up a larger percentage of total rpe cell area ( p < 0.05 ) . the total number of mitochondria was unchanged between all three groups as was their localization . a thickening of brm in cse mice was observed compared to never smokers ; however , this did not reach statistical significance . cigarette smoke exposure triggers the formation of ocl deposits in brm , which are maintained in cse mice treated with vehicle ( p < 0.01 ) but removed in those treated with cr2-fh . fenestrations counted along 100-m brm / cc sections revealed , on average , a 53% decline of the fenestrations in cse mice ( p = 0.002 ) , a deficit that was reduced to 17% in mice treated with cr2-fh , which is not statistically different from never smokers ( p = 0.06 ) . in addition , we measured the thickness of the onl , photoreceptor cell counts , and the area of the onl occupied by mller cells . smoke - exposed wt mice had a significantly thinner onl when compared to room air controls ( p < 0.05 ) , which is due to mller cell hypotrophy ( p < 0.01 ) as opposed to a loss in photoreceptors . in contrast , treatment with cr2-fh restored the thickness of the onl to normal levels ( p < 0.05 ) even though there was still evidence of mller cell hypotrophy ( p < 0.01 ) . quantification of morphologic structures from mice treated with alternative complement pathway inhibitor cr2-fh following cigarette smoke exposure ( cse ) while we confirmed and extended our previous data on retinal structure and function in mice following long - term smoke exposure , we additionally addressed two specific questions : first , are cse - induced changes reversible upon smoke cessation ; and second , since the pathologic changes are complement dependent , can we accelerate their resolution with complement inhibitory therapy ? first , are the structural and functional changes induced by cse reversible upon cessation of smoking ( smoke + pbs ) ? previously , we showed that while spatial acuity is retained in mice after 6 months of cse , contrast sensitivity is reduced by 60% . allowing mice to recover in room air for 3 months only marginally affected contrast sensitivity , as the threshold was still suppressed by 50% . similarly , the thinning of the onl observed after 6 months of smoke is retained after 3 months of recovery , as are mller cell atrophy , the presence of large numbers of ocl deposits in brm , and the increase in mitochondrial area . overall brm thickness after 3 months of room air was still increased in pbs mice by 30% ; however , this difference was no longer significant based on the large variability in brm thickness in cse mice . finally , the mislocalization of mitochondria observed after cse was no longer apparent following recovery in 3 months of room air . in patients , the question whether smoke - induced pathology is reversible has not yet been addressed . however , neuner and colleagues have posed a related question in the muenster aging and retina study . the group followed aged smokers and nonsmokers without amd over a median of 30.9 months and reported their adjusted risk ratios for incident amd . of the 9.6% of subjects who progressed to amd , the adjusted risk ratio in current smokers versus never smokers was 3.25 ( 95% confidence interval [ 1.507.06 ] ) , but was still significantly elevated by 1.28 ( 0.702.33 ) in former smokers versus never smokers . second , is continued complement activation required to maintain the structural and functional deficits observed in cse mice , or can damage be reversed with complement inhibition ? our group previously described a recombinant site - targeted inhibitor of the complement ap , cr2-fh . this fusion protein consists of the ap - inhibitory domain of mouse fh linked to a cr2 targeting fragment that binds membrane - bound complement activation products , and cr2-fh is orders of magnitude more effective at blocking the ap in vitro than native fh . cr2-fh has been shown to reduce complement activation , inflammation , and injury to the colon in a model of chronic colitis , to block the progression of both acute and chronic autoimmune demyelinating disease in an experimental encephalomyelitis study , and to attenuate deficits associated with cerebral and cardiac ischemia reperfusion injury . cr2-fh has also been used to inhibit cnv , a process requiring ap activation , as well as smoke - induced lipid deposition in cultured rpe cells . however , no studies have been conducted to investigate the efficacy of this ap inhibitory protein in a dry amd model , particularly using a therapeutic rather than preventative paradigm . here , we show the first conclusive evidence that cr2-fh is effective in reducing and reversing smoke - induced ocular pathology in a novel smoking cessation model . we previously showed that preventing ap activation ( fb ko mice ) prevented the development of all cse - mediated structural and functional alterations . here , we asked whether cr2-fh can accelerate the reversal of pathology when applied for a 12-week period post smoking cessation . remarkably , we found that smoke - induced decreases in contrast sensitivity can be completely reversed following treatment with cr2-fh . functional improvement was found to correlate with an attenuation of morphologic differences in rpe / brm / cc . not only did cr2-fh reverse the trend toward a thicker brm and increases in ocl deposit formation caused by cse , but it also reversed alterations in the cc , including restoration of fenestrations in areas of close proximity to ocl deposits . in addition , cr2-fh was effective in reversing all mitochondrial impairments observed under smoke conditions . our data suggest that inhibition of the complement ap is paramount to reversing ocular smoke pathology , and cr2-fh is a potent inhibitor of this pathway . further experiments need to be conducted to determine the therapeutic window , most effective dosage , and ideal delivery modality for this ap inhibitor following cse or other complement - dependent amd models . it will be of great interest to determine how these data relate to the ocular changes found in ga patients , in particular in light of the recent findings of the phase ii clinical trial for lampalizumab , in which an anti - fd monoclonal antibody applied intravitreally appeared to reduce geographic atrophy ( ga ) lesion size in a subpopulation of patients . how might smoke - induced ocular pathology be reversible with complement inhibition ? affected structures such as drusen , brm , rpe , and cc exhibit signs of complement activation and contain neoepitopes that continue to trigger complement activation . hence , in the absence of sufficient complement inhibition , de novo complement activation triggered by the continued presence of neoantigens and complement amplification through the complement ap will continue to drive complement activation and maintain the damaged structures even in the absence of the toxic stimulus , which in the present case is smoking . this hypothesis is supported by our gene expression study , indicating a rebound effect of ap inhibition , since both of the required activators of the ap are significantly upregulated upon cessation of smoking but return to normal levels in the presence of cr2-fh . it is plausible that in the absence of continued complement activation , complement - mediated damage leading to the growth of the lesions is reduced , and reparative mechanisms including removal of debris by macrophages , and repair of cellular metabolism by mitochondrial biogenesis , can occur . however , the exact mechanism whereby cr2-fh slows down or reverses the progression of disease requires further investigation . in summary , there is a growing body of evidence linking oxidative stress , cigarette smoking , and complement activation to the development and progression of amd . our data provided herein demonstrate that cse in mice causes behavioral and morphologic defects in the retina , rpe , brm , and cc , similar to those observed in patients with dry amd , and it persists post smoking cessation . cr2-fh administered to mice following cse provides the first evidence that complement - based therapy is effective in treating smoke - induced ocular pathology , and suggests that this line of therapy may be effective in treating dry amd in humans .

purposemorphologic and genetic evidence exists that an overactive complement system driven by the complement alternative pathway ( ap ) is involved in pathogenesis of age - related macular degeneration ( amd ) . smoking is the only modifiable risk factor for amd . as we have shown that smoke - related ocular pathology can be prevented in mice that lack an essential activator of ap , we ask here whether this pathology can be reversed by increasing inhibition in ap.methodsmice were exposed to either cigarette smoke ( cs ) or filtered air ( 6 hours / day , 5 days / week , 6 months ) . smoke - exposed animals were then treated with the ap inhibitor ( cr2-fh ) or vehicle control ( pbs ) for the following 3 months . spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months ; additional readouts included assessment of retinal morphology by electron microscopy ( em ) and gene expression analysis by quantitative rt-pcr.resultsthe cs mice treated with cr2-fh showed significant improvement in contrast threshold compared to pbs - treated mice , whereas spatial frequency was unaffected by cs or pharmacologic intervention . treatment with cr2-fh in cs animals reversed thinning of the retina observed in pbs - treated mice as analyzed by spectral - domain optical coherence tomography , and reversed most morphologic changes in rpe and bruch 's membrane seen in cs animals by em.conclusionstaken together , these findings suggest that ap inhibitors not only prevent , but have the potential to accelerate the clearance of complement - mediated ocular injury . improving our understanding of the regulation of the ap is paramount to developing novel treatment approaches for amd .

Materials and Methods Animals Exposure to Cigarette Smoke Optokinetic Response Test Tissue Preparation Ultrastructural Analysis Quantitative RT-PCR Statistics Results Effect of CR2-fH Treatment on Smoke-Induced Impairment of Visual Function Effect of CR2-fH Treatment on Smoke-Induced Changes in Gene Expression Effects of CR2-fH on Morphologic Alterations in RPE-BrM Caused by Constant Smoke Exposure Discussion

all experiments were approved by the medical university of south carolina institutional animal care and use committee and performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research . cigarette smoke exposure was carried out ( 5 hours per day , 5 days per week ) by burning 3r4f reference cigarettes ( university of kentucky , louisville , ky , usa ) using a smoking machine ( model te-10 ; teague enterprises , woodland , ca , usa ) for 6 months as published previously . following 6 months of cse , smoke - exposed animals were randomized into two groups ( 1 , control pbs ; 2 , cr2-fh treated ) , and mice were followed for 12 weeks post smoking cessation . all tests were conducted under photopic conditions with a mean luminance of 52 cd m. visual acuity was measured by finding the spatial frequency threshold of each animal at a constant speed ( 12/s ) and contrast ( 100% ) with a staircase procedure that systematically increased the spatial frequency of the grating until the animal no longer exhibited detectable responses . contrast sensitivity was determined by taking the reciprocal of the contrast threshold at a fixed spatial frequency ( 0.131 cyc / deg ) and speed ( 12/s ) . electron microscopy images were captured using a jeol jem 1400 ( peabody , ma , usa ) transmission electron microscope using serialem ( peabody , ma , usa ) software to automate image capture overnight with 1200 to 1500 images captured per section , yielding datasets that were then processed with the ncr toolset to generate image mosaics with corrections for image aberrations induced by electron microscopy . fold changes in qrt - pcr experiments were analyzed by z - test ( treatment versus never smokers ; p < 0.05 ) and t - test using the holm - sidak method ( cse mice , pbs versus cr2-fh , with = 5.0% ; p < 0.01 ) . the control group was kept in a filtered air environment , and the two experimental groups were subjected to cigarette smoke . cigarette smoke exposure was carried out ( 5 hours per day , 5 days per week ) by burning 3r4f reference cigarettes ( university of kentucky , louisville , ky , usa ) using a smoking machine ( model te-10 ; teague enterprises , woodland , ca , usa ) for 6 months as published previously . following 6 months of cse , smoke - exposed animals were randomized into two groups ( 1 , control pbs ; 2 , cr2-fh treated ) , and mice were followed for 12 weeks post smoking cessation . all tests were conducted under photopic conditions with a mean luminance of 52 cd m. visual acuity was measured by finding the spatial frequency threshold of each animal at a constant speed ( 12/s ) and contrast ( 100% ) with a staircase procedure that systematically increased the spatial frequency of the grating until the animal no longer exhibited detectable responses . contrast sensitivity was determined by taking the reciprocal of the contrast threshold at a fixed spatial frequency ( 0.131 cyc / deg ) and speed ( 12/s ) . electron microscopy images were captured using a jeol jem 1400 ( peabody , ma , usa ) transmission electron microscope using serialem ( peabody , ma , usa ) software to automate image capture overnight with 1200 to 1500 images captured per section , yielding datasets that were then processed with the ncr toolset to generate image mosaics with corrections for image aberrations induced by electron microscopy . , fold changes in qrt - pcr experiments were analyzed by z - test ( treatment versus never smokers ; p < 0.05 ) and t - test using the holm - sidak method ( cse mice , pbs versus cr2-fh , with = 5.0% ; p < 0.01 ) . there is ample evidence that morphologic and cellular alterations in dry amd lead to vision loss or impairment as measured by changes in visual acuity and contrast sensitivity . here , we generated amd - associated morphologic alterations and loss of contrast sensitivity in mice exposed to long - term smoke inhalation as reported previously . however , contrast sensitivity was significantly reduced in cse mice , which exhibited a significant decrease in contrast sensitivity compared to never smokers ( group 1 , to be treated with pbs : 4.57 0.97 ; or group 2 , to be treated with cr2-fh : 5.78 0.52 ; versus never smokers : 10.14 0.41 ; p < 0.01 ; fig . optomotor responses were analyzed in c57bl/6j mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with nothing , pbs , or alternative complement pathway inhibitor , cr2-fh . ( b ) contrast sensitivity was measured by taking the reciprocal of the contrast threshold at a fixed spatial frequency ( 0.131 cyc / deg ) and speed ( 12/s ) . after cse , mice showed a significant reduction in contrast sensitivity compared to controls , which was still maintained after 3 months of cse cessation . however , smoke - exposed mice treated with cr2-fh showed a contrast sensitivity that was similar to room air controls and significantly higher than in pbs - treated mice following cse . as expected , spatial acuity in cse mice treated with either pbs or cr2-fh did not differ from mice exposed to room air ( fig . on the other hand , cse mice treated with pbs still exhibited a robust decrease in contrast sensitivity ( smoke + pbs ; 4.89 1.14 ) compared to never smokers ( control ; 9.97 0.53 ; p < 0.001 ; fig . importantly , contrast sensitivity threshold in cse mice treated with cr2-fh recovered to levels similar to those of age - matched never smokers ( smoke + cr2-fh : 9.97 0.53 versus control never smokers : 9.14 0.39 ; p = 0.29 ) . to recap , after 6 months of cse , expression of genes whose products are involved in photoreceptor cell function , complement inhibition , and inhibition of angiogenic and lysosomal function was downregulated , whereas increases were observed for genes whose products are involved in complement activation , angiogenesis , oxidative stress , and mitochondrial fission and fusion . analysis of marker gene expression in smoke - exposed mice following cessation of cigarette smoke exposure ( cse ) and treatment with pbs or alternative complement pathway inhibitor , cr2-fh , using quantitative rt - pcr on cdna generated from retina and rpe / choroid / sclera fraction . data are expressed as mean sem ( n = 3 per condition ; * p < 0.05 analyzing fold changes between treatment groups versus never smokers ; # p < 0.01 analyzing fold change differences between cse mice treated with pbs versus cr2-fh ) . three months after smoking cessation , pbs - treated animals had normal levels of gene expression for photoreceptor function , pro- and antiangiogenic factors , oxidative stress , and autophagy . loss in contrast sensitivity following smoke exposure has previously been shown to be associated with specific morphologic alterations in rpe , brm , and mller cells . here , we asked whether these same features play a role in the recovery of visual function in cse mice treated with cr2-fh . electron micrographs of the outer retina obtained from age - matched mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with pbs ( b ) ( smoke + pbs ) , cr2-fh ( c ) ( smoke + alternative complement pathway inhibitor , cr2-fh ) , or 3 additional months of room air ( a ) ( control , never smokers ) were compared . electron micrographs of the retinal pigment epithelium / bruch 's membrane / choriocapillaris complex ( rpe / brm / cc ) obtained from age - matched mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with pbs ( smoke + pbs ) , cr2-fh ( smoke + alternative complement pathway inhibitor , cr2-fh ) , or 3 additional months of room air ( control , never smokers ) were compared , providing an rpe / brm / cc overview ( a c ) , or focusing on mitochondria ( d f ) and brm ( g i ) . ( c , i ) the rpe / brm / cc appears normal in mice treated with cr2-fh following cse ( smoke + cr2-fh ) . the morphologic features of mitochondria shown in mice exposed to smoke mitochondria have degraded outer membranes and disorganized cristae ( e ) , but normal appearance in never smokers ( d ) and mice treated with cr2-fh following cse ( f ) . bruch 's membrane is a pentalaminar structure consisting of the basement membrane of the rpe , the inner collagenous layer , the middle elastic layer , the ocl , and the basement membrane of the cc . taken together , these results are consistent with previous findings from our lab obtained from animals examined immediately following the completion of the 6-month cse period , with the exception of the lack of mitochondrial mislocalization . while we previously reported that cigarette smoke exposure ( cse ) affects the mitochondrial distribution in c57bl/6j mice after 6 months , with mitochondria exhibiting an apical shift from the basal to central compartment , mitochondrial distribution is back to normal 3 months after the cessation of cse ( smoke + pbs ) , and is not further affected by complement inhibition ( smoke + cr2-fh ) . cigarette smoke exposure triggers the formation of ocl deposits in brm , which are maintained in cse mice treated with vehicle ( p < 0.01 ) but removed in those treated with cr2-fh . fenestrations counted along 100-m brm / cc sections revealed , on average , a 53% decline of the fenestrations in cse mice ( p = 0.002 ) , a deficit that was reduced to 17% in mice treated with cr2-fh , which is not statistically different from never smokers ( p = 0.06 ) . in contrast , treatment with cr2-fh restored the thickness of the onl to normal levels ( p < 0.05 ) even though there was still evidence of mller cell hypotrophy ( p < 0.01 ) . quantification of morphologic structures from mice treated with alternative complement pathway inhibitor cr2-fh following cigarette smoke exposure ( cse ) there is ample evidence that morphologic and cellular alterations in dry amd lead to vision loss or impairment as measured by changes in visual acuity and contrast sensitivity . here , we generated amd - associated morphologic alterations and loss of contrast sensitivity in mice exposed to long - term smoke inhalation as reported previously . however , contrast sensitivity was significantly reduced in cse mice , which exhibited a significant decrease in contrast sensitivity compared to never smokers ( group 1 , to be treated with pbs : 4.57 0.97 ; or group 2 , to be treated with cr2-fh : 5.78 0.52 ; versus never smokers : 10.14 0.41 ; p < 0.01 ; fig . optomotor responses were analyzed in c57bl/6j mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with nothing , pbs , or alternative complement pathway inhibitor , cr2-fh . ( b ) contrast sensitivity was measured by taking the reciprocal of the contrast threshold at a fixed spatial frequency ( 0.131 cyc / deg ) and speed ( 12/s ) . after cse , mice showed a significant reduction in contrast sensitivity compared to controls , which was still maintained after 3 months of cse cessation . however , smoke - exposed mice treated with cr2-fh showed a contrast sensitivity that was similar to room air controls and significantly higher than in pbs - treated mice following cse . as expected , spatial acuity in cse mice treated with either pbs or cr2-fh did not differ from mice exposed to room air ( fig . on the other hand , cse mice treated with pbs still exhibited a robust decrease in contrast sensitivity ( smoke + pbs ; 4.89 1.14 ) compared to never smokers ( control ; 9.97 0.53 ; p < 0.001 ; fig . importantly , contrast sensitivity threshold in cse mice treated with cr2-fh recovered to levels similar to those of age - matched never smokers ( smoke + cr2-fh : 9.97 0.53 versus control never smokers : 9.14 0.39 ; p = 0.29 ) . to recap , after 6 months of cse , expression of genes whose products are involved in photoreceptor cell function , complement inhibition , and inhibition of angiogenic and lysosomal function was downregulated , whereas increases were observed for genes whose products are involved in complement activation , angiogenesis , oxidative stress , and mitochondrial fission and fusion . analysis of marker gene expression in smoke - exposed mice following cessation of cigarette smoke exposure ( cse ) and treatment with pbs or alternative complement pathway inhibitor , cr2-fh , using quantitative rt - pcr on cdna generated from retina and rpe / choroid / sclera fraction . data are expressed as mean sem ( n = 3 per condition ; * p < 0.05 analyzing fold changes between treatment groups versus never smokers ; # p < 0.01 analyzing fold change differences between cse mice treated with pbs versus cr2-fh ) . three months after smoking cessation , pbs - treated animals had normal levels of gene expression for photoreceptor function , pro- and antiangiogenic factors , oxidative stress , and autophagy . loss in contrast sensitivity following smoke exposure has previously been shown to be associated with specific morphologic alterations in rpe , brm , and mller cells . here , we asked whether these same features play a role in the recovery of visual function in cse mice treated with cr2-fh . electron micrographs of the outer retina obtained from age - matched mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with pbs ( b ) ( smoke + pbs ) , cr2-fh ( c ) ( smoke + alternative complement pathway inhibitor , cr2-fh ) , or 3 additional months of room air ( a ) ( control , never smokers ) were compared . electron micrographs of the retinal pigment epithelium / bruch 's membrane / choriocapillaris complex ( rpe / brm / cc ) obtained from age - matched mice after 6 months of cigarette smoke exposure ( cse ) or room air followed by 3 months of treatment with pbs ( smoke + pbs ) , cr2-fh ( smoke + alternative complement pathway inhibitor , cr2-fh ) , or 3 additional months of room air ( control , never smokers ) were compared , providing an rpe / brm / cc overview ( a c ) , or focusing on mitochondria ( d f ) and brm ( g i ) . ( c , i ) the rpe / brm / cc appears normal in mice treated with cr2-fh following cse ( smoke + cr2-fh ) . the morphologic features of mitochondria shown in mice exposed to smoke mitochondria have degraded outer membranes and disorganized cristae ( e ) , but normal appearance in never smokers ( d ) and mice treated with cr2-fh following cse ( f ) . bruch 's membrane is a pentalaminar structure consisting of the basement membrane of the rpe , the inner collagenous layer , the middle elastic layer , the ocl , and the basement membrane of the cc . while we previously reported that cigarette smoke exposure ( cse ) affects the mitochondrial distribution in c57bl/6j mice after 6 months , with mitochondria exhibiting an apical shift from the basal to central compartment , mitochondrial distribution is back to normal 3 months after the cessation of cse ( smoke + pbs ) , and is not further affected by complement inhibition ( smoke + cr2-fh ) . cigarette smoke exposure triggers the formation of ocl deposits in brm , which are maintained in cse mice treated with vehicle ( p < 0.01 ) but removed in those treated with cr2-fh . fenestrations counted along 100-m brm / cc sections revealed , on average , a 53% decline of the fenestrations in cse mice ( p = 0.002 ) , a deficit that was reduced to 17% in mice treated with cr2-fh , which is not statistically different from never smokers ( p = 0.06 ) . in addition , we measured the thickness of the onl , photoreceptor cell counts , and the area of the onl occupied by mller cells . in contrast , treatment with cr2-fh restored the thickness of the onl to normal levels ( p < 0.05 ) even though there was still evidence of mller cell hypotrophy ( p < 0.01 ) . quantification of morphologic structures from mice treated with alternative complement pathway inhibitor cr2-fh following cigarette smoke exposure ( cse ) while we confirmed and extended our previous data on retinal structure and function in mice following long - term smoke exposure , we additionally addressed two specific questions : first , are cse - induced changes reversible upon smoke cessation ; and second , since the pathologic changes are complement dependent , can we accelerate their resolution with complement inhibitory therapy ? previously , we showed that while spatial acuity is retained in mice after 6 months of cse , contrast sensitivity is reduced by 60% . similarly , the thinning of the onl observed after 6 months of smoke is retained after 3 months of recovery , as are mller cell atrophy , the presence of large numbers of ocl deposits in brm , and the increase in mitochondrial area . our group previously described a recombinant site - targeted inhibitor of the complement ap , cr2-fh . here , we show the first conclusive evidence that cr2-fh is effective in reducing and reversing smoke - induced ocular pathology in a novel smoking cessation model . remarkably , we found that smoke - induced decreases in contrast sensitivity can be completely reversed following treatment with cr2-fh . our data suggest that inhibition of the complement ap is paramount to reversing ocular smoke pathology , and cr2-fh is a potent inhibitor of this pathway . this hypothesis is supported by our gene expression study , indicating a rebound effect of ap inhibition , since both of the required activators of the ap are significantly upregulated upon cessation of smoking but return to normal levels in the presence of cr2-fh . it is plausible that in the absence of continued complement activation , complement - mediated damage leading to the growth of the lesions is reduced , and reparative mechanisms including removal of debris by macrophages , and repair of cellular metabolism by mitochondrial biogenesis , can occur . our data provided herein demonstrate that cse in mice causes behavioral and morphologic defects in the retina , rpe , brm , and cc , similar to those observed in patients with dry amd , and it persists post smoking cessation . cr2-fh administered to mice following cse provides the first evidence that complement - based therapy is effective in treating smoke - induced ocular pathology , and suggests that this line of therapy may be effective in treating dry amd in humans .

shoulder impingement syndrome ( sis ) is a common complaint for patients of all ages and different activity levels . it has been defined as compression and mechanical abrasion of the rotator cuff structures as they pass beneath the coracoacromial arch during elevation of the arm ( 1 ) . a wide range of different factors is involved in causing sis : including anatomic abnormalities of the coracoacromial arch or humeral head ( 2 ) , tension overload ischemia ( 3 ) , repetitive eccentric overload ( 2 - 3 ) , aberrant kinematic patterns due to poor rotator cuff or scapular muscles function ( 2,4 - 5 ) , and poor posture and scapular kinematic abnormalities ( 2 ) . people who are constantly doing repetitive overhead motions relate to their occupations or athletic activities are also at risk of sis ( 1 , 6 ) . pain and dysfunction of patient with sis occur when the shoulder is placed in positions of elevation , an activity that is commonplace during many sporting and daily living activities . thus , the patients are at risk of losing their physical independence and jobs which have important socioeconomic implications . the scapular position and motions on the thorax is a critical component of the normal glenohumeral function and plays great roles in facilitating optimal shoulder movements ( 6 - 8 ) . in normal upper - quarter function these muscles must dynamically position the glenoid so that efficient glenohumeral movement can occur . during all movements of the glenohumeral joint especially overhead elevation of the arm , it is of great importance that the scapular - stabilizing musculature should be strong enough to properly position the scapula . the main scapula stabilizers are the levator scapulae , rhomboids major and minor , serratus anterior , and trapezii . these muscle groups function through synergistic co - contraction with rotator cuff to control the scapular movement ( 9 ) . when weakness or dysfunction is present in the scapular musculature , normal scapular positioning and mechanics may become altered which result in abnormal stresses to the capsular structures , rotator cuff compression and reduced performance ( 7 ) . there are some evidences suggesting that kinematic of scapulothoracic motions were impaired in sis ( 2 , 4 , 9 - 10 ) . the scapulothoracic kinematics can be altered in response to inappropriate or incorrect movement pattern , macro and micro traumatic injuries , abnormal scapulohumeral rhythm and other shoulder pathologies ( 8,10 ) . altered muscle activity in the scapular muscles is commonly believed to be a crucial factor contributing to sis ( 2,11 - 12 ) . the scapulothoracic kinematics are also affected by abnormal posture of thoracic and cervical spine ( 13 ) . this winging pattern appears to represent scapular internal rotation and anterior tilting ( 14 ) . recently , 3-dimensional kinematic analysis has demonstrated decreased scapular posterior tilt , decreased upward rotation , and decreased scapular external rotation during glenohumeral elevation in sis patients ( 2,5 ) . the treatment of sis is % 90 -95 conservative and often includes rotator cuff strengthening exercises ( 6,15 ) , stretching exercises ( 16 ) , immobilization , passive , active and active assisted range of motion exercises ( rom ) , various mobilization techniques , home exercise programs ( 17 ) and various physical therapy methods such as heat , transcutaneous electrical nerve stimulation ( tens ) and ultrasound ( us ) and etc ( 18 - 19 ) . in a conservative approach , new insights in shoulder rehabilitation emphasize the dynamic stabilization of the scapula as an essential part of the management because the ability to position and control movements of the scapula is very important for optimal upper limb function . when the scapula fails to perform its stabilization role , shoulder function is inefficient , which can result not only in decreased neuromuscular performance , but also may predispose the individual to shoulder injuries ( 20 ) . this lack of attention may often lead into the incomplete treatment ( 7 ) , therefore , reestablishment of normal shoulder function and restoring normal scapular muscle activation patterns by scapular stabilization based exercises , in our view , are the keys to a successful rehabilitation program . the purpose of our investigation was to compare the effectiveness of two treatment approaches for impingement syndrome of the shoulder : ( 1 ) a 6-week scapular stabilization based exercises , and ( 2 ) a rom exercise program combined with physical modalities on pain , posture , flexibility and mobility of sis patients . ethical approval for this study was granted by the research ethics committee of tehran university of medical sciences ( grant number 90 - 9 - 28 - 1992 ) . subjects were provided with information booklets explaining the purpose of the study and signed informed consent documents prior to participation . subjects were free to withdraw from the study at any time . this study was conducted in hazrat rasool - e - akram hospital located in tehran ( iran ) between 2011and 2012 . subjects : a total of 98 subjects was initially recruited from hazrat rasool - e - akram hospital sports medicine and orthopedic clinics and judged to meet the criteria for the study . inclusion and exclusion criteria were assessed for each subject based on a clinical examination performed by the first author . the inclusion criteria were as follows : ( 1 ) male and female mentally fitted between the ages of 18 to 75 years ; ( 2 ) unilateral shoulder pain of more than one month localized ( anterior and/or anterolateral ) to the acromion ; ( 3 ) tenderness to palpation of the rotator cuff tendons ; ( 4 ) positive impingement tests , or a painful arc of movement ( 60120 ) ; ( 5 ) pain produced or increased during flexion and/or abduction of the symptomatic shoulder . forward shoulder translation measurement scapular protraction measurement all subjects tested positive for impingement tests ( which included the hawkins , neer , and empty can tests ) and underwent a full screening of cervical and shoulder rom , resisted motions , and special tests . therefore , to correctly identify patients with shoulder impingement , a combination of clinical tests is recommended . according to ure et al . findings , multiple tests were able to correctly distinguish sis from other shoulder pathologies in 86% of cases ( 21 - 22 ) . exclusion criteria were as follows : ( 1 ) cervical or shoulder symptoms reproduced by a cervical screening exam ; ( 2 ) abnormal results with reflex or thoracic outlet tests ; ( 3 ) symptoms of numbness or tingling in the upper extremity ; ( 4 ) pregnancy , or ( 5 ) a history of the followings : onset of symptoms due to traumatic injury , glenohumeral joint dislocation , acromioclavicular joint separation , shoulder fracture , surgery on the shoulder , fibromyalgia , use of any treatment within three months . main participants were 72 sis patients who were randomly allocated into two groups : ( 1 ) scapular stabilization based exercise therapy group ( et ) and ( 2 ) physical therapy group ( pt ) . random allocation of the subjects was done by using a random number table and block random sampling ; a : et ; b : pt ( a block size of 4 ) . the subjects who were included in the study signed university - approved informed consent forms and completed demographic data sheets . four subjects were excluded at the intervention stage and three subjects in et group due to irregular attendance at therapy sessions , travel and an accident and one subject in pt group due to irregular attendance at therapy sessions ( flowchart . in addition to inclusion - exclusion criteria , the clinical evaluation included assessment of pain , active shoulder external rotation and abduction range , forward head posture , mid - thoracic curve , forward shoulder translation , scapular protraction & rotation and pectoralis minor length were done pre and post intervention . the interventions for both groups were done by two clinicians that were unaware of the treatments groups . one supervised exercise therapy for et group on even days and the other did physical therapy for pt group on odd days . the power analysis of the study was performed to detect a 10% differences in pain and shoulder abduction with =0/05 and a power of 80% , a sample size of 36 per group was required . pain : subjects were asked to record their maximal pain during the movements based on visual analogue scale ( vas ) for pain . the vas used in the study was a 10-cm line where the 0 was marked as no pain and the 10 as the worst pain imaginable ( 23 ) . shoulder range of motion ( rom ) : the ranges of active external rotation and abduction were measured by a standard goniometer in both symptomatic and asymptomatic shoulders as follows : shoulder abduction was measured in the seated chair position , as in flexion , with the trunk upright . the arm was actively elevated in the strict coronal plane with the thumb pointed up toward the ceiling to allow the required external rotation necessary to avoid impingement of the greater tuberosity on the acromion process . shoulder external rotation was measured in supine with the hips and knees flexed to approximately 45 degrees . the tested arm was supported on the table in 90 degrees of abduction , elbow flexed to 90 degrees , the forearm in midway between pronation / supination and the wrist in neutral . a towel roll was placed under the humerus to ensure neutral horizontal positioning ; which required the humerus to be level to the acromion process based on visual inspection . once positioned , the participant was asked to rotate the arm into external rotation to the end available range without discomfort . once active end - range was achieved the measurement was recorded ( 24 ) . forward head posture ( fhp ) : to measure fhp , a lateral photograph was then taken of the cervicothoracic region , using a canon camera ( model : ixy digital 3000 is).the camera was placed 2 meters from the subject and mounted on a tripod , leveled with a bubble spirit level to control frontal and sagittal angles . this procedure has been used in previous published studies ( 25 - 27 ) . the method chosen to measure the fhp for the current investigation was direct measurement from lateral view photographs of head and shoulder posture . to measure the angles , an a4-sized sheet of graph paper the transparency film was then placed over the photograph and aligned so that one of the vertical lines was placed over the plumb line and the intersection of two vertical and horizontal lines coincided with the point the c7 marker came in contact with the skin . to calculate the position of the head in relation to c7 ( c7-tragus angle ) , the angle between the horizontal line and the line connecting tragus of the ear to spinous process of c7 , was measured with a goniometer and documented in degrees . mid - thoracic curve : this curvature was measured by palpating and marking the spinous processes of the second thoracic vertebra ( t2 ) and the twelfth thoracic vertebra ( t12 ) by counting spinous processes , beginning with c7 . the researcher placed the tip of the flexi ruler on t2 and conform it to the subject 's spine , marking the flexi ruler at t12 . the flexi ruler was transferred to a sheet of white paper , and the curve of the flexi ruler was traced . a metric ruler was used to measure the length and the flexi ruler was used to measure the height of the curve in centimeters . the following formula was used to determine mid - thoracic curvature : mid - thoracic curvature = = 4 [ arc tan ( 2 h / l ) ] where h = the height of the curve and l = the length of the curve ( fig 3 ) ( 25 ) . mid - thoracic curvature was measured and documented in degrees for each subject . anthropometric measurement of pectoralis forward shoulder translation(fst ) : fst was measured using a combination square ruler ( cl-01 model , e - base measuring tools co , taiwan ) consists of a 40-cm to measure the distance from the wall to the anterior tip of the subject 's acromion process . the anterior tip of acromion process was marked and the distance of the point and wall was measured and documented in millimeter to determine the amount of fst . this measurement was done three times for each shoulder and the average was recorded as fst ( 28 ) ( fig.1 ) . scapular protraction & rotation : scapular protraction and rotation were measured with the subject standing , subjects nodded the head and neck forward and backward five times , then inhaled and exhaled deeply to produce a natural , reproducible standing posture and head and neck position ( 25 ) . for determining scapular protraction , subjects were asked to adopt a comfortable and natural standing position . after palpation , non - allergenic adhesive markers 6 mm in diameter were attached to the following points ( fig . 2 ) : a= the root of the scapular spine b= a mark on the thoracic spine corresponding to the root of the scapular spine c= a mark on the thoracic spine corresponding to the inferior angle of the scapula d= the inferior angle of the scapula e= the tip of the acromion of the scapula . the following formula was used to determine scapular protraction : scapular protraction = baeae line bae = the distance from the mark on the thoracic spine corresponding to the root of the scapular spine to the tip of the acromion . line ae= the distance from the root of the scapular spine to the tip of the acromion . scapular rotation was measured by palpating and marking the inferior angle of the scapula ( d ) and the corresponding mark on the thoracic spine ( c ) . the following formula was used to determine scapular rotation : scapular rotation= tan =cdbc line cd= the distance between the inferior angle of the scapula and the corresponding mark on the thoracic spine . line bc= the distance between the marks on the thoracic spine corresponding to the root of the scapular spine and the inferior angle of the scapula ( fig . scapular rotation was measured and documented in degrees.scapular protraction and scapular rotation were measured bilaterally on each subject . the symmetry of the scapular was determined for each subject using the following formula : where l= the ratio of left scapular protraction to left scapular rotation , and r= the ratio of right scapular protraction to right scapular rotation . anthropometric measurement of pectoralis minor ( pm ) length : the anthropometric measurement of the pm length was performed according to the protocol , described by borstad ( 29 ) . before the pm length measurement , two anatomical landmarks were determined first the medial - inferior angle of the coracoid process and a second landmark just lateral to the sternocostal junction of the inferior aspect of the fourth rib . a caliper was used to measure the distance between both bony reference points ( fig . the pm length measurement was done three times for each method and the average was recorded . intervention protocols : following the evaluation , participants of et and pt groups began a six - week program ( three times per week ) . exercise therapy intervention - after a brief explanation about the exercises protocol , the et subjects participated in three supervised exercise sessions per week over a 6-week period ( 16 , 20 , 30 - 32 ) . this exercises protocol consisted of a 10 minute walking warm up on a treadmill ( dk city- dx3-b1 ) , flexibility , strengthening , scapular stabilization and postural exercises . the patients were asked to avoid any other exercises and severe daily activities during their treatment . at the first session , therabands ( the hygienic corp , akron , ohio ) are color - coded , with each color representing a different resistance . strength training with theraband consisted of exercises for rotator cuffs , scapular retractors , shoulder external rotators ( figs 4 , 5 ) , d2- pnf pattern ( fig .6 ) and serratus anterior punches ( fig .7 ) . the level of difficulty of exercises was increased based on quality of shoulder motion and perceived intensity of pain . the level of tubing resistance was adjusted accordingly for all subjects throughout the treatment process . at the first session , the et subjects were asked to do five repetitions of each of the tubing exercises to see if they were too hard or too easy ; then the appropriate theraband was chosen based on feedback from the subject , observation and palpation of the target muscles by the investigator . five subjects of the et group used tan ( ultrathin ) , sixteen used yellow ( thin ) , and the remaining subjects used red ( medium ) theraband . each tubing exercise was performed as 3 sets of 10 repetitions with a 60-second rest period between each set . at the end of every week , the subjects were evaluated and progressed to the next higher level of resistance using yellow ( thin ) , red ( medium ) , green ( heavy ) , blue ( extra - heavy ) and black ( special heavy ) therabands according to each subject . external rotation of shoulder with arm abducted to 90 and elbow flexed to 90 external rotation of shoulder with arm at side and elbow flexed to 90 proprioceptive neuromuscular facilitation ( pnf ) d2 pattern.(a)starting and ( b ) ending position . ( a)starting and ( b ) ending position another exercises program was applied in this study ( table 1 ) targeted the periscapular muscle to improve stabilization of the scapula ( fig .8 ) . t to y to w exercise on swiss ball scapular - clock exercise was the other training we used in this study to facilitate the scapular motions of elevation , depression , protraction , and retraction as well as joint kinesthesia and range of motion . the subject stood beside a plinth and put his hand on a ball and moved it to show 3 , 6 , 9 or 12 o'clock based on an imaginary clock he had on his mind ( fig .9 ) . this exercise also was done by pressing a ball and replacing it on a wall ( fig .10 ) . scapular - clock exercise on the table scapular - clock exercises on a wall the flexibility exercises of our program consisted of sleeper stretch ( fig .11 ) , crossed arm stretch ( fig .12 ) , corner stretch ( fig .13 ) and pectorals stretches ( fig .14 ) which aimed to elongate pectoralis major/ minor muscles and stretch of shoulder capsule . two sets of stretching exercises were performed with a minimum of 10 repetitions for 30 seconds . crossed arm stretch ( posterior capsule stretch ) corner stretch ( posterior capsule stretch ) pectorals stretches . ( left)starting and ( right ) ending position physical therapy intervention : a combination of physical modalities and range of motion exercises was chosen as a conventional physical therapy in this study for pt group . their protocol included pendulum and rom exercises ( scaption , abduction , flexion , extension , horizontal abduction , horizontal adduction and rotations ) , infrared therapy ( 33 ) ( 500 watt ir lamp , philips co , nederland ) ; ultrasound therapy and tens ( combined btl-4825 s topline , uk ) ( 32 ) which performed three times per week for 6 weeks . infrared lamp was in distance of 45 - 50 cm from pain area according to the patient tolerance . ultrasound therapy was applied on the subacromial region ( us frequency : 1 mhz ; us mode : continuous ; time : 5 minutes ; probe size : 5 cm2 ; and an intensity of 1 w / cm2).we also used two- electrode tens ( pulse width : 50 - 250 ms , pulse rate : 90 130 hz ) over painful area according to the patient with the most comfortable intensity level . obviously , these modalities were chosen to reduce pain , improve tissue extensibility and increase range of shoulder motions . the data were analyzed using the statistical package for the social sciences ( spss , version 19 , spss inc , and chicago , il ) . descriptive statistics ( mean , sd , range ) were computed for each study variable . normal distribution of data was determined by observing histograms and one - sample kolmogorov - smirnov test and parametric tests were used to analyze the data . a paired - sample t test was applied to determine the differences in each group . an independent sample t test was used to compare the baseline measurements between the groups at the beginning and at the end of training and also to analysis change scores of both groups after the test . the change scores of a group were defined as the increase or decrease of each variable from pre - test to post - test . the level of significance was set at p<0.05 . in order to study the improvement process of participants , the change score of all data were also calculated using the following equation : change score= post test average - pre test average the change scores will reveal the increase or decrease of each variable from pre - test to post - test . to assess the intra - tester reliability of objective tests , 10 sis subjects , who completed informed consent documentation , had repeated measurements a week apart in a pilot study . test - retest reliability of variables was assessed using paired match t - test ( p0.05 ) and the correlation between the first and second assessing was also obtained ( table 2 ) . the data were analyzed using the statistical package for the social sciences ( spss , version 19 , spss inc , and chicago , il ) . descriptive statistics ( mean , sd , range ) were computed for each study variable . normal distribution of data was determined by observing histograms and one - sample kolmogorov - smirnov test and parametric tests were used to analyze the data . a paired - sample t test was applied to determine the differences in each group . an independent sample t test was used to compare the baseline measurements between the groups at the beginning and at the end of training and also to analysis change scores of both groups after the test . the change scores of a group were defined as the increase or decrease of each variable from pre - test to post - test . the level of significance was set at p<0.05 . in order to study the improvement process of participants , the change score of all data were also calculated using the following equation : change score= post test average - pre test average the change scores will reveal the increase or decrease of each variable from pre - test to post - test . to assess the intra - tester reliability of objective tests , 10 sis subjects , who completed informed consent documentation , had repeated measurements a week apart in a pilot study . test - retest reliability of variables was assessed using paired match t - test ( p0.05 ) and the correlation between the first and second assessing was also obtained ( table 2 ) . there were no significant differences between pt and et groups for the demographic variables ( table 3 ) , indicating that the groups were well matched . there were 55 females and 13 males , with ages ranging from 21 to 78 years , 75.8% of subjects in et group and 87.7% of pt group were female . the majority of subjects in both groups were right dominant and right shoulder was affected in them ( table 4 ) . pre test results : independent- sample t test revealed no significant differences ( p<0.05 ) between two groups at the beginning of the study ( table 3 ) . post test results : there were significant differences in vas , abduction , external rotation , forward shoulder translation , pectoralis minor length in the pt group ( table 5 ) between pre and posttests ( p<0.05 ) in pt group . all variables showed significant differences in et group except scapular rotation and symmetry ( table 5 ) between pre and posttests ( p<0.05 ) . comparison between change scores in training groups : the improvement of shoulder abduction and external rotation ranges , postural parameters ( forward shoulder translation , forward head posture , mid - thoracic curve and pectoralis minor length ) in et group was significantly greater than that the pt group . moreover , scapular rotation and symmetry had no or very little changes between pt and et groups ( table 6 ) . the purpose of the current study was to investigate the effect of a 6-week supervised scapular stabilization exercise therapy on pain , scapular position , head and back posture , and shoulder mobility in comparison with physical therapy in patients with sis . our findings indicate that the exercise protocol suggested in this study significantly decreased pain , improved scapular protraction , head and back posture and increased shoulder mobility . however , we did not observe between - group differences for scapular rotation and symmetry following the exercise therapy . pain : an equal effectiveness of physiotherapy and exercise therapy in decreasing pain was obtained in this study and no significant difference was found in the vas score and the shoulder pain was decreased in subjects of both groups . the effect of exercise therapy in reducing pain alone or in combination with other treatments has been shown in the previous studies ( 15 , 34 - 36 ) . rotator cuff muscles stabilize the humeral head in the glenoid , causing humerus to rotate outside while protecting the distance between large tubercle and acromion and preventing compression . it was why that the resistance training ( theraband ) used in this study , was effective in reducing pain in et group subjects . furthermore , the stretching exercises of our program improved the flexibility of shoulder tight tissue that could be effective in pain decrease . it seems that our exercise therapy protocol is effective as a treatment for the reduction of pain in sis patients . in the present study , ultrasound and tens was used for the treatment of pt group subjects . despite the fact that ultrasound has been used in the treatment of shoulder pain for decades , there are evidences showed that us alone has no benefit in decreasing sis pain ( 18 , 34 , 37 - 39 ) and also no available evidence for efficacy of tens alone for patients with sis pain ( 38 ) . bakurt et al . did not find any statistically significant difference in studying the immediate effects of tens and heat on the pain related to stage one of sis . . found positive effects of continuous ultrasound with high frequency tens in decreasing pain and improving range of movement . ( 38 ) therefore , in this study , a therapeutic package of continuous ultrasound with high frequency tens and superficial heat ( ir ) was used which was effective in reducing pain between pre and post intervention in pt group due to the thermal effect of us and ir in combination with analgesic effect of tens . shoulder range of motion ( rom ) : the findings of this study demonstrated the significant differences of affected shoulder abduction and external rotation between pre and post intervention in both group . also there were significant differences in shoulder rom and its progression rate between pt and et groups ; which imply that et group had remarkable improvement in shoulder rom due to stretching exercises used in this study for decreasing tight shoulder capsule and shortened muscles especially pectoral muscles . also increasing shoulder rom our findings agree with those of other researchers ( 37,41 - 42 ) who have documented improvements in rom following an exercise program in patients with sis . posture : the effect of a six - week exercise intervention was evaluated in changing head , shoulder and back posture in comparison with physical modalities in this study . a decrease was observed in fhp , fst and mid - thoracic curve in both groups pre and post intervention but there was a significant difference between et and pt groups . the results revealed that the exercise protocol used in current study significantly decreased fhp , fst and mid - thoracic curve , suggesting that our exercises have improved patients ' posture . the flexibility exercises of this study were chosen based on the results that indicate tightness of levator scapulae , pectorals in sis ( 28 ) and also a previous study findings which has showed selective activation of deep cervical flexors , middle and lower trapezes and also serratus anterior in sis patient ( 43 ) . it seems that the supervised stretching of the tight anterior shoulder muscles with strengthening of the relatively weaker muscles in present protocol could have a significant synergistic effect on patients ' posture . our findings are similar to kluemper et al . and lynch et al . findings who have studied the effect exercise intervention on fhp and fsp in swimmers ( 16,28 ) . scapular protraction & rotation : another purpose of present study was to examine the effect of our exercise program on the position of scapula . the abnormal position of scapula in sis patients may lead to instability of shoulder joint . the results of our study revealed significant differences in the scapular protraction of the et group compared to the pt group within six - week exercise therapy , indeed there was no change in scapular protraction in pt group . but scapular rotation and symmetry did not indicate any significant difference between pre and post intervention in both group and also between groups . we expected to find more substantial changes in scapular rotation and symmetry after exercise based on alizadeh et al . study ( 30 ) . a significant changes in scapular symmetry and rotation might require a long term practice , perhaps due to short term duration of our exercise protocol or low accuracy of the measurement methods used in this study , scapular symmetry and rotation showed no significant changes . pectoralis minor length ( pml ) : abnormal kinematic of scapula in sis might be lead to pectoralis minor shortening and tightness , therefore in this study pml in patient involved shoulder was measured with anthropometric method .the results of this assessment indicate a significant difference in pml between pre and post intervention in both group and also between groups . pml was increased more in et group compared to pt group because of applying stretching exercises . our findings are similar to those of other researchers ( 44 - 46 ) . a major limitation of our work is that we have no follow - up period . may be if the subjects were followed - up , a significant difference in pain might be found after several weeks . also scapular and shoulder muscles strength was not assessed in this study and further research is needed , however , to evaluate shoulder girdle muscles strength . the scapula plays a vital role in shoulder function , thus this study highlights exercise prescription to enhance scapular stabilization during the sis rehabilitation . the scapular stabilization based exercise intervention was successful at increasing shoulder range of abduction and external rotation , decreasing forward head and shoulder postures and pectoralis minor flexibility . this study supports basis for scapular stabilization based exercise therapy in the rehabilitation of sis . also be noted that exercise therapy is effective as a treatment for the reduction of pain in these patients .

background : dysfunction in the kinetic chain caused by poor scapula stabilization can contribute to shoulder injuries and shoulder impingement syndrome ( sis ) . the purpose of this study was to compare the effectiveness of two treatment approaches scapular stabilization based exercise therapy and physical therapy in patients with sis . methods : the study is a randomized clinical trial in which 68 patients with sis were randomly assigned in two groups of exercise therapy ( et ) and physical therapy ( pt ) and received 18 sessions of treatment . pain , shoulders ' range of abduction and external rotation , shoulder protraction , scapular rotation and symmetry as well as postural assessment and pectoralis minor length were evaluated pre and post intervention . the paired - sample t test and the independent sample t test were applied respectively to determine the differences in each group and between two groups . results : our findings indicated significant differences in abduction and external rotation range , improvement of forward shoulder translation and increase in the flexibility of the involved shoulder between the two groups ( respectively ; p=0.024 , p=0.001 , p<0/0001 , p<0/0001 ) . no significant difference was detected in pain reduction between the groups ( p=0.576 ) . protraction of the shoulder ( p<0.0001 ) , forward head posture ( p<0/0001 ) and mid thoracic curvature ( p<0.0001 ) revealed a significant improvement in the et group . apparent changes occurred in scapular rotation and symmetry in both groups but no significant differences were observed between the two groups ( respectively ; p=0.183 , p=0.578 ) . conclusion : the scapular stabilization based exercise intervention was successful in increasing shoulder range , decreasing forward head and shoulder postures and pectoralis minor flexibility .

Introduction Methods Data Analysis Results Discussion Conclusion

shoulder impingement syndrome ( sis ) is a common complaint for patients of all ages and different activity levels . a wide range of different factors is involved in causing sis : including anatomic abnormalities of the coracoacromial arch or humeral head ( 2 ) , tension overload ischemia ( 3 ) , repetitive eccentric overload ( 2 - 3 ) , aberrant kinematic patterns due to poor rotator cuff or scapular muscles function ( 2,4 - 5 ) , and poor posture and scapular kinematic abnormalities ( 2 ) . pain and dysfunction of patient with sis occur when the shoulder is placed in positions of elevation , an activity that is commonplace during many sporting and daily living activities . the scapular position and motions on the thorax is a critical component of the normal glenohumeral function and plays great roles in facilitating optimal shoulder movements ( 6 - 8 ) . during all movements of the glenohumeral joint especially overhead elevation of the arm , it is of great importance that the scapular - stabilizing musculature should be strong enough to properly position the scapula . when weakness or dysfunction is present in the scapular musculature , normal scapular positioning and mechanics may become altered which result in abnormal stresses to the capsular structures , rotator cuff compression and reduced performance ( 7 ) . the treatment of sis is % 90 -95 conservative and often includes rotator cuff strengthening exercises ( 6,15 ) , stretching exercises ( 16 ) , immobilization , passive , active and active assisted range of motion exercises ( rom ) , various mobilization techniques , home exercise programs ( 17 ) and various physical therapy methods such as heat , transcutaneous electrical nerve stimulation ( tens ) and ultrasound ( us ) and etc ( 18 - 19 ) . when the scapula fails to perform its stabilization role , shoulder function is inefficient , which can result not only in decreased neuromuscular performance , but also may predispose the individual to shoulder injuries ( 20 ) . this lack of attention may often lead into the incomplete treatment ( 7 ) , therefore , reestablishment of normal shoulder function and restoring normal scapular muscle activation patterns by scapular stabilization based exercises , in our view , are the keys to a successful rehabilitation program . the purpose of our investigation was to compare the effectiveness of two treatment approaches for impingement syndrome of the shoulder : ( 1 ) a 6-week scapular stabilization based exercises , and ( 2 ) a rom exercise program combined with physical modalities on pain , posture , flexibility and mobility of sis patients . subjects were provided with information booklets explaining the purpose of the study and signed informed consent documents prior to participation . this study was conducted in hazrat rasool - e - akram hospital located in tehran ( iran ) between 2011and 2012 . forward shoulder translation measurement scapular protraction measurement all subjects tested positive for impingement tests ( which included the hawkins , neer , and empty can tests ) and underwent a full screening of cervical and shoulder rom , resisted motions , and special tests . therefore , to correctly identify patients with shoulder impingement , a combination of clinical tests is recommended . exclusion criteria were as follows : ( 1 ) cervical or shoulder symptoms reproduced by a cervical screening exam ; ( 2 ) abnormal results with reflex or thoracic outlet tests ; ( 3 ) symptoms of numbness or tingling in the upper extremity ; ( 4 ) pregnancy , or ( 5 ) a history of the followings : onset of symptoms due to traumatic injury , glenohumeral joint dislocation , acromioclavicular joint separation , shoulder fracture , surgery on the shoulder , fibromyalgia , use of any treatment within three months . main participants were 72 sis patients who were randomly allocated into two groups : ( 1 ) scapular stabilization based exercise therapy group ( et ) and ( 2 ) physical therapy group ( pt ) . in addition to inclusion - exclusion criteria , the clinical evaluation included assessment of pain , active shoulder external rotation and abduction range , forward head posture , mid - thoracic curve , forward shoulder translation , scapular protraction & rotation and pectoralis minor length were done pre and post intervention . the interventions for both groups were done by two clinicians that were unaware of the treatments groups . one supervised exercise therapy for et group on even days and the other did physical therapy for pt group on odd days . the power analysis of the study was performed to detect a 10% differences in pain and shoulder abduction with =0/05 and a power of 80% , a sample size of 36 per group was required . the vas used in the study was a 10-cm line where the 0 was marked as no pain and the 10 as the worst pain imaginable ( 23 ) . shoulder range of motion ( rom ) : the ranges of active external rotation and abduction were measured by a standard goniometer in both symptomatic and asymptomatic shoulders as follows : shoulder abduction was measured in the seated chair position , as in flexion , with the trunk upright . the arm was actively elevated in the strict coronal plane with the thumb pointed up toward the ceiling to allow the required external rotation necessary to avoid impingement of the greater tuberosity on the acromion process . the tested arm was supported on the table in 90 degrees of abduction , elbow flexed to 90 degrees , the forearm in midway between pronation / supination and the wrist in neutral . forward head posture ( fhp ) : to measure fhp , a lateral photograph was then taken of the cervicothoracic region , using a canon camera ( model : ixy digital 3000 is).the camera was placed 2 meters from the subject and mounted on a tripod , leveled with a bubble spirit level to control frontal and sagittal angles . to measure the angles , an a4-sized sheet of graph paper the transparency film was then placed over the photograph and aligned so that one of the vertical lines was placed over the plumb line and the intersection of two vertical and horizontal lines coincided with the point the c7 marker came in contact with the skin . to calculate the position of the head in relation to c7 ( c7-tragus angle ) , the angle between the horizontal line and the line connecting tragus of the ear to spinous process of c7 , was measured with a goniometer and documented in degrees . mid - thoracic curve : this curvature was measured by palpating and marking the spinous processes of the second thoracic vertebra ( t2 ) and the twelfth thoracic vertebra ( t12 ) by counting spinous processes , beginning with c7 . a metric ruler was used to measure the length and the flexi ruler was used to measure the height of the curve in centimeters . the following formula was used to determine mid - thoracic curvature : mid - thoracic curvature = = 4 [ arc tan ( 2 h / l ) ] where h = the height of the curve and l = the length of the curve ( fig 3 ) ( 25 ) . anthropometric measurement of pectoralis forward shoulder translation(fst ) : fst was measured using a combination square ruler ( cl-01 model , e - base measuring tools co , taiwan ) consists of a 40-cm to measure the distance from the wall to the anterior tip of the subject 's acromion process . the anterior tip of acromion process was marked and the distance of the point and wall was measured and documented in millimeter to determine the amount of fst . 2 ) : a= the root of the scapular spine b= a mark on the thoracic spine corresponding to the root of the scapular spine c= a mark on the thoracic spine corresponding to the inferior angle of the scapula d= the inferior angle of the scapula e= the tip of the acromion of the scapula . the following formula was used to determine scapular protraction : scapular protraction = baeae line bae = the distance from the mark on the thoracic spine corresponding to the root of the scapular spine to the tip of the acromion . line ae= the distance from the root of the scapular spine to the tip of the acromion . scapular rotation was measured by palpating and marking the inferior angle of the scapula ( d ) and the corresponding mark on the thoracic spine ( c ) . the following formula was used to determine scapular rotation : scapular rotation= tan =cdbc line cd= the distance between the inferior angle of the scapula and the corresponding mark on the thoracic spine . line bc= the distance between the marks on the thoracic spine corresponding to the root of the scapular spine and the inferior angle of the scapula ( fig . the symmetry of the scapular was determined for each subject using the following formula : where l= the ratio of left scapular protraction to left scapular rotation , and r= the ratio of right scapular protraction to right scapular rotation . anthropometric measurement of pectoralis minor ( pm ) length : the anthropometric measurement of the pm length was performed according to the protocol , described by borstad ( 29 ) . exercise therapy intervention - after a brief explanation about the exercises protocol , the et subjects participated in three supervised exercise sessions per week over a 6-week period ( 16 , 20 , 30 - 32 ) . this exercises protocol consisted of a 10 minute walking warm up on a treadmill ( dk city- dx3-b1 ) , flexibility , strengthening , scapular stabilization and postural exercises . strength training with theraband consisted of exercises for rotator cuffs , scapular retractors , shoulder external rotators ( figs 4 , 5 ) , d2- pnf pattern ( fig .6 ) and serratus anterior punches ( fig .7 ) . at the first session , the et subjects were asked to do five repetitions of each of the tubing exercises to see if they were too hard or too easy ; then the appropriate theraband was chosen based on feedback from the subject , observation and palpation of the target muscles by the investigator . five subjects of the et group used tan ( ultrathin ) , sixteen used yellow ( thin ) , and the remaining subjects used red ( medium ) theraband . at the end of every week , the subjects were evaluated and progressed to the next higher level of resistance using yellow ( thin ) , red ( medium ) , green ( heavy ) , blue ( extra - heavy ) and black ( special heavy ) therabands according to each subject . ( a)starting and ( b ) ending position another exercises program was applied in this study ( table 1 ) targeted the periscapular muscle to improve stabilization of the scapula ( fig .8 ) . t to y to w exercise on swiss ball scapular - clock exercise was the other training we used in this study to facilitate the scapular motions of elevation , depression , protraction , and retraction as well as joint kinesthesia and range of motion . scapular - clock exercise on the table scapular - clock exercises on a wall the flexibility exercises of our program consisted of sleeper stretch ( fig .11 ) , crossed arm stretch ( fig .12 ) , corner stretch ( fig .13 ) and pectorals stretches ( fig .14 ) which aimed to elongate pectoralis major/ minor muscles and stretch of shoulder capsule . ( left)starting and ( right ) ending position physical therapy intervention : a combination of physical modalities and range of motion exercises was chosen as a conventional physical therapy in this study for pt group . their protocol included pendulum and rom exercises ( scaption , abduction , flexion , extension , horizontal abduction , horizontal adduction and rotations ) , infrared therapy ( 33 ) ( 500 watt ir lamp , philips co , nederland ) ; ultrasound therapy and tens ( combined btl-4825 s topline , uk ) ( 32 ) which performed three times per week for 6 weeks . obviously , these modalities were chosen to reduce pain , improve tissue extensibility and increase range of shoulder motions . normal distribution of data was determined by observing histograms and one - sample kolmogorov - smirnov test and parametric tests were used to analyze the data . a paired - sample t test was applied to determine the differences in each group . an independent sample t test was used to compare the baseline measurements between the groups at the beginning and at the end of training and also to analysis change scores of both groups after the test . test - retest reliability of variables was assessed using paired match t - test ( p0.05 ) and the correlation between the first and second assessing was also obtained ( table 2 ) . normal distribution of data was determined by observing histograms and one - sample kolmogorov - smirnov test and parametric tests were used to analyze the data . a paired - sample t test was applied to determine the differences in each group . an independent sample t test was used to compare the baseline measurements between the groups at the beginning and at the end of training and also to analysis change scores of both groups after the test . test - retest reliability of variables was assessed using paired match t - test ( p0.05 ) and the correlation between the first and second assessing was also obtained ( table 2 ) . there were no significant differences between pt and et groups for the demographic variables ( table 3 ) , indicating that the groups were well matched . there were 55 females and 13 males , with ages ranging from 21 to 78 years , 75.8% of subjects in et group and 87.7% of pt group were female . the majority of subjects in both groups were right dominant and right shoulder was affected in them ( table 4 ) . pre test results : independent- sample t test revealed no significant differences ( p<0.05 ) between two groups at the beginning of the study ( table 3 ) . post test results : there were significant differences in vas , abduction , external rotation , forward shoulder translation , pectoralis minor length in the pt group ( table 5 ) between pre and posttests ( p<0.05 ) in pt group . all variables showed significant differences in et group except scapular rotation and symmetry ( table 5 ) between pre and posttests ( p<0.05 ) . comparison between change scores in training groups : the improvement of shoulder abduction and external rotation ranges , postural parameters ( forward shoulder translation , forward head posture , mid - thoracic curve and pectoralis minor length ) in et group was significantly greater than that the pt group . moreover , scapular rotation and symmetry had no or very little changes between pt and et groups ( table 6 ) . the purpose of the current study was to investigate the effect of a 6-week supervised scapular stabilization exercise therapy on pain , scapular position , head and back posture , and shoulder mobility in comparison with physical therapy in patients with sis . our findings indicate that the exercise protocol suggested in this study significantly decreased pain , improved scapular protraction , head and back posture and increased shoulder mobility . however , we did not observe between - group differences for scapular rotation and symmetry following the exercise therapy . pain : an equal effectiveness of physiotherapy and exercise therapy in decreasing pain was obtained in this study and no significant difference was found in the vas score and the shoulder pain was decreased in subjects of both groups . the effect of exercise therapy in reducing pain alone or in combination with other treatments has been shown in the previous studies ( 15 , 34 - 36 ) . it was why that the resistance training ( theraband ) used in this study , was effective in reducing pain in et group subjects . furthermore , the stretching exercises of our program improved the flexibility of shoulder tight tissue that could be effective in pain decrease . despite the fact that ultrasound has been used in the treatment of shoulder pain for decades , there are evidences showed that us alone has no benefit in decreasing sis pain ( 18 , 34 , 37 - 39 ) and also no available evidence for efficacy of tens alone for patients with sis pain ( 38 ) . ( 38 ) therefore , in this study , a therapeutic package of continuous ultrasound with high frequency tens and superficial heat ( ir ) was used which was effective in reducing pain between pre and post intervention in pt group due to the thermal effect of us and ir in combination with analgesic effect of tens . shoulder range of motion ( rom ) : the findings of this study demonstrated the significant differences of affected shoulder abduction and external rotation between pre and post intervention in both group . also there were significant differences in shoulder rom and its progression rate between pt and et groups ; which imply that et group had remarkable improvement in shoulder rom due to stretching exercises used in this study for decreasing tight shoulder capsule and shortened muscles especially pectoral muscles . also increasing shoulder rom our findings agree with those of other researchers ( 37,41 - 42 ) who have documented improvements in rom following an exercise program in patients with sis . posture : the effect of a six - week exercise intervention was evaluated in changing head , shoulder and back posture in comparison with physical modalities in this study . a decrease was observed in fhp , fst and mid - thoracic curve in both groups pre and post intervention but there was a significant difference between et and pt groups . the flexibility exercises of this study were chosen based on the results that indicate tightness of levator scapulae , pectorals in sis ( 28 ) and also a previous study findings which has showed selective activation of deep cervical flexors , middle and lower trapezes and also serratus anterior in sis patient ( 43 ) . it seems that the supervised stretching of the tight anterior shoulder muscles with strengthening of the relatively weaker muscles in present protocol could have a significant synergistic effect on patients ' posture . scapular protraction & rotation : another purpose of present study was to examine the effect of our exercise program on the position of scapula . the results of our study revealed significant differences in the scapular protraction of the et group compared to the pt group within six - week exercise therapy , indeed there was no change in scapular protraction in pt group . but scapular rotation and symmetry did not indicate any significant difference between pre and post intervention in both group and also between groups . we expected to find more substantial changes in scapular rotation and symmetry after exercise based on alizadeh et al . a significant changes in scapular symmetry and rotation might require a long term practice , perhaps due to short term duration of our exercise protocol or low accuracy of the measurement methods used in this study , scapular symmetry and rotation showed no significant changes . pectoralis minor length ( pml ) : abnormal kinematic of scapula in sis might be lead to pectoralis minor shortening and tightness , therefore in this study pml in patient involved shoulder was measured with anthropometric method .the results of this assessment indicate a significant difference in pml between pre and post intervention in both group and also between groups . may be if the subjects were followed - up , a significant difference in pain might be found after several weeks . also scapular and shoulder muscles strength was not assessed in this study and further research is needed , however , to evaluate shoulder girdle muscles strength . the scapula plays a vital role in shoulder function , thus this study highlights exercise prescription to enhance scapular stabilization during the sis rehabilitation . the scapular stabilization based exercise intervention was successful at increasing shoulder range of abduction and external rotation , decreasing forward head and shoulder postures and pectoralis minor flexibility . this study supports basis for scapular stabilization based exercise therapy in the rehabilitation of sis .

complications can be defined as a secondary disease , accident , or negative reaction that occur during the course of an illness and usually aggravate the illness . complications of pelvic fractures are always described in a routine way and as associated injuries of pelvic fractures . pelvic ring fractures require transmission of a tremendous amount of force , and often happen in severely injured patients with multiple organ injuries . the most common associated injuries are bleeding and retroperitoneal hematoma , intra - abdominal and urogenital injuries , and never injuries in addition to severe pelvic fractures.1 , 2 , 3 approximately 12% of trauma patients admitted to level 1 or 2 trauma centers in the united states exhibit pelvic ring injury , and injuries to intra - abdominal or urogenital organs are present in an estimated 16% of these patients . prompt diagnosis and effective management of these injuries is essential to a successful outcome.2 , 3 great efforts have been made to improve treatment of pelvic fracture and its complications , and substantial improvements have been achieved . however rare complications of pelvic fractures , such as ureteral obstruction caused by retroperitoneal hematoma induced abdominal compartment syndrome ( acs ) , bowel entrapment , external iliac artery injury , and open scrotal sac injury are not easily diagnosed and treated . unawareness of these rare conditions makes the diagnosis difficult , and failure to provide prompt treatment leads to poor outcomes . in order to increase the awareness of these rare complications of pelvic fracture and describe the correct diagnosis and effective treatment , we present four cases that exhibit the above - mentioned four different types of rare complications . this study was reviewed and approved by the ethical committee of the daping hospital , third military medical university , china . all four patients reported in this study have signed informed consent forms , agreeing that we could report their clinical courses . patients who suffered from pelvic fractures in our department from june 2009 to july 2012 were retrospectively reviewed . the mechanisms of injuries , the type of pelvic fracture , and the complications are analyzed . four cases exhibited the four types of rare complications , namely ureteral obstruction caused by retroperitoneal hematoma induced abdominal compartment syndrome , bowel entrapment , external iliac artery injury , and severe open scrotal sac injury . extensive literature search on medline / pubmed was performed , including articles related to complications of pelvic and acetabular fractures . we carefully reviewed articles related to the above - mentioned four types of rare complications . suggestions for diagnosis and recommendations for treatment of these rare complications of pelvic fractures were given . a total of 188 cases of pelvic fractures were treated in our department between june 2009 and june 2012 . there were 124 male and 64 female patients , with an average of 41.2 years of age . the mechanisms of injury included traffic injury ( 122 cases ) , high fall injury ( 35 cases ) , and heavy object pound injury ( 24 cases ) . of the 188 cases , 37 were complicated by or associated with chest injuries , 14 with brain injuries , 45 with upper extremities injuries , 36 with lower extremities injuries , 12 with ureteral injuries , 3 with bladder injuries , 6 with bowel injuries , and 3 with spleen disruption . there were four cases that exhibited four types of rare complications , namely ureteral obstruction caused by retroperitoneal hematoma induced abdominal compartment syndrome , bowel entrapment , external iliac artery injury , and severe open scrotal sac injury . a 48-year - old male cleaner fell from a 9 m high building when cleaning the outside window in our hospital and was transferred to the emergency room within 6 min following the accident . on admission , his blood pressure was 100/65 mmhg ; it decreased to 6572/4046 mmhg 10 min after admission . after the patient was hemodynamically stabilized by infusion of 3000 ml crystalloid fluid and 1200 ml suspended red blood cells , plain roentgenogram and three - dimensional computed tomography ( ct ) were performed and demonstrated type 61-b2.1 pelvic fracture according to arbeitsgemeinschaft fur osteosynthesefragen / orthopaedic trauma association ( ao / ota ) classification scheme ( fig . additional injuries included a compound fracture of the left proximal femur , fractures of the tenth to twelfth ribs on the left side with lung contusion and hemothorax , fracture of the distal radius and proximal ulna and radius on the left side , and laceration of the head ( fig . 1b , c , d and e ) . digital subtraction angiography ( dsa ) performed along with ct revealed no arterial bleeding of the internal iliac artery and its branches , and thus no need to perform embolism . the patient exhibited oliguria 4 h post - trauma , even following successful fluid resuscitation . the bladder pressure measured by a foley catheter increased from 8 mmhg to 22 mmhg . following consultation by experts in our trauma center and the department of urology , the patient underwent double j bilateral ureteral catheter placement , during which no urine was observed in the ureteral orifice into the bladder . immediately following the replacement of ureteral catheter , urine was observed in the urinary bag and urinary output continued at a rate of 50105 ml / h . bladder pressure increased slightly 1 h following the surgical operation , decreased slowly , and then reached approximately 8 mmhg 8 days following surgery . the ureteral catheter was then removed . following placement of the bilateral ureteral catheter , external fixation of the pelvis and left femur ten days after surgery , the patient was discharged from the intensive care unit with good renal function and open reduction and internal fixation for the fracture of distal end of the left radius , proximal end fracture of the left femur . the pelvic fracture was thought to be stabilized with external fixation and no open reduction and internal fixation ( orif ) was performed . one month following the injury , the patient was released from our department and transferred to the department of rehabilitation for functional recovery . four month after discharge , x - ray examination showed healing of the pelvic fracture ( fig . the second case involved in a 19-year old man who was injured in a traffic accident , but the victim failed to describe how he was injured . after injury , he was admitted to a level ii hospital and demonstrated pain of the left shoulder , waist , and pelvis . ct examination revealed ao / ota type 61.a2.2 pelvic fracture , left ao / ota type 62.a3.2 acetabulum fracture ( fig . pelvic external fixation and closed drainage of the thoracic cavity were performed . in the next few days the patient became febrile ( range from 37.8 c to 38.6 c ) ; the cause was considered to be absorbed fever of damaged tissues . however in addition , abdominal pain developed day 4 after injury and physical examination found no obvious signs of peritoneal irritation . abdominal x - ray revealed severe distention but no free gas or fluid . due to the unknown origin of fever and potential of infection , internal fixation and pelvic fixation were not performed , and the patient was transferred to our hospital for further treatment . again , x - ray radiology found severe bowel distention but no free gas and fluid in the abdomen ( fig . 2b ) ; however , ct examination showed that a small segment of bowel was entrapped in the fracture fragments ( fig . 2c ) . the entrapped bowel had perforated and was put outside of the abdomen ( fig . seven days after laparotomy , the high fever resolved and orif of the acetabulum fracture were performed through posterior approach . the next 3 months were uneventful and the patient was re - admitted for bowel reconstruction . a 25-year - old man was hit in the lower abdomen and groin region directly by a car with a speed of 50 miles per hour . he was admitted to a primary hospital close to the scene following the accident . at admission , the patient 's heart rate was 100 beats per minute , blood pressure 95/65 mmhg , and glasgow coma scale score 14 ; x - ray and ct scan revealed pelvic fracture ( data were not obtained from this hospital ) . eight hours later , he presented with severe pain , swelling , and weakened pulse of the left lower extremity . injury to the main arteries of the lower limb extremity was suspected , and the patient was transferred to our department 12 h following the injury . he exhibited high fever with normal heart beat , blood pressure , and breath rate . physical examination found contusion and ecchymosis in the groin region , and femoral and pedal pulse could not be detected . angiography along with ct showed disruption of external iliac artery and bilateral pubic ramus fracture ( ao / ota type 61 a2.3 , fig . 3a ) , and lab examination showed slight elevation of serum creatinine ( cre ) and blood urea nitrogen ( bun ) . exploration of the groin region was performed and a 2-cm long thrombus was observed in the left proximal external iliac artery . the embolic artery was then excised and artificial blood vessel prosthesis transplantation was performed ( fig . following surgery , the patient developed high fever with increased serum cre and blood bun . severe pain developed in the left lower extremity , followed by no sense of feeling . the thigh and leg became stiff and hence amputation was performed to save his life ( fig . a 57-year old man was lying in rest on a sloped lawn with his legs open when he was struck onto the groin region by the rear part of a backing truck with a speed of approximately 15 miles per hour . he was admitted to the closest hospital 1 h after injury . on admission , x - ray radiology revealed fracture of the left pubic rami and right acetabulum ( fig . debridement and suture of the wounded scrotum were performed and external fixation was applied to fix the pelvis . three days later , the man became febrile ( range from 38.1 c to 39.9 c ) and the groin region exhibited an unpleasant smell . eight days after injury , the patient was transferred to our hospital for further treatment . on admission , physical examination found high fever , normal blood pressure , high heart rate ( 96115 beat / minute ) and high breath rate ( 1625/min ) . part of the scrotum became necrotic , and abscess was observed in the sutured scrotum ( fig . a large quantity of abscesses was found in the sutured wound and was flushed out . the displaced fracture fragments of the pubic rami could be touched through the open wound ( fig . the patient 's high fever subsided gradually over the next 3 days following the operation . two more operations were performed to remove and replace the new vacuum sealing drainage 6 and 12 days following the first operation , respectively . six days later , the wound was clean , a large amount of granulated tissue developed , and the wound was closed . orif of the pelvic and acatabulem fracture was performed via the stoppa approach simultaneously ( fig . the patient recovered completely without any signs of infection and was discharged 10 days later . the most recent follow - up visit was 5 months after the last surgical operation , and the patient had not infection of the scrotum and wound healed completely ( fig . the patient told us that he had a normal erectile function and had normal sexual intercourse one week prior to the last follow - up . a 48-year - old male cleaner fell from a 9 m high building when cleaning the outside window in our hospital and was transferred to the emergency room within 6 min following the accident . on admission , his blood pressure was 100/65 mmhg ; it decreased to 6572/4046 mmhg 10 min after admission . after the patient was hemodynamically stabilized by infusion of 3000 ml crystalloid fluid and 1200 ml suspended red blood cells , plain roentgenogram and three - dimensional computed tomography ( ct ) were performed and demonstrated type 61-b2.1 pelvic fracture according to arbeitsgemeinschaft fur osteosynthesefragen / orthopaedic trauma association ( ao / ota ) classification scheme ( fig . additional injuries included a compound fracture of the left proximal femur , fractures of the tenth to twelfth ribs on the left side with lung contusion and hemothorax , fracture of the distal radius and proximal ulna and radius on the left side , and laceration of the head ( fig . 1b , c , d and e ) . digital subtraction angiography ( dsa ) performed along with ct revealed no arterial bleeding of the internal iliac artery and its branches , and thus no need to perform embolism . the patient exhibited oliguria 4 h post - trauma , even following successful fluid resuscitation . the bladder pressure measured by a foley catheter increased from 8 mmhg to 22 mmhg . following consultation by experts in our trauma center and the department of urology , the patient underwent double j bilateral ureteral catheter placement , during which no urine was observed in the ureteral orifice into the bladder . immediately following the replacement of ureteral catheter , urine was observed in the urinary bag and urinary output continued at a rate of 50105 ml / h . bladder pressure increased slightly 1 h following the surgical operation , decreased slowly , and then reached approximately 8 mmhg 8 days following surgery . the ureteral catheter was then removed . following placement of the bilateral ureteral catheter , external fixation of the pelvis and left femur ten days after surgery , the patient was discharged from the intensive care unit with good renal function and open reduction and internal fixation for the fracture of distal end of the left radius , proximal end fracture of the left femur . the pelvic fracture was thought to be stabilized with external fixation and no open reduction and internal fixation ( orif ) was performed . one month following the injury , the patient was released from our department and transferred to the department of rehabilitation for functional recovery . four month after discharge , x - ray examination showed healing of the pelvic fracture ( fig . the second case involved in a 19-year old man who was injured in a traffic accident , but the victim failed to describe how he was injured . after injury , he was admitted to a level ii hospital and demonstrated pain of the left shoulder , waist , and pelvis . ct examination revealed ao / ota type 61.a2.2 pelvic fracture , left ao / ota type 62.a3.2 acetabulum fracture ( fig . pelvic external fixation and closed drainage of the thoracic cavity were performed . in the next few days the patient became febrile ( range from 37.8 c to 38.6 c ) ; the cause was considered to be absorbed fever of damaged tissues . however , his high fever did not resolve in the next 27 days following injury . in addition , abdominal pain developed day 4 after injury and physical examination found no obvious signs of peritoneal irritation . abdominal x - ray revealed severe distention but no free gas or fluid . due to the unknown origin of fever and potential of infection , internal fixation and pelvic fixation were not performed , and the patient was transferred to our hospital for further treatment . again , x - ray radiology found severe bowel distention but no free gas and fluid in the abdomen ( fig . 2b ) ; however , ct examination showed that a small segment of bowel was entrapped in the fracture fragments ( fig . 2c ) . the entrapped bowel had perforated and was put outside of the abdomen ( fig . seven days after laparotomy , the high fever resolved and orif of the acetabulum fracture were performed through posterior approach . the next 3 months were uneventful and the patient was re - admitted for bowel reconstruction . a 25-year - old man was hit in the lower abdomen and groin region directly by a car with a speed of 50 miles per hour . he was admitted to a primary hospital close to the scene following the accident . at admission , the patient 's heart rate was 100 beats per minute , blood pressure 95/65 mmhg , and glasgow coma scale score 14 ; x - ray and ct scan revealed pelvic fracture ( data were not obtained from this hospital ) . eight hours later , he presented with severe pain , swelling , and weakened pulse of the left lower extremity . injury to the main arteries of the lower limb extremity was suspected , and the patient was transferred to our department 12 h following the injury . he exhibited high fever with normal heart beat , blood pressure , and breath rate . physical examination found contusion and ecchymosis in the groin region , and femoral and pedal pulse could not be detected . angiography along with ct showed disruption of external iliac artery and bilateral pubic ramus fracture ( ao / ota type 61 a2.3 , fig . 3a ) , and lab examination showed slight elevation of serum creatinine ( cre ) and blood urea nitrogen ( bun ) . exploration of the groin region was performed and a 2-cm long thrombus was observed in the left proximal external iliac artery . the embolic artery was then excised and artificial blood vessel prosthesis transplantation was performed ( fig . following surgery , the patient developed high fever with increased serum cre and blood bun . severe pain developed in the left lower extremity , followed by no sense of feeling . the thigh and leg became stiff and hence amputation was performed to save his life ( fig . a 57-year old man was lying in rest on a sloped lawn with his legs open when he was struck onto the groin region by the rear part of a backing truck with a speed of approximately 15 miles per hour . he was admitted to the closest hospital 1 h after injury . on admission , x - ray radiology revealed fracture of the left pubic rami and right acetabulum ( fig . debridement and suture of the wounded scrotum were performed and external fixation was applied to fix the pelvis . three days later , the man became febrile ( range from 38.1 c to 39.9 c ) and the groin region exhibited an unpleasant smell . eight days after injury , the patient was transferred to our hospital for further treatment . on admission , physical examination found high fever , normal blood pressure , high heart rate ( 96115 beat / minute ) and high breath rate ( 1625/min ) . part of the scrotum became necrotic , and abscess was observed in the sutured scrotum ( fig . a large quantity of abscesses was found in the sutured wound and was flushed out . the displaced fracture fragments of the pubic rami could be touched through the open wound ( fig . the patient 's high fever subsided gradually over the next 3 days following the operation . two more operations were performed to remove and replace the new vacuum sealing drainage 6 and 12 days following the first operation , respectively . six days later , the wound was clean , a large amount of granulated tissue developed , and the wound was closed . orif of the pelvic and acatabulem fracture was performed via the stoppa approach simultaneously ( fig . the patient recovered completely without any signs of infection and was discharged 10 days later . the most recent follow - up visit was 5 months after the last surgical operation , and the patient had not infection of the scrotum and wound healed completely ( fig . the patient told us that he had a normal erectile function and had normal sexual intercourse one week prior to the last follow - up . intra - abdominal hypertension ( iah ) is defined as intra - abdominal pressure ( iap ) > 12 mmhg . acs is defined as iap above 20 mmhg with the evidence of organ dysfunction / failure . acs / iah affects all body systems , in particular the cardiac , respiratory , renal , and neurologic systems , as a result of the blockage of blood flow . under these circumstances , abdominal decompression is indicated to reverse end - organ failure and minimize intra - abdominal hypertension - related morbidity and mortality.5 , 6 the origin of acs can be divided into retroperitoneal , intraperitoneal , parietal , and intestinal types . although it is less common , acs accompanying pelvic fracture has been previously reported.7 , 8 the prevention of acs in pelvic fracture is very important since acs leads to severe morbidity regardless of what measures are taken . combined measures have been established in our department to prevent the occurrence of acs in pelvic fractured patients . we routinely perform digital subtraction angiography ( dsa ) in patients with major pelvic fracture following successful fluid resuscitation . when bleeding from arterial branches is identified , transcatheter arterial embolization ( tae ) is performed , which can control arterial bleeding in most cases.3 , 9 external fixation is performed to minimize bleeding from unstable fracture fragments . regarding bleeding from venous vessels of the presacral plexus , a stable hemodynamics can generally be achieved using the tamponade effect of the retroperitoneum . in extremely rare conditions , if the retoperitonerum is damaged and the tamponade effect does not exist , retroperitoneal packing is indicated.3 , 10 also , in the resuscitation stage , the mean arterial pressure ( map ) was maintaind at approximately 80 mmhg , which is sufficient to provide blood supply to vital organs and reduce bleeding and the incidence of acs because large / aggressive volume fluid resuscitation is one of predisposing factors for iah / acs . utilizing the above - mentioned measures , we managed to reduce bleeding and limit the development of retroperitoneal hematoma . but even so , massive retroperitoneal hematoma and acs have been formed and lead to bilateral ureteral obstruction and renal failure as reported in the current study . under these circumstances , hessmann et al advocated evacuation of the retroperitoneal hematoma , whereas flint et al suggested that bilateral tube nephrostomy was a better option . flint and rothenberger et al believed that , because of the increased risk of sepsis and further hemorrhage , surgical exploration of the retroperitoneum to remove the hematoma should be avoided . in our cases , we selected bilateral ureteral catheter support to rescue renal function . mohmand et al suggested that acute kidney injury exhibits early and frequent consequences of iah / acs and can be present at relatively low levels of intra - abdominal pressure ( iap ) . it is also reported that at least several hours are required from the onset of acs before irreversible respiratory dysfunction , cardiac dysfunction , and ischemia of the intra - abdominal organs occur . thus , if iap does not increase continuously and remains less than 30 mmhg and no other systems or organs are affected by acs , ureteral catheter support is a good option to cope with postrenal renal failure . however , when iap continues to increase , or other organs are afflicted , opening the abdomen is warranted . in summary , combined measures , including digital subtraction angiography and embolism ( if necessary ) , external fixation of the pelvic ring , and controlling map at the proper level should be used to limit the development of retroperitoneal hematoma and to prevent the occurrence of acs . at the early stage of acs when only the urinary system is afflicted with iap less than 30 mmhg , and no signs of other organ injury or failure , bilateral ureteral catheter support to rescue renal function can be used to save renal function . however , when iap continues to increase , or other organs are afflicted , open the abdomen is mandatory . the typical symptoms included peritoneal irritation , shock , and severe sepsis , making them easily diagnosed . it is difficult to diagnose due to its clinical complexity , and the difficulty in distinguishing the adynamic ileus from bowel entrapment . in 24 such cases reported in the literature , the diagnosis were delayed in six cases.16 , 17 , 18 , 19 four cases of bowel entrapment were diagnosed during laparotomy , which was performed during the initial management of trauma . of the remaining cases , the average time to diagnosis was 14.9 days ( range 290 days ) . it is extremely difficult to diagnose when the entrapped bowel is only part of the bowel , as reported in the present case , due to the atypical symptoms . delayed diagnosis and treatment often lead to severe sepsis and death.16 , 17 , 18 , 19 ct is generally considered a sensitive tool for the diagnosis of bowel entrapment . combined use of oral and iv contrast in ct evaluation may increase diagnostic accuracy and reduce morbidity in patients with blunt abdominal injury.17 , 20 however , caution and sufficient awareness are the principal determining factors to establish the correct diagnosis of bowel entrapment . if progressive ileus in a patient with a pelvic fracture persists for over 3 days , an occult bowel injury such as entrapment at the fracture site , should be suspected and contrasted ct should accordingly be performed . immediate laparotomy is recommended as long as the diagnosis is determined to repair or resect the entrapped bowel . the pelvic fracture should usually be fixed externally at the initial stage due to risk of infection . most pelvic hemorrhages are thought to be caused by injury to small arteries or veins in the fractured cancellous pelvic bone or in the surrounding soft tissues , and only 6%18% of patients with unstable pelvic fractures exhibit hemorrhage from large arteries . when arterial injuries are present , the majority involve in branches of the internal iliac artery . there are only a few published reports of injuries to the branches of the external iliac artery , and fewer to the external iliac artery itself.22 , 23 , 24 injuries to the external iliac artery and its branches , such as the case described here , are thought to be extremely rare in pelvic fractures . the reason is due to the lower incidence of such complications , and diagnosis is sometimes delayed , thus increasing the risk of loss of both life and limb . recognition of the risk factors for injury of the external iliac artery and its branches in pelvic fractures is helpful to make a correct diagnosis . firstly , external iliac arterial injury should be highly suspected in some injury patterns of pelvic fractures . manson et al suggested that caudal displacement of the hemipelvis in rare conditions disrupts the pelvic floor and pelvic vasculature far more than a standard vertical shear injury . it is reported that caudal displacement of the hemipelvis leads to significant disruption of the external iliac artery and to surgical hemipelvectomy . pubic rami fracture is another pelvic fracture pattern in which the branch of external iliac artery ( inferior epigastric artery or corona mortis ) is highly susceptible to injury , even if the fracture is stable.23 , 24 secondly , anatomical variation is another factor for rare bleeding . the aberrant obturator artery ( aoa ) is a common arterial variant that occurs in more than half of the population and , if present in pelvic fractured patients , is commonly injured.25 , 26 thirdly , pre - injury medical conditions , such as anticoagulation treatment , are risk factors for external iliac artery injury . lastly , direct violence to the groin region as reported in the current study is a high risk factor for injury of the external iliac artery and its branches . although the incidence of external iliac artery injury is rare , the diagnosis is not difficult . delayed diagnosis leads to poor outcome . in our case , although the patient was grateful to be alive , we understood that the lost extremity could have been saved if early diagnosis had been established . when injury of the external iliac artery and its branches is confirmed , tae , endovascular blunting repair , open vascular reconstruction , and prosthetic bypass graft should be selectively performed.27 , 28 , 29 during wartime , the incidence of scrotal and/or testicular injury complicated by pelvic fracture is high . in contrast , the incidence of scrotal and/or testicular injury is rare in civilian injuries , and usually occurs in a situation called fuel tank injury . it is a rare condition involving motorcycle driver impact.31 , 32 , 33 , 34 in our case , the mechanism of injury was not fuel tank injury . it was caused by direct crash into the groin region when two legs of the patient were open . in open scrotal and/or testicular injury , the diagnosis is easy to establish ; however , in closed injury , high suspicion , thorough physical examination and doppler ultrasound are useful for correct diagnosis . the extent of soft tissue injury should be classified by multi - modality and multi - planar imaging so that correct treatment may be applied . injuries to the scrotum and its contents may cause impaired fertility , chronic pain , hypogonadism , and altered self - image ; thus , selection of treatment is highly important . if an open wound exists , primary suture of the wound is not recommended , and debridement and vacuum sealing drainage are suggested to avoid infection.37 , 38 however , aggressive debridement should be avoided if the patient is associated with pubic ramus fracture close to or connected to the scrotal wound , as reported in the current case . also , when vacuum sealing drainage is applied , the pressure should be monitored to avoid testis necrosis caused by too high pressure . external fixation of the pelvic fracture along with simple reduction of the significantly displaced pubic ramus fracture is recommended . in closed injury , dislocation of testis should be replaced . in both open and closed injuries , the testis should be excised if it has been necrotic . in north america , most european countries , and japan in asia , sophisticated trauma care systems have been established in the past couple of decades . in mainland china , greater attention is paid to the treatment of public health problems and trauma care remains in a nascent stage in small cities and rural and semi - urban areas . thus , many injured patients have a high chance of incorrect diagnosis and treatment when they are admitted to a lower level hospital . three of the four cases reported in the current study did not receive proper care at the initial stage after trauma . these situations may also be true in other developing counties , and may only be solved through the establishment of a sophisticated trauma care system . another issue for the care of rare complications of pelvic fractures is multi - disciplinary coordination . rare complications or associated injuries of pelvic fractures can be highly complex ; thus , coordination is warranted to achieve prompt and correct diagnosis and treatment . intra - abdominal hypertension ( iah ) is defined as intra - abdominal pressure ( iap ) > 12 mmhg . acs is defined as iap above 20 mmhg with the evidence of organ dysfunction / failure . acs / iah affects all body systems , in particular the cardiac , respiratory , renal , and neurologic systems , as a result of the blockage of blood flow . under these circumstances , abdominal decompression is indicated to reverse end - organ failure and minimize intra - abdominal hypertension - related morbidity and mortality.5 , 6 the origin of acs can be divided into retroperitoneal , intraperitoneal , parietal , and intestinal types . although it is less common , acs accompanying pelvic fracture has been previously reported.7 , 8 the prevention of acs in pelvic fracture is very important since acs leads to severe morbidity regardless of what measures are taken . combined measures have been established in our department to prevent the occurrence of acs in pelvic fractured patients . we routinely perform digital subtraction angiography ( dsa ) in patients with major pelvic fracture following successful fluid resuscitation . when bleeding from arterial branches is identified , transcatheter arterial embolization ( tae ) is performed , which can control arterial bleeding in most cases.3 , 9 external fixation is performed to minimize bleeding from unstable fracture fragments . regarding bleeding from venous vessels of the presacral plexus , a stable hemodynamics can generally be achieved using the tamponade effect of the retroperitoneum . in extremely rare conditions , if the retoperitonerum is damaged and the tamponade effect does not exist , retroperitoneal packing is indicated.3 , 10 also , in the resuscitation stage , the mean arterial pressure ( map ) was maintaind at approximately 80 mmhg , which is sufficient to provide blood supply to vital organs and reduce bleeding and the incidence of acs because large / aggressive volume fluid resuscitation is one of predisposing factors for iah / acs . utilizing the above - mentioned measures , we managed to reduce bleeding and limit the development of retroperitoneal hematoma . but even so , massive retroperitoneal hematoma and acs have been formed and lead to bilateral ureteral obstruction and renal failure as reported in the current study . under these circumstances , hessmann et al advocated evacuation of the retroperitoneal hematoma , whereas flint et al suggested that bilateral tube nephrostomy was a better option . flint and rothenberger et al believed that , because of the increased risk of sepsis and further hemorrhage , surgical exploration of the retroperitoneum to remove the hematoma should be avoided . in our cases , we selected bilateral ureteral catheter support to rescue renal function . mohmand et al suggested that acute kidney injury exhibits early and frequent consequences of iah / acs and can be present at relatively low levels of intra - abdominal pressure ( iap ) . it is also reported that at least several hours are required from the onset of acs before irreversible respiratory dysfunction , cardiac dysfunction , and ischemia of the intra - abdominal organs occur . thus , if iap does not increase continuously and remains less than 30 mmhg and no other systems or organs are affected by acs , ureteral catheter support is a good option to cope with postrenal renal failure . however , when iap continues to increase , or other organs are afflicted , opening the abdomen is warranted . in summary , combined measures , including digital subtraction angiography and embolism ( if necessary ) , external fixation of the pelvic ring , and controlling map at the proper level should be used to limit the development of retroperitoneal hematoma and to prevent the occurrence of acs . at the early stage of acs when only the urinary system is afflicted with iap less than 30 mmhg , and no signs of other organ injury or failure , bilateral ureteral catheter support to rescue renal function can be used to save renal function . however , when iap continues to increase , or other organs are afflicted , open the abdomen is mandatory . the typical symptoms included peritoneal irritation , shock , and severe sepsis , making them easily diagnosed . it is difficult to diagnose due to its clinical complexity , and the difficulty in distinguishing the adynamic ileus from bowel entrapment . in 24 such cases reported in the literature , the diagnosis were delayed in six cases.16 , 17 , 18 , 19 four cases of bowel entrapment were diagnosed during laparotomy , which was performed during the initial management of trauma . of the remaining cases , the average time to diagnosis was 14.9 days ( range 290 days ) . it is extremely difficult to diagnose when the entrapped bowel is only part of the bowel , as reported in the present case , due to the atypical symptoms . delayed diagnosis and treatment often lead to severe sepsis and death.16 , 17 , 18 , 19 ct is generally considered a sensitive tool for the diagnosis of bowel entrapment . combined use of oral and iv contrast in ct evaluation may increase diagnostic accuracy and reduce morbidity in patients with blunt abdominal injury.17 , 20 however , caution and sufficient awareness are the principal determining factors to establish the correct diagnosis of bowel entrapment . if progressive ileus in a patient with a pelvic fracture persists for over 3 days , an occult bowel injury such as entrapment at the fracture site , should be suspected and contrasted ct should accordingly be performed . immediate laparotomy is recommended as long as the diagnosis is determined to repair or resect the entrapped bowel . the pelvic fracture should usually be fixed externally at the initial stage due to risk of infection . most pelvic hemorrhages are thought to be caused by injury to small arteries or veins in the fractured cancellous pelvic bone or in the surrounding soft tissues , and only 6%18% of patients with unstable pelvic fractures exhibit hemorrhage from large arteries . when arterial injuries are present , the majority involve in branches of the internal iliac artery . there are only a few published reports of injuries to the branches of the external iliac artery , and fewer to the external iliac artery itself.22 , 23 , 24 injuries to the external iliac artery and its branches , such as the case described here , are thought to be extremely rare in pelvic fractures . the reason is due to the lower incidence of such complications , and diagnosis is sometimes delayed , thus increasing the risk of loss of both life and limb . recognition of the risk factors for injury of the external iliac artery and its branches in pelvic fractures is helpful to make a correct diagnosis . firstly , external iliac arterial injury should be highly suspected in some injury patterns of pelvic fractures . manson et al suggested that caudal displacement of the hemipelvis in rare conditions disrupts the pelvic floor and pelvic vasculature far more than a standard vertical shear injury . it is reported that caudal displacement of the hemipelvis leads to significant disruption of the external iliac artery and to surgical hemipelvectomy . pubic rami fracture is another pelvic fracture pattern in which the branch of external iliac artery ( inferior epigastric artery or corona mortis ) is highly susceptible to injury , even if the fracture is stable.23 , 24 secondly , anatomical variation is another factor for rare bleeding . the aberrant obturator artery ( aoa ) is a common arterial variant that occurs in more than half of the population and , if present in pelvic fractured patients , is commonly injured.25 , 26 thirdly , pre - injury medical conditions , such as anticoagulation treatment , are risk factors for external iliac artery injury . lastly , direct violence to the groin region as reported in the current study is a high risk factor for injury of the external iliac artery and its branches . although the incidence of external iliac artery injury is rare , the diagnosis is not difficult . delayed diagnosis leads to poor outcome . in our case , although the patient was grateful to be alive , we understood that the lost extremity could have been saved if early diagnosis had been established . when injury of the external iliac artery and its branches is confirmed , tae , endovascular blunting repair , open vascular reconstruction , and prosthetic bypass graft should be selectively performed.27 , 28 , 29 during wartime , the incidence of scrotal and/or testicular injury complicated by pelvic fracture is high . in contrast , the incidence of scrotal and/or testicular injury is rare in civilian injuries , and usually occurs in a situation called fuel tank injury . it is a rare condition involving motorcycle driver impact.31 , 32 , 33 , 34 in our case , the mechanism of injury was not fuel tank injury . it was caused by direct crash into the groin region when two legs of the patient were open . in open scrotal and/or testicular injury , the diagnosis is easy to establish ; however , in closed injury , high suspicion , thorough physical examination and doppler ultrasound are useful for correct diagnosis . the extent of soft tissue injury should be classified by multi - modality and multi - planar imaging so that correct treatment may be applied . injuries to the scrotum and its contents may cause impaired fertility , chronic pain , hypogonadism , and altered self - image ; thus , selection of treatment is highly important . if an open wound exists , primary suture of the wound is not recommended , and debridement and vacuum sealing drainage are suggested to avoid infection.37 , 38 however , aggressive debridement should be avoided if the patient is associated with pubic ramus fracture close to or connected to the scrotal wound , as reported in the current case . also , when vacuum sealing drainage is applied , the pressure should be monitored to avoid testis necrosis caused by too high pressure . external fixation of the pelvic fracture along with simple reduction of the significantly displaced pubic ramus fracture is recommended . in closed injury , dislocation of testis in north america , most european countries , and japan in asia , sophisticated trauma care systems have been established in the past couple of decades . in mainland china , greater attention is paid to the treatment of public health problems and trauma care remains in a nascent stage in small cities and rural and semi - urban areas . thus , many injured patients have a high chance of incorrect diagnosis and treatment when they are admitted to a lower level hospital . three of the four cases reported in the current study did not receive proper care at the initial stage after trauma . these situations may also be true in other developing counties , and may only be solved through the establishment of a sophisticated trauma care system . another issue for the care of rare complications of pelvic fractures is multi - disciplinary coordination . rare complications or associated injuries of pelvic fractures can be highly complex ; thus , coordination is warranted to achieve prompt and correct diagnosis and treatment . rare complications of pelvic fracture are difficult to diagnose , and failure to make a correct diagnosis usually results in poor outcome . high suspicion and sufficient awareness are the principal elements that lead to correct diagnosis and treatment . this study was supported by the national science foundation of china ( 81271935 ) , foundation of state key laboratory of trauma , burns and combined injury ( sklzz sklzz201124 ) , and the military medical research foundation of china ( aws11j008 ) .

purposeto enhance the awareness of rare complications of pelvic fracture and describe the correct diagnosis and effective treatment.methodsa total of 188 cases of pelvic fractures were retrospectively reviewed , and four patients who suffered from four types of rare pelvic fracture complications were described , namely ureteral obstruction caused by retroperitoneal hematoma - induced abdominal compartment syndrome ( acs ) , bowel entrapment , external iliac artery injury , and open scrotal sac injury.resultswe demonstrated that combined measures should be employed to prevent the occurrence of acs following major pelvic fractures . ureteral catheter support may be a good option at an early stage when acs occurred . contrasted computed tomography examination and sufficient awareness are keys to a correct diagnosis of bowel entrapment following pelvic fractures . recognition of risk factors , early diagnosis , and prompt treatment of suspected injury of the external iliac artery are keys to patient survival and to avoid limb loss . scrotal and/or testicular injury complicated by pelvic fractures should be carefully treated to maintain normal gonad function . additionally , establishment of a sophisticated trauma care system and multi - disciplinary coordination are important for correct diagnosis and treatment of rare complications in pelvic fractures.conclusionsrare complications of pelvic fractures are difficult to diagnose and negatively impact outcome . recognition of risk factors and sufficient awareness are essential for correct diagnosis and prompt treatment .

Introduction Materials and methods Results Case one Case two Case three Case four Discussion Combined measures should be employed to prevent the occurrence of ACS after pelvic fracture and ureteral catheter support is potentially a good option at the early stage of ACS when the kidney is the only affected organ Sufficient awareness and contrasted CT examination are keys to the diagnosis of bowel entrapment following pelvic fractures Sufficient awareness, recognition of risk factors, early diagnosis, and prompt treatment of suspected injury of the external iliac artery and its branches are keys to patient survival and to avoidance of limb loss Scrotal and/or testicular injury complicated by pelvic fracture is a rare condition that should be carefully treated to maintain normal gonad function Sophisticated trauma care system and multi-disciplinary coordination are important for the care of rare complications in pelvic fractures Conclusions Fund

complications of pelvic fractures are always described in a routine way and as associated injuries of pelvic fractures . the most common associated injuries are bleeding and retroperitoneal hematoma , intra - abdominal and urogenital injuries , and never injuries in addition to severe pelvic fractures.1 , 2 , 3 approximately 12% of trauma patients admitted to level 1 or 2 trauma centers in the united states exhibit pelvic ring injury , and injuries to intra - abdominal or urogenital organs are present in an estimated 16% of these patients . prompt diagnosis and effective management of these injuries is essential to a successful outcome.2 , 3 great efforts have been made to improve treatment of pelvic fracture and its complications , and substantial improvements have been achieved . however rare complications of pelvic fractures , such as ureteral obstruction caused by retroperitoneal hematoma induced abdominal compartment syndrome ( acs ) , bowel entrapment , external iliac artery injury , and open scrotal sac injury are not easily diagnosed and treated . unawareness of these rare conditions makes the diagnosis difficult , and failure to provide prompt treatment leads to poor outcomes . in order to increase the awareness of these rare complications of pelvic fracture and describe the correct diagnosis and effective treatment , we present four cases that exhibit the above - mentioned four different types of rare complications . patients who suffered from pelvic fractures in our department from june 2009 to july 2012 were retrospectively reviewed . the mechanisms of injuries , the type of pelvic fracture , and the complications are analyzed . four cases exhibited the four types of rare complications , namely ureteral obstruction caused by retroperitoneal hematoma induced abdominal compartment syndrome , bowel entrapment , external iliac artery injury , and severe open scrotal sac injury . extensive literature search on medline / pubmed was performed , including articles related to complications of pelvic and acetabular fractures . we carefully reviewed articles related to the above - mentioned four types of rare complications . suggestions for diagnosis and recommendations for treatment of these rare complications of pelvic fractures were given . a total of 188 cases of pelvic fractures were treated in our department between june 2009 and june 2012 . the mechanisms of injury included traffic injury ( 122 cases ) , high fall injury ( 35 cases ) , and heavy object pound injury ( 24 cases ) . of the 188 cases , 37 were complicated by or associated with chest injuries , 14 with brain injuries , 45 with upper extremities injuries , 36 with lower extremities injuries , 12 with ureteral injuries , 3 with bladder injuries , 6 with bowel injuries , and 3 with spleen disruption . there were four cases that exhibited four types of rare complications , namely ureteral obstruction caused by retroperitoneal hematoma induced abdominal compartment syndrome , bowel entrapment , external iliac artery injury , and severe open scrotal sac injury . after the patient was hemodynamically stabilized by infusion of 3000 ml crystalloid fluid and 1200 ml suspended red blood cells , plain roentgenogram and three - dimensional computed tomography ( ct ) were performed and demonstrated type 61-b2.1 pelvic fracture according to arbeitsgemeinschaft fur osteosynthesefragen / orthopaedic trauma association ( ao / ota ) classification scheme ( fig . digital subtraction angiography ( dsa ) performed along with ct revealed no arterial bleeding of the internal iliac artery and its branches , and thus no need to perform embolism . following placement of the bilateral ureteral catheter , external fixation of the pelvis and left femur ten days after surgery , the patient was discharged from the intensive care unit with good renal function and open reduction and internal fixation for the fracture of distal end of the left radius , proximal end fracture of the left femur . after injury , he was admitted to a level ii hospital and demonstrated pain of the left shoulder , waist , and pelvis . injury to the main arteries of the lower limb extremity was suspected , and the patient was transferred to our department 12 h following the injury . exploration of the groin region was performed and a 2-cm long thrombus was observed in the left proximal external iliac artery . the most recent follow - up visit was 5 months after the last surgical operation , and the patient had not infection of the scrotum and wound healed completely ( fig . digital subtraction angiography ( dsa ) performed along with ct revealed no arterial bleeding of the internal iliac artery and its branches , and thus no need to perform embolism . following placement of the bilateral ureteral catheter , external fixation of the pelvis and left femur ten days after surgery , the patient was discharged from the intensive care unit with good renal function and open reduction and internal fixation for the fracture of distal end of the left radius , proximal end fracture of the left femur . four month after discharge , x - ray examination showed healing of the pelvic fracture ( fig . after injury , he was admitted to a level ii hospital and demonstrated pain of the left shoulder , waist , and pelvis . angiography along with ct showed disruption of external iliac artery and bilateral pubic ramus fracture ( ao / ota type 61 a2.3 , fig . 3a ) , and lab examination showed slight elevation of serum creatinine ( cre ) and blood urea nitrogen ( bun ) . exploration of the groin region was performed and a 2-cm long thrombus was observed in the left proximal external iliac artery . part of the scrotum became necrotic , and abscess was observed in the sutured scrotum ( fig . the most recent follow - up visit was 5 months after the last surgical operation , and the patient had not infection of the scrotum and wound healed completely ( fig . acs / iah affects all body systems , in particular the cardiac , respiratory , renal , and neurologic systems , as a result of the blockage of blood flow . although it is less common , acs accompanying pelvic fracture has been previously reported.7 , 8 the prevention of acs in pelvic fracture is very important since acs leads to severe morbidity regardless of what measures are taken . combined measures have been established in our department to prevent the occurrence of acs in pelvic fractured patients . we routinely perform digital subtraction angiography ( dsa ) in patients with major pelvic fracture following successful fluid resuscitation . but even so , massive retroperitoneal hematoma and acs have been formed and lead to bilateral ureteral obstruction and renal failure as reported in the current study . under these circumstances , hessmann et al advocated evacuation of the retroperitoneal hematoma , whereas flint et al suggested that bilateral tube nephrostomy was a better option . it is also reported that at least several hours are required from the onset of acs before irreversible respiratory dysfunction , cardiac dysfunction , and ischemia of the intra - abdominal organs occur . thus , if iap does not increase continuously and remains less than 30 mmhg and no other systems or organs are affected by acs , ureteral catheter support is a good option to cope with postrenal renal failure . in summary , combined measures , including digital subtraction angiography and embolism ( if necessary ) , external fixation of the pelvic ring , and controlling map at the proper level should be used to limit the development of retroperitoneal hematoma and to prevent the occurrence of acs . at the early stage of acs when only the urinary system is afflicted with iap less than 30 mmhg , and no signs of other organ injury or failure , bilateral ureteral catheter support to rescue renal function can be used to save renal function . it is difficult to diagnose due to its clinical complexity , and the difficulty in distinguishing the adynamic ileus from bowel entrapment . in 24 such cases reported in the literature , the diagnosis were delayed in six cases.16 , 17 , 18 , 19 four cases of bowel entrapment were diagnosed during laparotomy , which was performed during the initial management of trauma . it is extremely difficult to diagnose when the entrapped bowel is only part of the bowel , as reported in the present case , due to the atypical symptoms . delayed diagnosis and treatment often lead to severe sepsis and death.16 , 17 , 18 , 19 ct is generally considered a sensitive tool for the diagnosis of bowel entrapment . combined use of oral and iv contrast in ct evaluation may increase diagnostic accuracy and reduce morbidity in patients with blunt abdominal injury.17 , 20 however , caution and sufficient awareness are the principal determining factors to establish the correct diagnosis of bowel entrapment . most pelvic hemorrhages are thought to be caused by injury to small arteries or veins in the fractured cancellous pelvic bone or in the surrounding soft tissues , and only 6%18% of patients with unstable pelvic fractures exhibit hemorrhage from large arteries . when arterial injuries are present , the majority involve in branches of the internal iliac artery . there are only a few published reports of injuries to the branches of the external iliac artery , and fewer to the external iliac artery itself.22 , 23 , 24 injuries to the external iliac artery and its branches , such as the case described here , are thought to be extremely rare in pelvic fractures . recognition of the risk factors for injury of the external iliac artery and its branches in pelvic fractures is helpful to make a correct diagnosis . firstly , external iliac arterial injury should be highly suspected in some injury patterns of pelvic fractures . it is reported that caudal displacement of the hemipelvis leads to significant disruption of the external iliac artery and to surgical hemipelvectomy . pubic rami fracture is another pelvic fracture pattern in which the branch of external iliac artery ( inferior epigastric artery or corona mortis ) is highly susceptible to injury , even if the fracture is stable.23 , 24 secondly , anatomical variation is another factor for rare bleeding . the aberrant obturator artery ( aoa ) is a common arterial variant that occurs in more than half of the population and , if present in pelvic fractured patients , is commonly injured.25 , 26 thirdly , pre - injury medical conditions , such as anticoagulation treatment , are risk factors for external iliac artery injury . lastly , direct violence to the groin region as reported in the current study is a high risk factor for injury of the external iliac artery and its branches . although the incidence of external iliac artery injury is rare , the diagnosis is not difficult . when injury of the external iliac artery and its branches is confirmed , tae , endovascular blunting repair , open vascular reconstruction , and prosthetic bypass graft should be selectively performed.27 , 28 , 29 during wartime , the incidence of scrotal and/or testicular injury complicated by pelvic fracture is high . in contrast , the incidence of scrotal and/or testicular injury is rare in civilian injuries , and usually occurs in a situation called fuel tank injury . in open scrotal and/or testicular injury , the diagnosis is easy to establish ; however , in closed injury , high suspicion , thorough physical examination and doppler ultrasound are useful for correct diagnosis . the extent of soft tissue injury should be classified by multi - modality and multi - planar imaging so that correct treatment may be applied . if an open wound exists , primary suture of the wound is not recommended , and debridement and vacuum sealing drainage are suggested to avoid infection.37 , 38 however , aggressive debridement should be avoided if the patient is associated with pubic ramus fracture close to or connected to the scrotal wound , as reported in the current case . also , when vacuum sealing drainage is applied , the pressure should be monitored to avoid testis necrosis caused by too high pressure . in north america , most european countries , and japan in asia , sophisticated trauma care systems have been established in the past couple of decades . in mainland china , greater attention is paid to the treatment of public health problems and trauma care remains in a nascent stage in small cities and rural and semi - urban areas . thus , many injured patients have a high chance of incorrect diagnosis and treatment when they are admitted to a lower level hospital . these situations may also be true in other developing counties , and may only be solved through the establishment of a sophisticated trauma care system . another issue for the care of rare complications of pelvic fractures is multi - disciplinary coordination . rare complications or associated injuries of pelvic fractures can be highly complex ; thus , coordination is warranted to achieve prompt and correct diagnosis and treatment . although it is less common , acs accompanying pelvic fracture has been previously reported.7 , 8 the prevention of acs in pelvic fracture is very important since acs leads to severe morbidity regardless of what measures are taken . combined measures have been established in our department to prevent the occurrence of acs in pelvic fractured patients . but even so , massive retroperitoneal hematoma and acs have been formed and lead to bilateral ureteral obstruction and renal failure as reported in the current study . flint and rothenberger et al believed that , because of the increased risk of sepsis and further hemorrhage , surgical exploration of the retroperitoneum to remove the hematoma should be avoided . in our cases , we selected bilateral ureteral catheter support to rescue renal function . it is also reported that at least several hours are required from the onset of acs before irreversible respiratory dysfunction , cardiac dysfunction , and ischemia of the intra - abdominal organs occur . thus , if iap does not increase continuously and remains less than 30 mmhg and no other systems or organs are affected by acs , ureteral catheter support is a good option to cope with postrenal renal failure . in summary , combined measures , including digital subtraction angiography and embolism ( if necessary ) , external fixation of the pelvic ring , and controlling map at the proper level should be used to limit the development of retroperitoneal hematoma and to prevent the occurrence of acs . at the early stage of acs when only the urinary system is afflicted with iap less than 30 mmhg , and no signs of other organ injury or failure , bilateral ureteral catheter support to rescue renal function can be used to save renal function . it is difficult to diagnose due to its clinical complexity , and the difficulty in distinguishing the adynamic ileus from bowel entrapment . in 24 such cases reported in the literature , the diagnosis were delayed in six cases.16 , 17 , 18 , 19 four cases of bowel entrapment were diagnosed during laparotomy , which was performed during the initial management of trauma . it is extremely difficult to diagnose when the entrapped bowel is only part of the bowel , as reported in the present case , due to the atypical symptoms . delayed diagnosis and treatment often lead to severe sepsis and death.16 , 17 , 18 , 19 ct is generally considered a sensitive tool for the diagnosis of bowel entrapment . combined use of oral and iv contrast in ct evaluation may increase diagnostic accuracy and reduce morbidity in patients with blunt abdominal injury.17 , 20 however , caution and sufficient awareness are the principal determining factors to establish the correct diagnosis of bowel entrapment . if progressive ileus in a patient with a pelvic fracture persists for over 3 days , an occult bowel injury such as entrapment at the fracture site , should be suspected and contrasted ct should accordingly be performed . most pelvic hemorrhages are thought to be caused by injury to small arteries or veins in the fractured cancellous pelvic bone or in the surrounding soft tissues , and only 6%18% of patients with unstable pelvic fractures exhibit hemorrhage from large arteries . there are only a few published reports of injuries to the branches of the external iliac artery , and fewer to the external iliac artery itself.22 , 23 , 24 injuries to the external iliac artery and its branches , such as the case described here , are thought to be extremely rare in pelvic fractures . recognition of the risk factors for injury of the external iliac artery and its branches in pelvic fractures is helpful to make a correct diagnosis . firstly , external iliac arterial injury should be highly suspected in some injury patterns of pelvic fractures . it is reported that caudal displacement of the hemipelvis leads to significant disruption of the external iliac artery and to surgical hemipelvectomy . pubic rami fracture is another pelvic fracture pattern in which the branch of external iliac artery ( inferior epigastric artery or corona mortis ) is highly susceptible to injury , even if the fracture is stable.23 , 24 secondly , anatomical variation is another factor for rare bleeding . the aberrant obturator artery ( aoa ) is a common arterial variant that occurs in more than half of the population and , if present in pelvic fractured patients , is commonly injured.25 , 26 thirdly , pre - injury medical conditions , such as anticoagulation treatment , are risk factors for external iliac artery injury . lastly , direct violence to the groin region as reported in the current study is a high risk factor for injury of the external iliac artery and its branches . although the incidence of external iliac artery injury is rare , the diagnosis is not difficult . when injury of the external iliac artery and its branches is confirmed , tae , endovascular blunting repair , open vascular reconstruction , and prosthetic bypass graft should be selectively performed.27 , 28 , 29 during wartime , the incidence of scrotal and/or testicular injury complicated by pelvic fracture is high . in contrast , the incidence of scrotal and/or testicular injury is rare in civilian injuries , and usually occurs in a situation called fuel tank injury . it was caused by direct crash into the groin region when two legs of the patient were open . in open scrotal and/or testicular injury , the diagnosis is easy to establish ; however , in closed injury , high suspicion , thorough physical examination and doppler ultrasound are useful for correct diagnosis . the extent of soft tissue injury should be classified by multi - modality and multi - planar imaging so that correct treatment may be applied . if an open wound exists , primary suture of the wound is not recommended , and debridement and vacuum sealing drainage are suggested to avoid infection.37 , 38 however , aggressive debridement should be avoided if the patient is associated with pubic ramus fracture close to or connected to the scrotal wound , as reported in the current case . also , when vacuum sealing drainage is applied , the pressure should be monitored to avoid testis necrosis caused by too high pressure . in closed injury , dislocation of testis in north america , most european countries , and japan in asia , sophisticated trauma care systems have been established in the past couple of decades . thus , many injured patients have a high chance of incorrect diagnosis and treatment when they are admitted to a lower level hospital . these situations may also be true in other developing counties , and may only be solved through the establishment of a sophisticated trauma care system . another issue for the care of rare complications of pelvic fractures is multi - disciplinary coordination . rare complications or associated injuries of pelvic fractures can be highly complex ; thus , coordination is warranted to achieve prompt and correct diagnosis and treatment . rare complications of pelvic fracture are difficult to diagnose , and failure to make a correct diagnosis usually results in poor outcome . high suspicion and sufficient awareness are the principal elements that lead to correct diagnosis and treatment .

acute respiratory distress syndrome ( ards ) is a clinical entity characterized by acute onset respiratory failure , diffuse pulmonary opacities , and severe hypoxemia , in the absence of obvious cardiac dysfunction . based on the severity of hypoxemia , ards is categorized as mild ( pao2/fio2 ratio > 200 and 300 mmhg ) , moderate ( pao2/fio2 ratio > 100 and 200 mmhg ) , and severe ( pao2/fio2 ratio 100 mmhg ) . with increasing severity of ards , the mortality increases from 27% in mild to 45% in severe ards . apart from treatment of the underlying cause , the management of ards involves correction of hypoxemia either by supplemental oxygen or by the provision of positive pressure ventilation . positive pressure ventilation can be delivered either invasively ( endotracheal airway ) or noninvasively ( face or nasal mask ) . invasive mechanical ventilation is the standard of care for the management of ards and has been demonstrated to reduce mortality . however , invasive ventilation is associated with several complications including a higher incidence of ventilator - associated pneumonia , barotrauma , volutrauma , and others . niv has an important role in the management of acute respiratory failure , especially those secondary to acute exacerbations of chronic obstructive pulmonary disease and acute cardiogenic pulmonary edema . in hypoxemic respiratory failure , niv improves oxygenation , reduce dyspnea , unload respiratory muscle , and hence may help in avoiding invasive mechanical ventilation . a recent meta - analysis of six randomized controlled trials involving subjects with ards suggested that the use of niv avoided intubation , but not mortality . however , in the meta - analysis , only three studies ( n = 89 ) involved patients with ards and the analysis also included postsurgical subjects with atelectasis . in another meta - analysis involving only subjects with ards , it was shown that niv avoids intubation in approximately 50% of the patients , provided the patients are judiciously chosen . we hypothesized that the use of niv may help in avoiding intubation in subjects with mild to moderate ards . in this study , we report our experience with the use of niv in ards . this was a prospective observational study assessing the efficacy of niv in combination with standard therapy in patients with ards . the study protocol was approved by the institute 's ethics committee and a written informed consent was obtained from all the patients or their next of kin . the study was conducted between june 2011 and may 2012 in the intensive care unit ( icu ) of the department of pulmonary medicine at the author 's institute . the icu is a 10-bedded unit catering to the care of medical - surgical critically ill patients . it comprises a team of seven physicians ( two consultants and five residents specialized in pulmonary and intensive care ) . two physicians are present round the clock and are supported by the nursing staff with a nurse to patient ratio of 1:2 . consecutive patients aged 18 years with a diagnosis of ards were eligible for inclusion in the study . ards was diagnosed based on the american - european consensus conference : acute onset respiratory failure of < 7 days ; bilateral infiltrates on chest radiograph ; pao2/fio2 < 300 ; and no clinical evidence of left heart failure or left atrial size < 40 mm on echocardiography . patients with any of the following were excluded : pao2/fio2 100 , age < 18 years , cardiogenic pulmonary edema , more than 2 organ failure , traumatic lung injury , hemodynamic instability , chronic carbon dioxide retention ( paco2 > 50 mmhg ) at presentation , severe upper gastrointestinal bleed , altered mentation with a glasgow coma scale < 8 , and facial abnormality . a sample of convenience , comprising 50 subjects with a diagnosis of ards was planned . subjects enrolled in the study were treated with niv using the icu ventilators ( servo - i universal , maquet , getinge ) . niv was started with initial inspiratory positive airway pressure ( ipap ) of 68 cm of h2o and was gradually increased by 2 cm to achieve clinical response in the form of relief of dyspnea , respiratory rate of 30 breaths / min and a tidal volume of 68 ml / kg ideal body weight , or a maximum ipap of 20 cm of h2o . expiratory positive airway pressure ( epap ) was initiated at 34 cm of h2o and increased by 1 cm of h2o to achieve a spo2 92% or a maximum epap of 10 cm of h2o . during the initial 24 h of niv , patients were weaned off niv if they were able to maintain spo2 92% on fio2 of 30% and the respiratory rate was 30 breaths / min . worsening of dyspnea , worsening of or lack of improvement in hypoxemia , persistence of respiratory rate > 35 breaths / min , appearance of respiratory acidosis , circulatory shock , or altered sensorium were defined as the features of niv failure , and the patient was qualified for endotracheal intubation . the ultimate decision to intubate was , however , left to the discretion of the icu physician . baseline demographic and clinical characteristics including the underlying etiology for ards were recorded for all the study participants . all subjects were monitored for clinical ( respiratory rate , blood pressure , sensorium , and others ) and laboratory parameters ( partial pressure of oxygen , partial pressure of carbon dioxide , arterial ph , and others ) . arterial blood gases ( abg ) , respiratory rate , mental status , mean arterial blood pressure , and pao2/fio2 values were recorded at the initiation of niv and then at 1 h , 4 h , and at the end of therapy . liver and renal function tests , complete blood count , and acute physiology and chronic health evaluation ii ( apache ii ) score ( at 24 h ) were performed in all the subjects . the diagnosis of malaria was based on the demonstration of malarial parasite in peripheral blood film or malaria antigen by commercially available rapid diagnostic test kit ( malaria antigen [ pf / pan ] detection test device , mediclone biotech private limited , india ) performed at least twice . the diagnosis of scrub typhus was made on the demonstration of positive igm antibody against orientia tsutsugamushi derived recombinant antigens by elisa method ( scrub typhus detect igm elisa , inbios india ) . icterohemorrhagiae , pomona , hebdomanis , australis , canicola , grippotyphosa , patoc , autumnalis , and cynopteri ) detected by microscopic agglutination testing ( mat ) led to a diagnosis of leptospirosis . h1n1 swine flu was diagnosed if influenza virus rna was detected in the nasopharyngeal swab by reverse transcriptase polymerase chain reaction . all the patients received deep venous thrombosis and stress ulcer prophylaxis as per the icu protocol . the primary outcome was the success of niv defined as the number of patients in whom endotracheal intubation was avoided . the secondary outcomes were duration of niv , icu stay , hospital mortality , and improvement in clinical and blood gas values at 1 and 4 h. factors predicting the success of niv in avoiding intubation were also analyzed . complications of niv , such as claustrophobia , intolerance , abdominal distention , pressure sores , and nasal bridge trauma , were also noted . changes at 1 h from baseline for pao2/fio2 ratio , paco2 , ph , mean arterial pressure , and respiratory rate were calculated . results are presented in a descriptive fashion as number and percentage or mean standard deviation ( sd ) , unless otherwise stated . improvement in clinical ( respiratory rate and mean arterial blood pressure ) and abg parameters ( ph , pao2 , and paco2 ) was analyzed using multifactorial repeated measures analysis of variance , with bonferroni adjustment for multiple comparisons ; the within - groups factor was time ( baseline , 1 , and 4 h ) , and the between - groups factor was niv success . for within - group analysis , univariate approach was used , as mauchly 's test of sphericity was found to be significant ( > 0.05 ) . post - hoc analysis for calculating the difference in means within - group a univariate logistic regression analysis was performed to derive adjusted odds ratios and 95% confidence intervals to analyze the factors predicting niv failure . this was a prospective observational study assessing the efficacy of niv in combination with standard therapy in patients with ards . the study protocol was approved by the institute 's ethics committee and a written informed consent was obtained from all the patients or their next of kin . the study was conducted between june 2011 and may 2012 in the intensive care unit ( icu ) of the department of pulmonary medicine at the author 's institute . the icu is a 10-bedded unit catering to the care of medical - surgical critically ill patients . it comprises a team of seven physicians ( two consultants and five residents specialized in pulmonary and intensive care ) . two physicians are present round the clock and are supported by the nursing staff with a nurse to patient ratio of 1:2 . consecutive patients aged 18 years with a diagnosis of ards were eligible for inclusion in the study . ards was diagnosed based on the american - european consensus conference : acute onset respiratory failure of < 7 days ; bilateral infiltrates on chest radiograph ; pao2/fio2 < 300 ; and no clinical evidence of left heart failure or left atrial size < 40 mm on echocardiography . patients with any of the following were excluded : pao2/fio2 100 , age < 18 years , cardiogenic pulmonary edema , more than 2 organ failure , traumatic lung injury , hemodynamic instability , chronic carbon dioxide retention ( paco2 > 50 mmhg ) at presentation , severe upper gastrointestinal bleed , altered mentation with a glasgow coma scale < 8 , and facial abnormality . a sample of convenience , comprising 50 subjects with a diagnosis of ards was planned . subjects enrolled in the study were treated with niv using the icu ventilators ( servo - i universal , maquet , getinge ) . niv was started with initial inspiratory positive airway pressure ( ipap ) of 68 cm of h2o and was gradually increased by 2 cm to achieve clinical response in the form of relief of dyspnea , respiratory rate of 30 breaths / min and a tidal volume of 68 ml / kg ideal body weight , or a maximum ipap of 20 cm of h2o . expiratory positive airway pressure ( epap ) was initiated at 34 cm of h2o and increased by 1 cm of h2o to achieve a spo2 92% or a maximum epap of 10 cm of h2o . during the initial 24 h of niv , patients were weaned off niv if they were able to maintain spo2 92% on fio2 of 30% and the respiratory rate was 30 breaths / min . worsening of dyspnea , worsening of or lack of improvement in hypoxemia , persistence of respiratory rate > 35 breaths / min , appearance of respiratory acidosis , circulatory shock , or altered sensorium were defined as the features of niv failure , and the patient was qualified for endotracheal intubation . the ultimate decision to intubate was , however , left to the discretion of the icu physician . baseline demographic and clinical characteristics including the underlying etiology for ards were recorded for all the study participants . all subjects were monitored for clinical ( respiratory rate , blood pressure , sensorium , and others ) and laboratory parameters ( partial pressure of oxygen , partial pressure of carbon dioxide , arterial ph , and others ) . arterial blood gases ( abg ) , respiratory rate , mental status , mean arterial blood pressure , and pao2/fio2 values were recorded at the initiation of niv and then at 1 h , 4 h , and at the end of therapy . liver and renal function tests , complete blood count , and acute physiology and chronic health evaluation ii ( apache ii ) score ( at 24 h ) were performed in all the subjects . the diagnosis of malaria was based on the demonstration of malarial parasite in peripheral blood film or malaria antigen by commercially available rapid diagnostic test kit ( malaria antigen [ pf / pan ] detection test device , mediclone biotech private limited , india ) performed at least twice . the diagnosis of scrub typhus was made on the demonstration of positive igm antibody against orientia tsutsugamushi derived recombinant antigens by elisa method ( scrub typhus detect igm elisa , inbios india ) . icterohemorrhagiae , pomona , hebdomanis , australis , canicola , grippotyphosa , patoc , autumnalis , and cynopteri ) detected by microscopic agglutination testing ( mat ) led to a diagnosis of leptospirosis . h1n1 swine flu was diagnosed if influenza virus rna was detected in the nasopharyngeal swab by reverse transcriptase polymerase chain reaction . all the patients received deep venous thrombosis and stress ulcer prophylaxis as per the icu protocol . the primary outcome was the success of niv defined as the number of patients in whom endotracheal intubation was avoided . the secondary outcomes were duration of niv , icu stay , hospital mortality , and improvement in clinical and blood gas values at 1 and 4 h. factors predicting the success of niv in avoiding intubation were also analyzed . complications of niv , such as claustrophobia , intolerance , abdominal distention , pressure sores , and nasal bridge trauma , were also noted . changes at 1 h from baseline for pao2/fio2 ratio , paco2 , ph , mean arterial pressure , and respiratory rate were calculated . results are presented in a descriptive fashion as number and percentage or mean standard deviation ( sd ) , unless otherwise stated . improvement in clinical ( respiratory rate and mean arterial blood pressure ) and abg parameters ( ph , pao2 , and paco2 ) was analyzed using multifactorial repeated measures analysis of variance , with bonferroni adjustment for multiple comparisons ; the within - groups factor was time ( baseline , 1 , and 4 h ) , and the between - groups factor was niv success . for within - group analysis , univariate approach was used , as mauchly 's test of sphericity was found to be significant ( > 0.05 ) . post - hoc analysis for calculating the difference in means within - group a univariate logistic regression analysis was performed to derive adjusted odds ratios and 95% confidence intervals to analyze the factors predicting niv failure . this was a prospective observational study assessing the efficacy of niv in combination with standard therapy in patients with ards . the study protocol was approved by the institute 's ethics committee and a written informed consent was obtained from all the patients or their next of kin . the study was conducted between june 2011 and may 2012 in the intensive care unit ( icu ) of the department of pulmonary medicine at the author 's institute . the icu is a 10-bedded unit catering to the care of medical - surgical critically ill patients . it comprises a team of seven physicians ( two consultants and five residents specialized in pulmonary and intensive care ) . two physicians are present round the clock and are supported by the nursing staff with a nurse to patient ratio of 1:2 . consecutive patients aged 18 years with a diagnosis of ards were eligible for inclusion in the study . ards was diagnosed based on the american - european consensus conference : acute onset respiratory failure of < 7 days ; bilateral infiltrates on chest radiograph ; pao2/fio2 < 300 ; and no clinical evidence of left heart failure or left atrial size < 40 mm on echocardiography . patients with any of the following were excluded : pao2/fio2 100 , age < 18 years , cardiogenic pulmonary edema , more than 2 organ failure , traumatic lung injury , hemodynamic instability , chronic carbon dioxide retention ( paco2 > 50 mmhg ) at presentation , severe upper gastrointestinal bleed , altered mentation with a glasgow coma scale < 8 , and facial abnormality . a sample of convenience , comprising 50 subjects with a diagnosis of ards was planned . subjects enrolled in the study were treated with niv using the icu ventilators ( servo - i universal , maquet , getinge ) . niv was started with initial inspiratory positive airway pressure ( ipap ) of 68 cm of h2o and was gradually increased by 2 cm to achieve clinical response in the form of relief of dyspnea , respiratory rate of 30 breaths / min and a tidal volume of 68 ml / kg ideal body weight , or a maximum ipap of 20 cm of h2o . expiratory positive airway pressure ( epap ) was initiated at 34 cm of h2o and increased by 1 cm of h2o to achieve a spo2 92% or a maximum epap of 10 cm of h2o . during the initial 24 h of niv , patients were weaned off niv if they were able to maintain spo2 92% on fio2 of 30% and the respiratory rate was 30 breaths / min . worsening of dyspnea , worsening of or lack of improvement in hypoxemia , persistence of respiratory rate > 35 breaths / min , appearance of respiratory acidosis , circulatory shock , or altered sensorium were defined as the features of niv failure , and the patient was qualified for endotracheal intubation . the ultimate decision to intubate was , however , left to the discretion of the icu physician . baseline demographic and clinical characteristics including the underlying etiology for ards were recorded for all the study participants . all subjects were monitored for clinical ( respiratory rate , blood pressure , sensorium , and others ) and laboratory parameters ( partial pressure of oxygen , partial pressure of carbon dioxide , arterial ph , and others ) . arterial blood gases ( abg ) , respiratory rate , mental status , mean arterial blood pressure , and pao2/fio2 values were recorded at the initiation of niv and then at 1 h , 4 h , and at the end of therapy . liver and renal function tests , complete blood count , and acute physiology and chronic health evaluation ii ( apache ii ) score ( at 24 h ) were performed in all the subjects . the diagnosis of malaria was based on the demonstration of malarial parasite in peripheral blood film or malaria antigen by commercially available rapid diagnostic test kit ( malaria antigen [ pf / pan ] detection test device , mediclone biotech private limited , india ) performed at least twice . the diagnosis of scrub typhus was made on the demonstration of positive igm antibody against orientia tsutsugamushi derived recombinant antigens by elisa method ( scrub typhus detect igm elisa , inbios india ) . presence of igm antibody against leptospira antigen ( nine serovars ; icterohemorrhagiae , pomona , hebdomanis , australis , canicola , grippotyphosa , patoc , autumnalis , and cynopteri ) detected by microscopic agglutination testing ( mat ) led to a diagnosis of leptospirosis . swine flu was diagnosed if influenza virus rna was detected in the nasopharyngeal swab by reverse transcriptase polymerase chain reaction . all the patients received deep venous thrombosis and stress ulcer prophylaxis as per the icu protocol . the primary outcome was the success of niv defined as the number of patients in whom endotracheal intubation was avoided . the secondary outcomes were duration of niv , icu stay , hospital mortality , and improvement in clinical and blood gas values at 1 and 4 h. factors predicting the success of niv in avoiding intubation were also analyzed . complications of niv , such as claustrophobia , intolerance , abdominal distention , pressure sores , and nasal bridge trauma , were also noted . changes at 1 h from baseline for pao2/fio2 ratio , paco2 , ph , mean arterial pressure , and respiratory rate were calculated . results are presented in a descriptive fashion as number and percentage or mean standard deviation ( sd ) , unless otherwise stated . improvement in clinical ( respiratory rate and mean arterial blood pressure ) and abg parameters ( ph , pao2 , and paco2 ) was analyzed using multifactorial repeated measures analysis of variance , with bonferroni adjustment for multiple comparisons ; the within - groups factor was time ( baseline , 1 , and 4 h ) , and the between - groups factor was niv success . for within - group analysis , univariate approach was used , as mauchly 's test of sphericity was found to be significant ( > 0.05 ) . post - hoc analysis for calculating the difference in means within - group was done by scheffe 's test . for between - groups a univariate logistic regression analysis was performed to derive adjusted odds ratios and 95% confidence intervals to analyze the factors predicting niv failure . there were 250 admissions during the study period , of which 50 patients had a diagnosis of ards at presentation . nine patients were excluded ( cardiogenic pulmonary edema , n = 2 ; severe ards , hypotension or poor mental status , n = 7 ) and 41 patients were included in the study . the mean sd age of the study population was 30.9 11.4 years ; 27 ( 65.9% ) were women . tropical infections followed by abdominal sepsis were the most common causes of ards [ table 1 ] . the median ( interquartile range [ iqr ] ) duration of illness prior to icu presentation was 3 ( 24 ) days . extrapulmonary ards ( n = 31 , 75.6% ) was encountered in the vast majority . the use of niv was successful in 18 ( 44% ) patients , while 23 subjects failed the initial trial of niv and required endotracheal intubation . the median ( iqr ) time to intubation was 3 ( 14 ) h. the indications for intubation were refractory hypoxemia ( n = 9 ; 22% ) , hypotension ( n = 7 ; 17.1% ) , altered sensorium ( n = 6 ; 14.6% ) , and uncorrected respiratory acidosis ( n = 1 ; 2.4% ) . overall , there were 19 ( 46.3% ) deaths in the study population , all in those failing niv . clinical and demographic profile of the study population the subjects were divided into two groups namely those with the successful application of niv and those failing niv [ table 2 ] . there was no difference in terms of age , gender distribution , and duration of illness before admission to icu between the two groups . the underlying etiology , severity ( mild or moderate ) , and type of ards ( pulmonary or extrapulmonary ) were equally represented in the two groups . the niv failure arm had a significantly higher apache ii score at baseline ( 23.7 vs. 13.2 ; p < 0.0001 ) in comparison to niv success . the median pao2/fio2 ratio at baseline was significantly higher in those requiring invasive mechanical ventilation . the duration of ventilation ( noninvasive and invasive ) was significantly higher in those requiring endotracheal intubation [ table 3 ] . clinical characteristics of the niv success and niv failure population outcome parameters of the study groups there was significant reduction in the respiratory rate in both groups at the end of 1 h and 4 h compared to baseline ; however , this was not different between the two groups [ table 4 ] . there was an increase in pao2/fio2 at 1 h in those with niv success in contrast to those with niv failure [ table 4 ] . the pao2/fio2 value of < 150 at 1 h had the best specificity in predicting niv failure compared to other values [ table 5 ] . on univariate logistic regression analysis , failure of improvement in pao2/fio2 at 1 h and higher baseline apache ii scores were the predictors of niv failure [ table 6 ] . serial clinical and arterial blood gas parameters during icu course of the two groups impact of pao2/fio2 ratio at 1 h and baseline apache > 17 factors predicting niv failure : univariate logistic regression analysis the results of this study suggest that the use of niv helped in avoiding intubation in 44% of the subjects with mild to moderate ards . baseline apache ii score > 17 and lack of improvement in pao2/fio2 ratio > 150 after 1 h of niv predicted niv failure . the successful use of niv results in the improvement of oxygenation in subjects with ards . epap with niv , akin to positive end - expiratory pressure opens collapsed alveoli with resultant increase in functional residual capacity and reduction of right to left shunt . this causes improvement in oxygenation , relief in dyspnea , and reduction in the respiratory muscle workload . in a recent study describing the early use of niv in mild ards , although in a meta - analysis of thirteen studies describing the use of niv in mild - to - moderate ards , niv prevented intubation in 50% of the patients . this was also observed in the current study where the use of niv prevented intubation in 44% of the subjects . tropical infections followed by abdominal sepsis were the most common causes of ards in the current study . the predominant type of ards was extrapulmonary ards , although the type of ards ( extrapulmonary or pulmonary ) did not affect the outcome of niv use in the current study , similar to a previous report . in another study with pulmonary ards secondary to h1n1 , this suggests that irrespective of the type of ards ( extrapulmonary or pulmonary ) , the use of niv helps in avoiding intubation in roughly half of the patients . niv use should be judicious in patients with ards . even in a highly selected group of patients with ards , that is , only those with mild to moderate ards with two or less organ system involvement and the absence of shock at presentation , niv achieved success in only 44% . in addition , in the present study , 19 of the 23 subjects in the niv failure group died , while there were no deaths in the niv success group . this finding is consistent with previously reported experience where the hospital mortality was significantly higher in the niv failure group ( 53.8100% ) in comparison to the niv success group . the most important reason for high mortality in ards patients managed with niv , apart from refractory hypoxemia is the severe systemic illness as reflected by higher disease severity scores such as apache ii . however , one can not rule out the possibility that overzealous use of niv led to an inadvertent delay in invasive mechanical ventilation and might have contributed to a higher mortality in this subgroup of patients . although the median time to intubation was 3 h , elective intubation in subjects with a pao2/fio2 ratio of < 150 at 1 h could have resulted in lesser deaths , and delay in intubation have been associated with poor outcomes . the use of niv was associated with a significant improvement in mean arterial blood pressure , paco2 , and pao2/fio2 ratio at 1 and 4 h of niv use in the niv success group . in a study comprising 54 subjects with ards , presence of hypotension and lack of improvement in oxygenation at 1 h were associated with the failure of niv to avoid intubation . in the study by antonelli et al . , pao2/fio2 ratio of 175 predicted niv failure , while in another study , pao2/fio2 ratio < 200 at 1-h predicted niv failure in subjects with ards . we evaluated 4 different values of pao2/fio2 ratio at 1 h based on previous studies [ table 5 ] . a value of pao2/fio2 ratio of < 150 at 1 h had the best combination of sensitivity ( 34.8% ) and specificity ( 94.4% ) . further , in the univariate analysis , change in pao2/fio2 ratio at 1 h and a high baseline apache ii score predicted failure of niv in avoiding intubation in subjects with ards . these findings suggest that patients with pao2/fio2 ratio of < 150 after 1 h of niv should be monitored even more closely . the study is a single center observational study comprising a small sample size and hence the results of the current study need to be confirmed in a multicenter study with a larger sample size . the study comprised predominantly of extrapulmonary ards and did not include subjects with severe ards ( pao2/fio2 ratio 100 ) . however , a randomized trial may not be feasible in patients with moderate - to - severe ards as they are likely to need some form of ventilatory support ( noninvasive or invasive ) rather than oxygen supplementation alone . judicious use of niv in subjects with mild to moderate ards helped in avoiding intubation in 44% of the subjects in the current study . a baseline apache ii score of > 17 and a pao2/fio2 ratio < 150 at 1 h predicted failure of niv in preventing intubation in the study population .

aim : there is sparse data on the role of noninvasive ventilation ( niv ) in acute respiratory distress syndrome ( ards ) from india . herein , we report our experience with the use of niv in mild to moderate ards.materials and methods : this was a prospective observational study involving consecutive subjects of ards treated with niv using an oronasal mask . patients were monitored clinically with serial arterial blood gas analysis . the success of niv , duration of niv use , intensive care unit stay , hospital mortality , and improvement in clinical and blood gas parameters were assessed . the success of niv was defined as prevention of endotracheal intubation.results:a total of 41 subjects ( 27 women , mean age : 30.9 years ) were included in the study . tropical infections followed by abdominal sepsis were the most common causes of ards . the use of niv was successful in 18 ( 44% ) subjects , while 23 subjects required intubation . the median time to intubation was 3 h. overall , 19 ( 46.3% ) deaths were encountered , all in those requiring invasive ventilation . the mean duration of ventilation was significantly higher in the intubated patients ( 7.1 vs. 2.6 days , p = 0.004 ) . univariate analysis revealed a lack of improvement in pao2/fio2 at 1 h and high baseline acute physiology and chronic health evaluation ii ( apache ii ) as predictors of niv failure.conclusions:use of niv in mild to moderate ards helped in avoiding intubation in about 44% of the subjects . a baseline apache ii score of > 17 and a pao2/fio2 ratio < 150 at 1 h predicts niv failure .

Introduction Materials and Methods None Study design Setting Patients Study protocol Study procedures Study outcomes Statistical analysis Results Discussion Conclusion None Financial support and sponsorship Conflicts of interest

acute respiratory distress syndrome ( ards ) is a clinical entity characterized by acute onset respiratory failure , diffuse pulmonary opacities , and severe hypoxemia , in the absence of obvious cardiac dysfunction . based on the severity of hypoxemia , ards is categorized as mild ( pao2/fio2 ratio > 200 and 300 mmhg ) , moderate ( pao2/fio2 ratio > 100 and 200 mmhg ) , and severe ( pao2/fio2 ratio 100 mmhg ) . with increasing severity of ards , the mortality increases from 27% in mild to 45% in severe ards . apart from treatment of the underlying cause , the management of ards involves correction of hypoxemia either by supplemental oxygen or by the provision of positive pressure ventilation . niv has an important role in the management of acute respiratory failure , especially those secondary to acute exacerbations of chronic obstructive pulmonary disease and acute cardiogenic pulmonary edema . a recent meta - analysis of six randomized controlled trials involving subjects with ards suggested that the use of niv avoided intubation , but not mortality . we hypothesized that the use of niv may help in avoiding intubation in subjects with mild to moderate ards . in this study , we report our experience with the use of niv in ards . this was a prospective observational study assessing the efficacy of niv in combination with standard therapy in patients with ards . the study protocol was approved by the institute 's ethics committee and a written informed consent was obtained from all the patients or their next of kin . the study was conducted between june 2011 and may 2012 in the intensive care unit ( icu ) of the department of pulmonary medicine at the author 's institute . consecutive patients aged 18 years with a diagnosis of ards were eligible for inclusion in the study . patients with any of the following were excluded : pao2/fio2 100 , age < 18 years , cardiogenic pulmonary edema , more than 2 organ failure , traumatic lung injury , hemodynamic instability , chronic carbon dioxide retention ( paco2 > 50 mmhg ) at presentation , severe upper gastrointestinal bleed , altered mentation with a glasgow coma scale < 8 , and facial abnormality . subjects enrolled in the study were treated with niv using the icu ventilators ( servo - i universal , maquet , getinge ) . niv was started with initial inspiratory positive airway pressure ( ipap ) of 68 cm of h2o and was gradually increased by 2 cm to achieve clinical response in the form of relief of dyspnea , respiratory rate of 30 breaths / min and a tidal volume of 68 ml / kg ideal body weight , or a maximum ipap of 20 cm of h2o . during the initial 24 h of niv , patients were weaned off niv if they were able to maintain spo2 92% on fio2 of 30% and the respiratory rate was 30 breaths / min . worsening of dyspnea , worsening of or lack of improvement in hypoxemia , persistence of respiratory rate > 35 breaths / min , appearance of respiratory acidosis , circulatory shock , or altered sensorium were defined as the features of niv failure , and the patient was qualified for endotracheal intubation . all subjects were monitored for clinical ( respiratory rate , blood pressure , sensorium , and others ) and laboratory parameters ( partial pressure of oxygen , partial pressure of carbon dioxide , arterial ph , and others ) . arterial blood gases ( abg ) , respiratory rate , mental status , mean arterial blood pressure , and pao2/fio2 values were recorded at the initiation of niv and then at 1 h , 4 h , and at the end of therapy . liver and renal function tests , complete blood count , and acute physiology and chronic health evaluation ii ( apache ii ) score ( at 24 h ) were performed in all the subjects . the primary outcome was the success of niv defined as the number of patients in whom endotracheal intubation was avoided . the secondary outcomes were duration of niv , icu stay , hospital mortality , and improvement in clinical and blood gas values at 1 and 4 h. factors predicting the success of niv in avoiding intubation were also analyzed . complications of niv , such as claustrophobia , intolerance , abdominal distention , pressure sores , and nasal bridge trauma , were also noted . changes at 1 h from baseline for pao2/fio2 ratio , paco2 , ph , mean arterial pressure , and respiratory rate were calculated . improvement in clinical ( respiratory rate and mean arterial blood pressure ) and abg parameters ( ph , pao2 , and paco2 ) was analyzed using multifactorial repeated measures analysis of variance , with bonferroni adjustment for multiple comparisons ; the within - groups factor was time ( baseline , 1 , and 4 h ) , and the between - groups factor was niv success . this was a prospective observational study assessing the efficacy of niv in combination with standard therapy in patients with ards . the study protocol was approved by the institute 's ethics committee and a written informed consent was obtained from all the patients or their next of kin . the study was conducted between june 2011 and may 2012 in the intensive care unit ( icu ) of the department of pulmonary medicine at the author 's institute . consecutive patients aged 18 years with a diagnosis of ards were eligible for inclusion in the study . patients with any of the following were excluded : pao2/fio2 100 , age < 18 years , cardiogenic pulmonary edema , more than 2 organ failure , traumatic lung injury , hemodynamic instability , chronic carbon dioxide retention ( paco2 > 50 mmhg ) at presentation , severe upper gastrointestinal bleed , altered mentation with a glasgow coma scale < 8 , and facial abnormality . subjects enrolled in the study were treated with niv using the icu ventilators ( servo - i universal , maquet , getinge ) . niv was started with initial inspiratory positive airway pressure ( ipap ) of 68 cm of h2o and was gradually increased by 2 cm to achieve clinical response in the form of relief of dyspnea , respiratory rate of 30 breaths / min and a tidal volume of 68 ml / kg ideal body weight , or a maximum ipap of 20 cm of h2o . during the initial 24 h of niv , patients were weaned off niv if they were able to maintain spo2 92% on fio2 of 30% and the respiratory rate was 30 breaths / min . worsening of dyspnea , worsening of or lack of improvement in hypoxemia , persistence of respiratory rate > 35 breaths / min , appearance of respiratory acidosis , circulatory shock , or altered sensorium were defined as the features of niv failure , and the patient was qualified for endotracheal intubation . arterial blood gases ( abg ) , respiratory rate , mental status , mean arterial blood pressure , and pao2/fio2 values were recorded at the initiation of niv and then at 1 h , 4 h , and at the end of therapy . liver and renal function tests , complete blood count , and acute physiology and chronic health evaluation ii ( apache ii ) score ( at 24 h ) were performed in all the subjects . the primary outcome was the success of niv defined as the number of patients in whom endotracheal intubation was avoided . the secondary outcomes were duration of niv , icu stay , hospital mortality , and improvement in clinical and blood gas values at 1 and 4 h. factors predicting the success of niv in avoiding intubation were also analyzed . complications of niv , such as claustrophobia , intolerance , abdominal distention , pressure sores , and nasal bridge trauma , were also noted . changes at 1 h from baseline for pao2/fio2 ratio , paco2 , ph , mean arterial pressure , and respiratory rate were calculated . improvement in clinical ( respiratory rate and mean arterial blood pressure ) and abg parameters ( ph , pao2 , and paco2 ) was analyzed using multifactorial repeated measures analysis of variance , with bonferroni adjustment for multiple comparisons ; the within - groups factor was time ( baseline , 1 , and 4 h ) , and the between - groups factor was niv success . this was a prospective observational study assessing the efficacy of niv in combination with standard therapy in patients with ards . the study protocol was approved by the institute 's ethics committee and a written informed consent was obtained from all the patients or their next of kin . the study was conducted between june 2011 and may 2012 in the intensive care unit ( icu ) of the department of pulmonary medicine at the author 's institute . consecutive patients aged 18 years with a diagnosis of ards were eligible for inclusion in the study . patients with any of the following were excluded : pao2/fio2 100 , age < 18 years , cardiogenic pulmonary edema , more than 2 organ failure , traumatic lung injury , hemodynamic instability , chronic carbon dioxide retention ( paco2 > 50 mmhg ) at presentation , severe upper gastrointestinal bleed , altered mentation with a glasgow coma scale < 8 , and facial abnormality . subjects enrolled in the study were treated with niv using the icu ventilators ( servo - i universal , maquet , getinge ) . niv was started with initial inspiratory positive airway pressure ( ipap ) of 68 cm of h2o and was gradually increased by 2 cm to achieve clinical response in the form of relief of dyspnea , respiratory rate of 30 breaths / min and a tidal volume of 68 ml / kg ideal body weight , or a maximum ipap of 20 cm of h2o . during the initial 24 h of niv , patients were weaned off niv if they were able to maintain spo2 92% on fio2 of 30% and the respiratory rate was 30 breaths / min . worsening of dyspnea , worsening of or lack of improvement in hypoxemia , persistence of respiratory rate > 35 breaths / min , appearance of respiratory acidosis , circulatory shock , or altered sensorium were defined as the features of niv failure , and the patient was qualified for endotracheal intubation . all subjects were monitored for clinical ( respiratory rate , blood pressure , sensorium , and others ) and laboratory parameters ( partial pressure of oxygen , partial pressure of carbon dioxide , arterial ph , and others ) . arterial blood gases ( abg ) , respiratory rate , mental status , mean arterial blood pressure , and pao2/fio2 values were recorded at the initiation of niv and then at 1 h , 4 h , and at the end of therapy . liver and renal function tests , complete blood count , and acute physiology and chronic health evaluation ii ( apache ii ) score ( at 24 h ) were performed in all the subjects . the primary outcome was the success of niv defined as the number of patients in whom endotracheal intubation was avoided . the secondary outcomes were duration of niv , icu stay , hospital mortality , and improvement in clinical and blood gas values at 1 and 4 h. factors predicting the success of niv in avoiding intubation were also analyzed . complications of niv , such as claustrophobia , intolerance , abdominal distention , pressure sores , and nasal bridge trauma , were also noted . changes at 1 h from baseline for pao2/fio2 ratio , paco2 , ph , mean arterial pressure , and respiratory rate were calculated . improvement in clinical ( respiratory rate and mean arterial blood pressure ) and abg parameters ( ph , pao2 , and paco2 ) was analyzed using multifactorial repeated measures analysis of variance , with bonferroni adjustment for multiple comparisons ; the within - groups factor was time ( baseline , 1 , and 4 h ) , and the between - groups factor was niv success . there were 250 admissions during the study period , of which 50 patients had a diagnosis of ards at presentation . nine patients were excluded ( cardiogenic pulmonary edema , n = 2 ; severe ards , hypotension or poor mental status , n = 7 ) and 41 patients were included in the study . the mean sd age of the study population was 30.9 11.4 years ; 27 ( 65.9% ) were women . tropical infections followed by abdominal sepsis were the most common causes of ards [ table 1 ] . the median ( interquartile range [ iqr ] ) duration of illness prior to icu presentation was 3 ( 24 ) days . the use of niv was successful in 18 ( 44% ) patients , while 23 subjects failed the initial trial of niv and required endotracheal intubation . the median ( iqr ) time to intubation was 3 ( 14 ) h. the indications for intubation were refractory hypoxemia ( n = 9 ; 22% ) , hypotension ( n = 7 ; 17.1% ) , altered sensorium ( n = 6 ; 14.6% ) , and uncorrected respiratory acidosis ( n = 1 ; 2.4% ) . overall , there were 19 ( 46.3% ) deaths in the study population , all in those failing niv . clinical and demographic profile of the study population the subjects were divided into two groups namely those with the successful application of niv and those failing niv [ table 2 ] . the underlying etiology , severity ( mild or moderate ) , and type of ards ( pulmonary or extrapulmonary ) were equally represented in the two groups . the niv failure arm had a significantly higher apache ii score at baseline ( 23.7 vs. 13.2 ; p < 0.0001 ) in comparison to niv success . the median pao2/fio2 ratio at baseline was significantly higher in those requiring invasive mechanical ventilation . the duration of ventilation ( noninvasive and invasive ) was significantly higher in those requiring endotracheal intubation [ table 3 ] . clinical characteristics of the niv success and niv failure population outcome parameters of the study groups there was significant reduction in the respiratory rate in both groups at the end of 1 h and 4 h compared to baseline ; however , this was not different between the two groups [ table 4 ] . there was an increase in pao2/fio2 at 1 h in those with niv success in contrast to those with niv failure [ table 4 ] . the pao2/fio2 value of < 150 at 1 h had the best specificity in predicting niv failure compared to other values [ table 5 ] . on univariate logistic regression analysis , failure of improvement in pao2/fio2 at 1 h and higher baseline apache ii scores were the predictors of niv failure [ table 6 ] . serial clinical and arterial blood gas parameters during icu course of the two groups impact of pao2/fio2 ratio at 1 h and baseline apache > 17 factors predicting niv failure : univariate logistic regression analysis the results of this study suggest that the use of niv helped in avoiding intubation in 44% of the subjects with mild to moderate ards . baseline apache ii score > 17 and lack of improvement in pao2/fio2 ratio > 150 after 1 h of niv predicted niv failure . the successful use of niv results in the improvement of oxygenation in subjects with ards . this causes improvement in oxygenation , relief in dyspnea , and reduction in the respiratory muscle workload . in a recent study describing the early use of niv in mild ards , although in a meta - analysis of thirteen studies describing the use of niv in mild - to - moderate ards , niv prevented intubation in 50% of the patients . this was also observed in the current study where the use of niv prevented intubation in 44% of the subjects . tropical infections followed by abdominal sepsis were the most common causes of ards in the current study . the predominant type of ards was extrapulmonary ards , although the type of ards ( extrapulmonary or pulmonary ) did not affect the outcome of niv use in the current study , similar to a previous report . in another study with pulmonary ards secondary to h1n1 , this suggests that irrespective of the type of ards ( extrapulmonary or pulmonary ) , the use of niv helps in avoiding intubation in roughly half of the patients . even in a highly selected group of patients with ards , that is , only those with mild to moderate ards with two or less organ system involvement and the absence of shock at presentation , niv achieved success in only 44% . in addition , in the present study , 19 of the 23 subjects in the niv failure group died , while there were no deaths in the niv success group . this finding is consistent with previously reported experience where the hospital mortality was significantly higher in the niv failure group ( 53.8100% ) in comparison to the niv success group . the most important reason for high mortality in ards patients managed with niv , apart from refractory hypoxemia is the severe systemic illness as reflected by higher disease severity scores such as apache ii . although the median time to intubation was 3 h , elective intubation in subjects with a pao2/fio2 ratio of < 150 at 1 h could have resulted in lesser deaths , and delay in intubation have been associated with poor outcomes . the use of niv was associated with a significant improvement in mean arterial blood pressure , paco2 , and pao2/fio2 ratio at 1 and 4 h of niv use in the niv success group . in a study comprising 54 subjects with ards , presence of hypotension and lack of improvement in oxygenation at 1 h were associated with the failure of niv to avoid intubation . , pao2/fio2 ratio of 175 predicted niv failure , while in another study , pao2/fio2 ratio < 200 at 1-h predicted niv failure in subjects with ards . we evaluated 4 different values of pao2/fio2 ratio at 1 h based on previous studies [ table 5 ] . a value of pao2/fio2 ratio of < 150 at 1 h had the best combination of sensitivity ( 34.8% ) and specificity ( 94.4% ) . further , in the univariate analysis , change in pao2/fio2 ratio at 1 h and a high baseline apache ii score predicted failure of niv in avoiding intubation in subjects with ards . these findings suggest that patients with pao2/fio2 ratio of < 150 after 1 h of niv should be monitored even more closely . the study is a single center observational study comprising a small sample size and hence the results of the current study need to be confirmed in a multicenter study with a larger sample size . the study comprised predominantly of extrapulmonary ards and did not include subjects with severe ards ( pao2/fio2 ratio 100 ) . judicious use of niv in subjects with mild to moderate ards helped in avoiding intubation in 44% of the subjects in the current study . a baseline apache ii score of > 17 and a pao2/fio2 ratio < 150 at 1 h predicted failure of niv in preventing intubation in the study population .

the p53 protein is an important tumor suppressor and is frequently mutated in cancer cells ( 1,2 ) . dna damage activates and stabilizes wild - type p53 , which results in increased transcription of multiple p53 target genes involved in cell cycle arrest or apoptotic cell death ( 3,4 ) . it has been estimated that over 50% of all cancers harbor a mutation in the p53 gene , and this interferes with the ability of the protein to effectively induce cell death ( 1 ) . loss of p53 function has been associated with increased resistance to chemotherapeutic agents ( 5 ) . in addition , loss of p53 or its downstream target , p21 , disrupts the g1/s checkpoint in response to dna damage ( 6,7 ) . lack of a g1/s checkpoint causes cells to depend entirely on their intra - s and g2/m checkpoints to ensure genomic integrity ( 8) . the ataxia - telangiectasia and rad3-related ( atr1 ) and checkpoint protein 1 ( chk1 ) pathways regulate genome fidelity at the g2/m transition and are especially important to cells lacking a functional p53 checkpoint pathway ( 9,10 ) . the phosphatidylinositol 3-kinase - related kinase atr , a dna damage and replication stress response protein , is part of a complex network of checkpoint proteins which are activated in response to deleterious lesions that affect replication fork progression ( 9,11 ) . in response to dna interstrand cross - links and other dna lesions , atr phosphorylates chk1 on two critical residues , ser-317 and ser-345 ( 12,13 ) . when activated , chk1 delays entry into mitosis by phosphorylating and inactivating cdc25a and cdc25c , two phosphatases required for cell cycle progression ( 14 ) . dna damage recognition activities of the fanconi anemia core complex cooperate with atr to signal for interstrand cross - link repair and chk1 phosphorylation ( 1517 ) . in the absence of chk1 , cells with dna damage continue through mitosis culminating in cell death by mitotic catastrophe due to the lack of a g2/m checkpoint ( 1820 ) . disruption of the chk1 g2/m checkpoint kinase is a provocative death target , especially for cells with compromised p53 since these cells lack an efficient g1/s checkpoint ( 2124 ) . 1abbreviations : atr , ataxia - telangiectasia and rad3-related ; chk1 , checkpoint 1 ; chk2 , checkpoint 2 ; atm , ataxia telangiectasia mutated ; mc , mitomycin c ; dmc , decarbamoyl mitomycin c ; mtt , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ; fbs , fetal bovine serum ; g418 , gentamycin ; dapi , 4,6-diamidino-2-phenylindole ; rt - pcr , reverse transcriptase - polymerase chain reaction ; anti - bpde , anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene . abbreviations : atr , ataxia - telangiectasia and rad3-related ; chk1 , checkpoint 1 ; chk2 , checkpoint 2 ; atm , ataxia telangiectasia mutated ; mc , mitomycin c ; dmc , decarbamoyl mitomycin c ; mtt , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ; fbs , fetal bovine serum ; g418 , gentamycin ; dapi , 4,6-diamidino-2-phenylindole ; rt - pcr , reverse transcriptase - polymerase chain reaction ; anti - bpde , anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene . mitomycin c ( mc ) , a bioreductive dna alkylating agent , is a well - known antitumor , antibiotic , and chemotherapeutic agent ( 2527 ) . within the intracellular compartment , mc is metabolized by reductive enzymes to generate reactive dna alkylating species and oxygen radicals through redox cycling ( 26 ) . activated mc alkylates guanine at the n - position to form dna monoadducts and dna intrastrand and interstrand cross - link adducts ( 28 ) . 10-decarbamoyl mitomycin c ( dmc ) , a derivative of mc , has also been shown to bind dna forming a similar but not identical array of dna adducts ( 29,30 ) . we recently demonstrated that equimolar concentrations of dmc produce more dna adducts in human cells than mc , and most of the adducts have altered stereochemistry ( 30 ) . specifically , the chirality of the mitosene linkage of mc to guanine - n of dna is opposite of that of dmc ( mitosene-1- vs mitosene-1- ) ( figure 1 ) . the critical cytotoxic lesion produced by chemotherapeutic dna damaging agents such as mitomycin c has been proposed to be the interstrand cross - link adducts ( 31,32 ) . these interstrand cross - link dna modifications , in part , inhibit strand separation during replication and transcription , which in turn activates atr and ataxia telangiectasia mutated ( atm ) checkpoint pathways ( 10 ) . although both mc and dmc produce these cross - link dna adducts , interestingly , dmc induces death of cells more rapidly than mc ( 29,33,34 ) . chemical structures of dna adducts : 1a , 1- mc monoadduct ; 2a , 1- dmc monoadduct ; 2b , 1- dmc monoadduct ; 3a , 1- interstrand cross - link ; 3b , 1- interstrand cross - link . we previously reported that dmc induces the death of many different cell types more rapidly than mc both in the presence and absence of wild - type p53 ( 33,34 ) . we hypothesized that this difference in cytotoxicity may be based on differential signaling of dna adducts of mc and dmc . the recent investigation of the nature of the cell death pathways induced by the two drugs suggested that the chk1 pathway might be involved in differential cytotoxicity ( 33 ) . moreover , we documented more dna adduct formation following the treatment of multiple different types of human cancer cells with dmc than with mc ( 30 ) . the rationale of the present study was to determine whether the mitomycin analogue dmc might be useful for clinical development for cancers that lack wild - type p53 because at equimolar concentrations , dmc is more cytotoxic to p53-deficient cells than mc and thus could be used at lower concentrations to induce cell death . we compared the amount and persistence of the dna damage induced by the two drugs and compared the cellular and molecular responses of human cancer cells with and without wild - type p53 treated with mc and dmc . we then focused our studies on cells without wild - type p53 ( dld-1 ) and correlated the dmc induced depletion of the g2 checkpoint control protein chk1 with increased cell death and characterized the involvement of the ubiquitin proteolysis pathway in the chk1 depletion . mitomycin c was obtained from bristol - myers squibb co. , wallingford , ct and from kyowa hakko kogyo co. ltd . the procedure used for the synthesis of 10-decarbamoyl mitomycin c was as described previously ( 35 ) . growth medium and penicillinstreptomycin solution were obtained from mediatech inc . , herndon , va and fetal bovine serum ( fbs ) and gentamycin ( g418 ) were purchased from gemini bio - products , west sacramento , ca . 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , and mg115 ( 2-llnval ) were purchased from sigma , allentown , pa . mcf-7 human breast cancer cells ( wild - type p53 ) , obtained from american type culture collection , were grown in 90% rpmi/10% fetal bovine serum and penicillin ( 50 units / ml)streptomycin ( 50 g / ml ) solution . the isogenic colon cancer cell lines used were the well characterized dld-1 and da-2 lines ; they were a generous gift from bert vogelstein ( johns hopkins school of medicine ) , whose group used a two - step procedure to establish a tetracycline - off system for controlled wild - type p53 expression in da-2 cells ( 36 ) . dld-1 cells do not express wild - type p53 , and da-2 cells maintained in the presence of doxycycline express a trace amount of wild - type p53 . dld-1 cells were grown in 90% mccoys/10% fetal bovine serum and penicillin ( 50 units / ml)streptomycin ( 50 g / ml ) solution , while da-2 cells were maintained in an additional 0.4 mg / ml g418 , 20 ng / ml doxycycline , and 0.25 mg / ml hygromycin b. all cell culture experiments were carried out at 37 c in an atmosphere of 95% air/5% co2 . the pcs3 - 6myc plasmid expressing myc - chk1 protein was kindly provided by you - wei zhang ( salk institute , la jolla , ca ) . comparative quantitation of mitochondrial and nuclear genome dna damage was carried out by quantitative pcr ( qpcr ) as previously described ( 3739 ) . briefly , mcf-7 cells were treated for 1 h with either mc or dmc ( 10 m ) . following incubation , cells were washed 2 with hanks buffer and either immediately harvested or allowed to recover in fresh media for 12 h at 37 c . dna was isolated from treated and untreated cells and subjected to qpcr for the measurement of nuclear and mitochondrial dna damage and repair . this assay functions by quantifying the degree of inhibition of pcr amplification observed in damaged samples , compared to control samples , that results from the presence of dna lesions that block or inhibit the progression of dna polymerase . thus , the qpcr assay has the advantage of measuring all polymerase - blocking lesions but the disadvantage of not identifying the nature of the dna damage . specifically , primers 2372 and 3927 were used to amplify a 12.2-kb portion of the dna polymerase gene , and primers 14841 and 5999 were used to amplify an 8.9-kb portion of the mitochondrial genome , which encompasses roughly 50% of the mitochondrial genome ( and thus many genes ) . additionally , a small fragment ( 221 bp ) of the mitochondrial genome was amplified using primers 14841 and 14620 ( amplifying a small region of the dna polymerase gene ) , and used to monitor the mtdna copy number as well as to normalize the results obtained from the large mitochondrial fragment . all primer sequences as well as qpcr assay conditions were as indicated by santos et al . ( 38 ) amplifications of treated controls were compared to those of untreated controls to calculate the relative amplification and determine the lesion frequency in treated samples above the basal levels in nontreated controls . results presented are the mean of at least two sets of pcrs for each amplicon for each sample ; n = 3 per sample . five micrograms of rna was used for cdna synthesis using the high capacity cdna archive kit ( applied biosystems ) . the primer - probes for chk1 , p21 , and mdm2 ( applied biosystems , celera discovery systems assays on demand ) and actin ( applied biosystems predeveloped assay reagents ) were utilized for taqman pcr using the applied biosystems 7500 sequence detection system ( perkin - elmer ) as follows : one cycle , 2 min ( 50 c ) ; one cycle , 10 min ( 94 c ) ; and 40 cycles , 15 s ( 94 c ) and 1 min ( 60 c ) . for sirna experiments , cells were seeded at 60% confluence in media without penicillinstreptomycin and allowed to attach overnight . cells were transfected with 25 nm of each of the specified sirna ( all from dharmacon ) for 24 h using lipofectamine 2000 ( invitrogen ) . at the end of the incubation period , fresh media without penicillinstreptomycin was added to cells for 2 h , after which cells were either left untreated or treated with 10 m mc or 10 m dmc for an additional 24 h. protein extracts were obtained from cells for western blot analysis . for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) analysis , after sirna transfections , cells were incubated for an additional 2 h with mtt solution and analyzed for proliferation . for overexpression experiments , cells were plated at 90% confluence and transfected with 4 g of each indicated plasmid for 24 h using lipofectamine 2000 . cells were then either left untreated or treated with 10 m mc or 10 m dmc for an additional 24 h followed by protein extraction or mtt analysis for proliferation . cells were washed twice with phosphate - buffered saline ( pbs ) containing 2% bovine serum albumin and 0.1% nan3 . the viabilities of the cell lines following treatment with dna damaging agents were determined using the tetrazolium dye - based microtitration assay as described ( 33 ) . briefly , at the end of the incubation period , cells were harvested , washed 2 in ice - cold pbs at ph 7.5 , and spun down after each wash at 300 g . cell pellets were dissolved in ripa buffer ( 0.1% w / v sds , 0.5% w / v deoxycholate , 150 mm nacl , 1 mm edta , 0.5 mm egta , 50 mm tris at ph 8.0 , 1 mm pmsf , 20 m aprotonin , and 20 m leupeptin and supplemented with phosphatase inhibitor cocktail ( sigma ) as suggested by the manufacturer ) and incubated on ice for 10 min with periodic shaking . protein samples were size - fractionated by electrophoresis in 10% sds denaturing poly acrylamide gels and electrotransferred to nitrocellulose membranes ( amersham ) . for ubiquitinated products , samples were separated on a 10% and 8% ( bottom and top , respectively ) denaturing gel prior to transfer . the resulting blots were incubated with the following primary antibodies : p53 specific monoclonal antibodies [ 1:1:1 mixture of 421 , 240 , and 1801 antibodies ) as described ( 40,41 ) ] , anti - chk1 ( santa cruz biotech . , the membranes were then incubated in antimouse or antirabbit secondary antibodies ( sigma ) , and the signals were visualized by chemiluminescence . after drug treatment , cells were fixed with 2% paraformaldehyde in pbs for 15 min at room temperature . cells were permeabilized with pbs containing 0.2% triton x-100 v / v and 1% fetal bovine serun v / v for 5 min at 20 c and washed 3 in 1% fbs - pbs . coverslips were immediately mounted onto slides with 4 , 6-diamidino-2-phenylindole ( dapi ; vector laboratories , inc . ) . briefly , cells were washed 2 in pbs and lysed in buffer a ( 50 mm tris - hcl ph 7.4 , 5 mm mgcl2 , 5 mm atp , 1 mm dtt , and 10% glycerol v / v ) . extracts were homogenized on ice and spun down at 4 c for 15 min at 15,000 g . thirty micrograms of proteins were analyzed on a nondenaturing gradient page gel ( 5 , 4 , and 3% from bottom up ) with rhinohide polyacrylamide strengthener ( molecular probes ) . gels were then covered with 0.4 mm of the proteasome substrate , suc - leu - leu - val - tyr - amc ( bachem ) diluted in buffer b ( buffer a modified to contain 1 mm atp ) , and kept at 37 c for 30 min on a rocker . proteasome activity bands were visualized when gels were exposed to uv ( 360 nm ) . we previously reported that dmc has increased cytotoxicity compared to mc in the presence or absence of wild - type p53 with 24 h of drug treatment ( 33,34 ) and have shown that the frequency of dna adduct formation in the mcf-7 wild - type p53 breast cancer cell line is higher for dmc than mc ( see table 1 ) ( 30 ) . the predominant stereochemistry of the 1-mitosene linkage of the dmc adducts to dna was 1- ( 2b and 3b ) , opposite to the predominant 1- stereochemistry of the mc adducts ( 1a , 2a , and 3a ) ( figure 1 and table 1 ) . the morphology of mcf-7 cells treated with mc for 24 h showed a barely detectable change in nuclear size as compared to the untreated cells ( figure 2a and b , mcf-7 ) . however , the mcf-7 cells treated with dmc showed a reproducible and statistically significant reduction in size as compared to the untreated population . mcf-7 cells were left untreated or treated for 24 h with 10 m mc or 10 m dmc . data represent three independent injections of dna adducts from the same experiment ( 30 ) . from data in ref ( 30 ) ; error limits are omitted . mcf-7 , da-2 , and dld-1 cells were left untreated or treated for 24 h with 10 m mc or 10 m dmc . ( a ) representative region of the dapi stained slide with nuclei from 3 independent experiments is depicted . ( b ) nuclei sizes were determined after staining cells grown on a coverslip with dapi . the number of nuclei measured for each treatment condition was mcf-7 ( 29 ) ; da-2 ( 32 ) ; and dld-1 ( 21 ) . error bars denote sem , and the significance was determined using a one - tailed paired t test for the drug treated samples compared to the untreated controls . p - values of 0.05 or less resulted for da-2 cells treated with dmc ; p - value = 0.03 ; dld-1 cells treated with dmc , p - value = 0.05 ; and dld-1 cells treated with mc , p - value = 0.05 . p - value = paired t test . to assess if the variable outcome of mc and dmc treatment of mcf-7 cells was cell type specific and if it occurred in the absence of wild - type p53 , we compared the nuclear morphology of isogenic colon cancer cells with or without wild - type p53 ( da-2 and dld-1 , respectively ) treated with the two drugs ( figure 2a and b ) . analysis of nuclear size as compared to the untreated cell population supported the hypothesis that dmc worked through different mechanisms of action than mc , although the different cell types demonstrated variability in their nuclear phenotypes following drug treatment . the nuclear morphology of da-2 cells treated with mc for 24 h showed a slight increase in size as compared to the same cells that were not treated with the drug ( figure 2a and b , da-2 ) . however , following 24 h of dmc treatment , da-2 cells showed a reproducible and statistically significant reduction in their nuclear size as compared to their untreated counterpart ( figure 2a and b , da-2 ) . in the dld-1 cells that have no functional p53 , mc treatment caused a reproducible and statistically significant increase in nuclear size , while dmc treatment caused a reproducible and statistically significant reduction in the nuclear size . nuclear shrinkage is often correlated with apoptosis , while swelling is associated with necrosis ( 43 ) . we also tested whether the variable outcome of dmc vs mc in affecting nuclear morphology correlated with a similar greater potency of dmc to induce damage in the nuclear genome as well as in the mitochondrial genome in the mcf-7 cell line ( figure 3a and b ) . more dna damage was caused by dmc than mc to both the mitochondrial genome and the nuclear genome of mcf-7 cells treated with the drugs for 1 h ( figure 3a and b ) . furthermore , the most detectable dmc induced mitochondrial dna damage was removed following 12 h without the drug ( figure 3a ) , but nuclear dna adducts caused by dmc persisted 12 h after drug removal ( figure 3b ) . these results do not indicate that mc caused no dna damage but rather that under conditions in which dmc caused significant and persistent dna damage , mc caused no detectable damage . the detection limit of this assay is approximately 1 lesion per 10 bases ( 38 ) . cells were either harvested immediately after treatment ( 0 h ) or allowed to recover for 12 h. isolated dna was subjected to qpcr , and the amplification of each treated sample was compared to untreated controls to determine the relative amplification . these values were then used to calculate the average number of lesions obtained per 10 kb of the genome . we were specifically interested in the p53-independent mechanism of cell death induced by dmc and for this reason focused on the dld-1 cell line for the chk1 studies . dmc treatment results in the depletion of chk1 protein , while mc treatment does not ( 33 ) . to determine if the previously identified chk1 down - regulation was mediated through a p53-independent ubiquitinproteasome pathway , we exposed dld-1 cells to proteasome inhibitors . total ubiquitinated protein products increased when cells were treated with mg115 ( z - llnval ) ( figure 4a , compare lanes 1 and 2 ) or with mg132 ( z - lllal , data not shown ) . as expected , dmc , but not mc , treatment caused the depletion of chk1 protein ( figure 4a , lanes 3 and 5 ) . importantly , chk1 levels and total ubiquitinated proteins were recovered in the presence of mg115 ( figure 4a , compare lanes 5 and 6 ) . the recovery of chk1 upon successful inhibition of the proteasome pathway implicates the proteasome as the primary mechanism for chk1 degradation in dmc treated cells ( figure 4a , lanes 5 and 6 ) . this mechanism of chk1 regulation in dmc treated dld-1 cells , which do not express wild - type p53 , was also observed in da-2 cells which express wild - type p53 ( data not shown ) . ( a ) whole cell extracts obtained from dld-1 cells were left untreated ( lane 1 ) , treated with 4 m mg115 ( lane 2 ) , 10 m mc or 10 m dmc ( lanes 3 and 5 ) , and 10 m mc or 10 m dmc with 4 m mg115 ( lanes 4 and 6 ) , and were analyzed by western blotting for chk1 protein ( top panels ) and accumulation of ubiquitinated proteins ( bottom panels ) . numbers at the top of each sample lane on the ubiquitin blot represent the relative chk1 protein normalized to actin . dld-1 cells were left untreated ( lane 1 ) and treated with 4 m mg115 ( lane 2 ) , 10 m mc ( lane 3 ) , or 10 m dmc ( lane 4 ) . extracts were obtained from cells and analyzed on a native polyacrylamide gel for proteasome activity ( left panel ) and western blot for proteasome subunits ( right panel ) . ( c ) fold change mrna levels for chk1 rna were analyzed by quantitative real - time reverse transcriptase - polymerase chain reaction pcr ( rt - pcr ) . ( d ) phase contrast image of dld-1 cells treated with mc and dmc as indicated . ( d ) ( d ) facs analysis of dld-1 cells treated with either 1 m or 10 m of mc or dmc . ( e ) whole cell extracts obtained from dld-1 cells treated with either 1 m or 10 m of mc or dmc were analyzed by western blotting for chk1 protein . while chk1 depletion was clearly facilitated by the ubiquititin proteosome pathway , we had not determined the signaling mechanism associated with its down - regulation . in our current investigation , we observed concomitant depletion of total ubiquitinated protein ( figure 4a , lane 5 ) . increased degradation of total ubiquitinated protein suggested that perhaps dna adducts of dmc signaled for an increase in proteasome activity . we used an established in - gel assay to detect , in cell lysates , the cleavage of the fluorogenic peptide substrate suc - leu - leu - val - tyr - amc , by the proteasome core particle with both regulatory particles ( rp2cp ) , core particle with one regulatory particle ( rp1cp ) , and only the core particle ( cp ) ( 42 ) . the inhibitor , mg115 decreased proteasome activity in dld-1 cells ( without wild - type p53 ) ( figure 4b , left panel , lane 2 ) . however , mc and dmc treatment did not increase proteasome activity compared to that in the untreated samples ( figure 4b , compare lanes 3 and 4 to lane 1 ) . the increased degradation of ubiquitinated substrates in dmc treated cells was not due to a change in proteasome levels or proteasome activity since dmc treated and untreated cells showed similar proteasome levels and activity , respectively ( figure 4b , compare the left panel to the right panel ) . the observed decrease in ubiquitinated products in the absence of an increase in proteasome activity suggested that dna adducts of dmc might increase signaling to the proteolysis machinery . to determine if the rescue of chk1 protein levels by mg115 was due to protein stabilization and not an increase in chk1 rna , we examined the levels of total chk1 mrna before and after the inhibition of the proteasome ( figure 4c ) . mg115 treatment did not significantly change the levels of the chk1 transcript under any of the test conditions ( figure 4c ) . dmc treatment resulted in the reduction of total chk1 mrna ; however , the rescue in chk1 protein accumulation seen in figure 4a was clearly due to a change in chk1 stability and not mrna level as the chk1 mrna level was not increased upon mg115 treatment . the chemical composition of the two mitomycin agents is similar enough that variable toxicity to human cells at equimolar concentrations , in combination with our previous adduct structure data , suggests that the two drugs possess different modes of action . however , comparing equimolar versus equitoxic doses is also important for addressing the question of different pathways of activation . because of the high toxicity of dmc , it was not simple to achieve equal cytotoxicity to mc . we determined that decreasing the concentration of dmc by 10-fold resulted in cytotoxicity comparable to that seen with mc , resulting in sufficient live cells to examine the biology ( figure 4d ) . when concentrations of 1 m to 50 m of the two agents were compared for the p53-deficient cells , the cell morphology and mtt data indicated a similar cytotoxicity of the two drugs at 1 m dmc and 10 m mc ( figure 4d and d ) . at these comparable concentrations , we then asked , how does the signaling of the two agents compare at these concentrations when very few cells were dead ? classification of checkpoint pathways is nontrivial , and the methods used to characterize the cellular changes must be superimposed on the drug treatment itself . we examined the cell cycle distribution of the p53-deficient cells as an indicator of how the two agents influenced the cell cycle populations when the cells showed similar mitochondrial activity . we found a striking difference in the cell cycle profiles with the two drugs at equitoxic concentrations ( figure 4d ) . the p53-deficient cells treated with 1 m dmc piled up in the s and g2/m stages , while the 10 m mc treated cells were found mostly in g1 ( figure 4d facs ) . we also examined the chk1 protein level in these treated cell populations and found that chk1 was not reduced during this early differential cell cycle response ( figure 4e ) . when the cells were equally alive , the chk1 levels were similar , but the cell cycle distribution was very different , suggesting that a different signaling pathway was engaged . our hypothesis is that mc and dmc can mechanistically influence p53-deficient human cells in different ways and that chk1 depletion is a later stage event that correlates with cell death . since we observed differential regulation of chk1 during mc- and dmc- induced cellular cytotoxicity , we hypothesized that the chk1 protein might play a role in the differential cytotoxicity observed for the two drugs . to address this hypothesis , we depleted the levels of endogenous chk1 using sirna and observed the outcome of drug treatment ( figure 5a , see lanes 79 ) . in the absence of chk1 sirna , down - regulation of chk1 protein was only seen in dmc treated cells ( figure 5a , see lanes 3 and 6 ) . in the presence of chk1 sirna , we observed the dramatic reduction of chk1 protein in mc treated cells and investigated if this , as predicted , increased the cytotoxicity of mc ( figure 5a see lanes 79 ) . we observed a statistically significant decrease in cell proliferation in mc treated samples when chk1 was depleted by chk1 sirna ( figure 5b , comparing nonspecific sirna to chk1-sirna ) . cells that had been treated with mc showed the most significant decrease in percent viable cells with sirna to chk1 as compared to the nonspecific sirna ( figure 5b ) . moreover , the cellular cytotoxicity of mc was not changed when cells were treated with mdm2 sirna used as an unrelated target control . as predicted , chk1 depletion increased mc induced death suggesting that any conditions that reduce the amount of chk1 also reduce cell viability . the fact that dmc treatment reduces chk1 , before the addition of chk1 sirna , renders the sirna addition here less significant . chk1 down - regulation increased mc cytotoxicity in the absence of wild - type p53 . ( a ) dld-1 cells were left untransfected or transfected with 25 nm nonspecific sirna , 25 nm mdm2 sirna , or 25 nm chk1 sirna for 24 h. after the transfection incubation period , cells were either left untreated or treated with 10 m mc or 10 m dmc for an additional 24 h. whole cell extracts were obtained from these cells and analyzed for chk1 expression . ( b ) to determine the percent proliferation , cells were treated as indicated above . mtt solution was added to each sample and incubated for an additional 2 h. values were obtained from 3 independent experiments , and significance was determined for the sirna treatment as compared to the drug treated control . p - values below 0.05 were obtained for all samples treated with sirna to chk1 . ( c ) for overexpression , cells were transfected with 4 g of either empty vector or myc - chk1 for 24 h. after the transfection incubation period , cells were either left untreated or treated with 10 m mc or 10 m dmc for an additional 24 h. whole cell extracts were obtained from these cells and analyzed for chk1 expression . actin was used as a loading control . to evaluate the proliferation of cells overexpressing myc - chk1 after treatment with mc or dmc , mtt solution was added to each sample , and cells were incubated for an additional 2 h. values were obtained from 3 independent experiments . error bars denote sem . to address the potential of stabilized chk1 to attenuate dmc cytotoxicity , we overexpressed a myc - tagged chk1 protein , which was successfully induced under all treatment conditions ( figure 5c ) . however , only the endogenous chk1 protein was selectively down - regulated by dmc , while the exogenous myc - chk1 protein was not ( figure 5c , lane 6 ) . the myc - tagged chk1 protein used in our experiments has been reported to induce phosphorylation of its downstream target cdc25c in vitro , but this exogenous chk1 has been shown to be less sensitive to atr mediated phosphorylation , activation , and proteasome mediated degradation ( 44 ) . treatment with mc did not dramatically influence either endogenous or exogenous chk1 ( figure 5c , compare lanes 2 and 4 ) . we examined the role of stabilized myc - chk1 in mitomycin induced cellular cytotoxicity ( figure 5c , graph ) . when exogenous chk1 was introduced , viability increased only slightly above the vector control ( figure 5c , graph ) . the limited rescue observed may be explained by the decreased activation for this exogenous chk1 by the atr mediated phosphorylation documented previously . however , the slight increase in viability observed with the addition of exogenous chk1 might be explained by a limited increase in activation of the g2/m checkpoint . while our studies have focused on the p53-independent activity of mc and dmc in dld-1 cells , the possibility existed that the persistent nuclear dna damage for 24 h with dmc might influence wild - type p53 mediated activation of transcription differently than mc . therefore , the outcome of 24 h treatment with mc and dmc on dna damage induced transcriptional activity of wild - type p53 was assessed in the p53-proficient mcf-7 cells and da-2 cells . both mc and dmc treatment stabilized p53 in both cell lines as determined by western blot analysis ( figure 6a and b ) . the stabilized wild - type p53 after 24 h of 10 m treatment with mc robustly activated transcription of the p53 target genes mdm2 and p21 . however , following 24 h of 10 m dmc treatment , the stabilized p53 resulted in less activation of p21 than mc and did not result in transactivation of mdm2 ( in fact , a decrease was observed ) ( figure 6a and b ) . the possibility exists that the increased frequency of dna adducts by dmc or perhaps the mitosene-1--dna adduct stereochemistry produced by dmc interferes with the ability of the basal transcription machinery to produce transcripts from the mdm2 gene and reduced transcription from the p21 gene . we have previously noted that at early time points ( 3 and 6 h ) after 10 m mc and dmc treatment transcription of the p53 target genes is similar ( 34 ) ; the decrease seen here could also be due to increased induction of cell death after 24 h. nevertheless , the different outcomes of mc and dmc treatment on p53-proficient cells and on the p53-pathway are another indication that dmc and mc can signal to the cellular machinery differently when used at similar concentrations . when we compared the p53-proficient mcf-7 cells treated with the two drugs at concentrations ranging from 1 m to 50 m , a 10-fold difference for the cytotoxic concentration was observed by mtt analysis and visualization of matrix attachment ( figure 6c and c ) . interestingly , while 1 m dmc treatment was equally cytotoxic to 10 m mc , the p53 stabilization achieved at the two concentrations was significantly different ( figure 6c , compare lanes 3 and 4 ) ; 10 m mc resulted in a more robust stabilization of p53 than 1 m dmc treatment . in addition , the equal p53 response at the 1 m dose in the p53-proficient cells clearly showed highly different cytotoxicity levels for the two drugs ( figure 6c , lanes 2 and 3 ) . this is yet another indication that increased cytotoxicity of dmc as compared to mc is through a p53-independent pathway . ( a ) mcf-7 and ( b ) da-2 cells were left untreated ( lane 1 ) or treated for 24 h with 10 m mc ( lane 2 ) or 10 m dmc ( lane 3 ) . after treatments , whole cell extracts were obtained from cells and analyzed for p53 protein stability . , cells were left untreated or treated for 24 h with 10 m mc or 10 m dmc . transcriptional activity of p53 target genes , p21 and mdm2 mrna , were analyzed by quantitative real - time reverse transcriptase - polymerase chain reaction pcr ( rt - pcr ) . ( c ) phase contrast image of mcf-7 cells treated with mc and dmc as indicated . ( c ) whole cell extracts obtained from mcf-7 cells treated with either 1 m or 10 m of mc or dmc were analyzed by western blotting for p53 protein . ( d ) model : dmc treatment generates increased amounts of the mitosene-1- dna adducts , while the mc treatment produces moderate amounts of mitosene-1- dna adducts . the increased dna adducts produced by dmc after 24 h interfere with p53 transcriptional activity , signal for the rapid depletion of endogenous chk1 by the proteasome , which may disrupt the g2/m cell cycle checkpoint and lead to nuclear condensation . the inability of cells to arrest at the g2/m checkpoint due to the lack of chk1 potentiates cytotoxicity of dmc . mc dna adducts activate chk1 , which may prolong the intra s and g2 checkpoints , increase nuclear size , and activate p53 transcriptional activity , while blocking replication stress induced cell death . down - regulation of chk1 has been described as an alternative pathway for inducing cell death when the more common apoptotic pathways have been sabotaged in cancer cells ( 22,45 ) . we observed chk1 down - regulation and increased cell death , especially in the absence of a p53 checkpoint , when cells were treated with dmc ( 33 ) . dmc is more cytotoxic than mc ( 29 ) , and this increased cytotoxicity does not depend on the function of wild - type p53 , which makes dmc a promising drug for the many cancers that contain mutant p53 ( 33,34 ) . in view of the close structural similarity between mc and dmc , the differences in their biological effects in human tumor cell cultures were originally unexpected . in this article , we further characterize these effects and propose to correlate them with the known structural differences of the dna adducts , the amount of adduct formation , and cellular signaling . interstrand cross - link ( icl ) dna adducts are the clinically relevant cytotoxic lesions produced upon exposure to mc and dmc ( 29 ) . the intrinsic ability of the cellular machinery to recognize and excise icl lesions from the genome and activate cell death when genomic integrity is beyond repair underlies the sensitivity of various cell lines to dna cross - linking ( 32 ) . the increased production of mitosene-1--dna adducts following dmc treatment or their persistence in the nuclear genome might account for the reduction in nuclear size that correlates with the increased cytotoxicity observed with this mitomycin derivative . the nature of the mitosene-1- interstrand cross - links and how they contribute to cytotoxicity are just beginning to emerge and will provide useful tools for dissecting how different dna adduct structures signal to alternative cell death pathways . such information could be useful in the design of compounds to target cancers containing a compromised p53 pathway . our model for how dmc - damaged dna signals differently from mc - damaged dna is presented in figure 6d . dmc treatment of cells caused a reduction in nuclear size which is consistent with a number of types of cell death including mitotic catastrophe and apoptosis ( 46 ) , while swelling is associated with necrosis ( 43 ) . the dld-1cells do not have functional p53 and showed nuclear shrinkage with dmc treatment and swelling with mc treatment . this difference in morphological outcome when the cells are treated at the same concentration supports the possibility that the structural difference of drugs can cause different types of cell death , especially in the absence of p53 function . we documented previously that caspase-3 and -9 activation and parp cleavage are limited in dmc treated cells lacking p53 ( 33 ) , but we have not examined the activation of caspase-2 . an alternative apoptotic program , which requires caspase-2 , can be engaged in cells with depleted chk1 , and this pathway is not affected by p53 loss ( 47 ) . our data demonstrate that chk1 depletion can increase the extent of mc induced cell death and is correlated with dmc induced cell death ; but our data do not indicate if the mechanism is through mitotic catastrophe or a caspase 2-dependent cell death . it should be noted that micronuclei formation has been reported as a characteristic of cells undergoing dna damage induced mitotic catastrophe ( 46,48 ) . the possibility of cytokinesis in the absence of complete dna replication due to increased cross - links and loss of chk1 dependent checkpoint could account for the observed micronuclei . while we do not have direct evidence for this , our confocal microscopy images of dapi stained nuclei ( figure 2 ) show brightly stained condensed nuclear architecture after the cells were treated with dmc for 24 h. it is known that icls disrupt dna replication and transcription ( 32 ) . icls are mostly cytotoxic to cells in the s phase , which are actively replicating , because these aberrant dna structures inhibit replication by stalling the polymerase . the ability of chk1 to arrest the cell cycle has been shown to prolong cell survival after dna damage ( 49,50 ) . it is shown here that increased production of mitosene-1--dna adducts by dmc correlates with a signaling mechanism that results in the down - regulation of chk1 by the ubiquitin proteolysis pathway . in the absence of chk1 recently , it was reported that atr / chk1 signaling is highly specific to replication blockers as opposed to the alternative atm / chk2 regulatory pathway ( 45,51 ) . this suggests that the primary pathway activated by replication blockers to induce recovery from dna damage and prolong cell survival is through the atr / chk1 pathway . the regulation of chk1 we have observed correlates well with the differential activation of this pathway by mc and dmc . sporadically , we see that 24 h treatment of cells with mc results in increased chk1 protein levels ; however , we never see a reduction of chk1 at the 24 h time point ( 33 ) . we previously showed that increasing the time of mc treatment to 72 h caused a reduction in the chk1 level ; here , we show that increasing the mc concentration by 10-fold ( to 100 m ) causes a reduction in chk1 . therefore , when the treatment conditions with mc are pushed to cytotoxic limits we do see a reduction in chk1 that correlates with cytotoxicity in cells without wild - type p53 . when chk1 was down - regulated by sirna , similar cytotoxicity of equimolar mc and dmc was achieved . reported that disruption of the chk1 g2 checkpoint using ucn-01 , a chk1 inhibitor , increased the cytotoxicity of mc , and this effect was independent of the wild - type p53 ( 52 ) . however , overexpressing chk1 caused only a slight decrease in the cytotoxicity of the two mitomycins at 24 h post - treatment . the activity of the exogenous chk1 is limited , and therefore , the slight increase in viability detected is most likely due to this fact ( 44 ) . it is possibile that the exogenous chk1 lacked significant kinase activity or that the lack of upstream or downstream factors that influence chk1 activity were down - regulated by dmc dna adduct signaling . down - regulation of these factors might render exogenous chk1 insufficient to significantly affect the cellular outcome after overexpression . however , since we observed a slight increase in survival after chk1 overexpression , it remains possible that overexpressed chk1 could rescue survival but requires additional factors . the regulation of chk1 has been implicated in the response of cells to replication blockers . phosphorylation of chk1 by atr at ser345 as opposed to ser317 preferentially destabilizes chk1 by marking it for ubiquitin mediated proteolysis . we observed that when the proteasome activity was inhibited by mg115 , chk1 protein levels were recovered even when cells were treated with dmc . our data support the hypothesis that dmc down - regulates chk1 by causing increased ubiquitination and subsequent degradation by the proteasome . importantly , we saw that this mechanism of chk1 down - regulation happened in the absence of increased proteasome activation . we did not observe any increase in proteasome activity in dmc treated samples but observed a decrease in total ubiquitinated protein products upon treatment with dmc . this supports the hypothesis that dmc dna adducts signal for altered protein post - translational modifications that cause rapid degradation of the tagged polypeptides . this was substantiated by preliminary protein microarray data showing that dmc treatment caused a dramatic reduction in the levels of many proteins on the sigma panorama cell signaling antibody array ( data not shown ) . when we examined the p53 transcriptional activity , we observed that while both mc and dmc could stabilize p53 , the p53 in 24 h dmc treated cells did not correlate with increased levels of the mdm2 gene transcript and resulted in lower p21 transcription than that seen in mc treated cells . the reduced p53 transcriptional activity following dmc treatment may be due to the fact that the persistent dna adducts of dmc , over time , negatively influence the transcription machinery . this suggests that even in the presence of wild - type p53 protein , dmc might activate a parallel p53-independent pathway because the p53 target genes are not as robustly turned on by dmc in spite of stabilization of the p53 protein . inhibition of mdm2 transcription by dna damage is not novel , for example , etoposide induced dna damage has also been reported to disrupt transcription of the p53 target gene mdm2(54 ) . the parallel death pathways of p53 activation and p53-independent cell death by dmc will require further investigation . we observed that dmc induces p53-independent cell death in cancer cells in a manner that correlated with ubiquitin mediated degradation of chk1 in the presence of persistent dna damage . we see that chk1 degradation is correlated with the predominant formation of mitosene-1- guanine adducts of dmc , in contrast to the mitosene-1- adducts formed with mc . this alteration of the chirality of the drug linkage to dna undoubtedly changes the alignment of dmc adducts , and this may be related to the basis of the different dna damage signaling mechanisms ( see model in figure 6 ) . the alignment of the mc mitosene-1- monoadduct and of the 1- cross - link in oligonucleotide duplexes has been well characterized ( 5557 ) . the alignment of the mitosene 1- adducts of dmc have not been studied yet . the mitomycins represent a case in which dna linkage chirality is proposed to modulate the biological response to dna damage . well - studied precedents of a relationship of dna adduct chirality to biological activity exist , most notably the case of the adducts of racemic anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ( anti - bpde ) . the highly tumorigenic ( + ) -anti - bpde enantiomer mostly forms ( 90% ) the ( + ) -trans - anti [ bp]-n - dg adduct , while the nontumorigenic ( )-anti - bpde enantiomer forms ( )-trans - anti- and ( )-cis - anti-[bp]-n - dg adducts . the latter two adducts are linked to the n atom of dg with chirality opposite to that of the ( + ) -trans - anti-[bp ] adduct ( 58 ) . the available data of the present investigation do not allow us to distinguish with certainty whether the observed differential signaling by mc and dmc is due to the difference in their adduct frequency ( see also table 1 ) or to intrinsic structural differences of their adducts critical for signaling . with respect to the first alternative , it is likely that the more intense dna alkylating activity of dmc compared to that of mc in vivo is facilitated by its lack of the 10-carbamoyl group . this group may hinder the attack by mc , especially of the production of the mc 1--dg adducts which are formed generally at very low frequency in vivo and in vitro(29,30,59 ) . in conclusion , hypoxia , which has been shown to protect cancer cells , was also reported to activate the atr / chk1 pathway to increase cell survival and protection against ros mediated dna damage ( 60,61 ) . dmc , which has been shown to have increased dna adduct formation under hypoxic conditions ( 29 ) , can possibly have therapeutic use due to the ability of its adducts to signal for rapid chk1 degradation , thereby counteracting the cancer - protective effects of hypoxia and should be considered for further investigation into its clinical relevance as a chemotherapeutic .

the mitomycin derivative 10-decarbamoyl mitomycin c ( dmc ) more rapidly activates a p53-independent cell death pathway than mitomycin c ( mc ) . we recently documented that an increased proportion of mitosene1--adduct formation occurs in human cells treated with dmc in comparison to those treated with mc . here , we compare the cellular and molecular response of human cancer cells treated with mc and dmc . we find the increase in mitosene 1--adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53 . dmc caused more dna damage than mc in the nuclear and mitochondrial genomes . checkpoint 1 protein ( chk1 ) was depleted following dmc , and the depletion of chk1 by dmc was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against chk1 depletion . gene silencing of chk1 by sirna increased the cytotoxicity of mc . dmc treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity , suggesting that dmc mediated dna adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis . thus , the mitosene-1- stereoisomeric dna adducts produced by the dmc signal for a p53-independent mode of cell death correlated with reduced nuclear size , persistent dna damage , increased ubiquitin proteolysis and reduced chk1 protein .

Introduction Materials and Methods Results Discussion

it has been estimated that over 50% of all cancers harbor a mutation in the p53 gene , and this interferes with the ability of the protein to effectively induce cell death ( 1 ) . in the absence of chk1 , cells with dna damage continue through mitosis culminating in cell death by mitotic catastrophe due to the lack of a g2/m checkpoint ( 1820 ) . abbreviations : atr , ataxia - telangiectasia and rad3-related ; chk1 , checkpoint 1 ; chk2 , checkpoint 2 ; atm , ataxia telangiectasia mutated ; mc , mitomycin c ; dmc , decarbamoyl mitomycin c ; mtt , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ; fbs , fetal bovine serum ; g418 , gentamycin ; dapi , 4,6-diamidino-2-phenylindole ; rt - pcr , reverse transcriptase - polymerase chain reaction ; anti - bpde , anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene . mitomycin c ( mc ) , a bioreductive dna alkylating agent , is a well - known antitumor , antibiotic , and chemotherapeutic agent ( 2527 ) . 10-decarbamoyl mitomycin c ( dmc ) , a derivative of mc , has also been shown to bind dna forming a similar but not identical array of dna adducts ( 29,30 ) . we recently demonstrated that equimolar concentrations of dmc produce more dna adducts in human cells than mc , and most of the adducts have altered stereochemistry ( 30 ) . although both mc and dmc produce these cross - link dna adducts , interestingly , dmc induces death of cells more rapidly than mc ( 29,33,34 ) . we previously reported that dmc induces the death of many different cell types more rapidly than mc both in the presence and absence of wild - type p53 ( 33,34 ) . we hypothesized that this difference in cytotoxicity may be based on differential signaling of dna adducts of mc and dmc . moreover , we documented more dna adduct formation following the treatment of multiple different types of human cancer cells with dmc than with mc ( 30 ) . the rationale of the present study was to determine whether the mitomycin analogue dmc might be useful for clinical development for cancers that lack wild - type p53 because at equimolar concentrations , dmc is more cytotoxic to p53-deficient cells than mc and thus could be used at lower concentrations to induce cell death . we compared the amount and persistence of the dna damage induced by the two drugs and compared the cellular and molecular responses of human cancer cells with and without wild - type p53 treated with mc and dmc . we then focused our studies on cells without wild - type p53 ( dld-1 ) and correlated the dmc induced depletion of the g2 checkpoint control protein chk1 with increased cell death and characterized the involvement of the ubiquitin proteolysis pathway in the chk1 depletion . thus , the qpcr assay has the advantage of measuring all polymerase - blocking lesions but the disadvantage of not identifying the nature of the dna damage . we previously reported that dmc has increased cytotoxicity compared to mc in the presence or absence of wild - type p53 with 24 h of drug treatment ( 33,34 ) and have shown that the frequency of dna adduct formation in the mcf-7 wild - type p53 breast cancer cell line is higher for dmc than mc ( see table 1 ) ( 30 ) . the morphology of mcf-7 cells treated with mc for 24 h showed a barely detectable change in nuclear size as compared to the untreated cells ( figure 2a and b , mcf-7 ) . however , the mcf-7 cells treated with dmc showed a reproducible and statistically significant reduction in size as compared to the untreated population . p - values of 0.05 or less resulted for da-2 cells treated with dmc ; p - value = 0.03 ; dld-1 cells treated with dmc , p - value = 0.05 ; and dld-1 cells treated with mc , p - value = 0.05 . to assess if the variable outcome of mc and dmc treatment of mcf-7 cells was cell type specific and if it occurred in the absence of wild - type p53 , we compared the nuclear morphology of isogenic colon cancer cells with or without wild - type p53 ( da-2 and dld-1 , respectively ) treated with the two drugs ( figure 2a and b ) . analysis of nuclear size as compared to the untreated cell population supported the hypothesis that dmc worked through different mechanisms of action than mc , although the different cell types demonstrated variability in their nuclear phenotypes following drug treatment . the nuclear morphology of da-2 cells treated with mc for 24 h showed a slight increase in size as compared to the same cells that were not treated with the drug ( figure 2a and b , da-2 ) . in the dld-1 cells that have no functional p53 , mc treatment caused a reproducible and statistically significant increase in nuclear size , while dmc treatment caused a reproducible and statistically significant reduction in the nuclear size . we also tested whether the variable outcome of dmc vs mc in affecting nuclear morphology correlated with a similar greater potency of dmc to induce damage in the nuclear genome as well as in the mitochondrial genome in the mcf-7 cell line ( figure 3a and b ) . more dna damage was caused by dmc than mc to both the mitochondrial genome and the nuclear genome of mcf-7 cells treated with the drugs for 1 h ( figure 3a and b ) . furthermore , the most detectable dmc induced mitochondrial dna damage was removed following 12 h without the drug ( figure 3a ) , but nuclear dna adducts caused by dmc persisted 12 h after drug removal ( figure 3b ) . these results do not indicate that mc caused no dna damage but rather that under conditions in which dmc caused significant and persistent dna damage , mc caused no detectable damage . we were specifically interested in the p53-independent mechanism of cell death induced by dmc and for this reason focused on the dld-1 cell line for the chk1 studies . dmc treatment results in the depletion of chk1 protein , while mc treatment does not ( 33 ) . as expected , dmc , but not mc , treatment caused the depletion of chk1 protein ( figure 4a , lanes 3 and 5 ) . the recovery of chk1 upon successful inhibition of the proteasome pathway implicates the proteasome as the primary mechanism for chk1 degradation in dmc treated cells ( figure 4a , lanes 5 and 6 ) . ( a ) whole cell extracts obtained from dld-1 cells were left untreated ( lane 1 ) , treated with 4 m mg115 ( lane 2 ) , 10 m mc or 10 m dmc ( lanes 3 and 5 ) , and 10 m mc or 10 m dmc with 4 m mg115 ( lanes 4 and 6 ) , and were analyzed by western blotting for chk1 protein ( top panels ) and accumulation of ubiquitinated proteins ( bottom panels ) . ( d ) phase contrast image of dld-1 cells treated with mc and dmc as indicated . ( d ) ( d ) facs analysis of dld-1 cells treated with either 1 m or 10 m of mc or dmc . ( e ) whole cell extracts obtained from dld-1 cells treated with either 1 m or 10 m of mc or dmc were analyzed by western blotting for chk1 protein . in our current investigation , we observed concomitant depletion of total ubiquitinated protein ( figure 4a , lane 5 ) . increased degradation of total ubiquitinated protein suggested that perhaps dna adducts of dmc signaled for an increase in proteasome activity . we used an established in - gel assay to detect , in cell lysates , the cleavage of the fluorogenic peptide substrate suc - leu - leu - val - tyr - amc , by the proteasome core particle with both regulatory particles ( rp2cp ) , core particle with one regulatory particle ( rp1cp ) , and only the core particle ( cp ) ( 42 ) . however , mc and dmc treatment did not increase proteasome activity compared to that in the untreated samples ( figure 4b , compare lanes 3 and 4 to lane 1 ) . the increased degradation of ubiquitinated substrates in dmc treated cells was not due to a change in proteasome levels or proteasome activity since dmc treated and untreated cells showed similar proteasome levels and activity , respectively ( figure 4b , compare the left panel to the right panel ) . the observed decrease in ubiquitinated products in the absence of an increase in proteasome activity suggested that dna adducts of dmc might increase signaling to the proteolysis machinery . to determine if the rescue of chk1 protein levels by mg115 was due to protein stabilization and not an increase in chk1 rna , we examined the levels of total chk1 mrna before and after the inhibition of the proteasome ( figure 4c ) . dmc treatment resulted in the reduction of total chk1 mrna ; however , the rescue in chk1 protein accumulation seen in figure 4a was clearly due to a change in chk1 stability and not mrna level as the chk1 mrna level was not increased upon mg115 treatment . when concentrations of 1 m to 50 m of the two agents were compared for the p53-deficient cells , the cell morphology and mtt data indicated a similar cytotoxicity of the two drugs at 1 m dmc and 10 m mc ( figure 4d and d ) . our hypothesis is that mc and dmc can mechanistically influence p53-deficient human cells in different ways and that chk1 depletion is a later stage event that correlates with cell death . since we observed differential regulation of chk1 during mc- and dmc- induced cellular cytotoxicity , we hypothesized that the chk1 protein might play a role in the differential cytotoxicity observed for the two drugs . in the absence of chk1 sirna , down - regulation of chk1 protein was only seen in dmc treated cells ( figure 5a , see lanes 3 and 6 ) . in the presence of chk1 sirna , we observed the dramatic reduction of chk1 protein in mc treated cells and investigated if this , as predicted , increased the cytotoxicity of mc ( figure 5a see lanes 79 ) . cells that had been treated with mc showed the most significant decrease in percent viable cells with sirna to chk1 as compared to the nonspecific sirna ( figure 5b ) . moreover , the cellular cytotoxicity of mc was not changed when cells were treated with mdm2 sirna used as an unrelated target control . the fact that dmc treatment reduces chk1 , before the addition of chk1 sirna , renders the sirna addition here less significant . to evaluate the proliferation of cells overexpressing myc - chk1 after treatment with mc or dmc , mtt solution was added to each sample , and cells were incubated for an additional 2 h. values were obtained from 3 independent experiments . while our studies have focused on the p53-independent activity of mc and dmc in dld-1 cells , the possibility existed that the persistent nuclear dna damage for 24 h with dmc might influence wild - type p53 mediated activation of transcription differently than mc . therefore , the outcome of 24 h treatment with mc and dmc on dna damage induced transcriptional activity of wild - type p53 was assessed in the p53-proficient mcf-7 cells and da-2 cells . however , following 24 h of 10 m dmc treatment , the stabilized p53 resulted in less activation of p21 than mc and did not result in transactivation of mdm2 ( in fact , a decrease was observed ) ( figure 6a and b ) . the possibility exists that the increased frequency of dna adducts by dmc or perhaps the mitosene-1--dna adduct stereochemistry produced by dmc interferes with the ability of the basal transcription machinery to produce transcripts from the mdm2 gene and reduced transcription from the p21 gene . we have previously noted that at early time points ( 3 and 6 h ) after 10 m mc and dmc treatment transcription of the p53 target genes is similar ( 34 ) ; the decrease seen here could also be due to increased induction of cell death after 24 h. nevertheless , the different outcomes of mc and dmc treatment on p53-proficient cells and on the p53-pathway are another indication that dmc and mc can signal to the cellular machinery differently when used at similar concentrations . this is yet another indication that increased cytotoxicity of dmc as compared to mc is through a p53-independent pathway . ( c ) phase contrast image of mcf-7 cells treated with mc and dmc as indicated . ( d ) model : dmc treatment generates increased amounts of the mitosene-1- dna adducts , while the mc treatment produces moderate amounts of mitosene-1- dna adducts . the increased dna adducts produced by dmc after 24 h interfere with p53 transcriptional activity , signal for the rapid depletion of endogenous chk1 by the proteasome , which may disrupt the g2/m cell cycle checkpoint and lead to nuclear condensation . mc dna adducts activate chk1 , which may prolong the intra s and g2 checkpoints , increase nuclear size , and activate p53 transcriptional activity , while blocking replication stress induced cell death . down - regulation of chk1 has been described as an alternative pathway for inducing cell death when the more common apoptotic pathways have been sabotaged in cancer cells ( 22,45 ) . we observed chk1 down - regulation and increased cell death , especially in the absence of a p53 checkpoint , when cells were treated with dmc ( 33 ) . in view of the close structural similarity between mc and dmc , the differences in their biological effects in human tumor cell cultures were originally unexpected . in this article , we further characterize these effects and propose to correlate them with the known structural differences of the dna adducts , the amount of adduct formation , and cellular signaling . the increased production of mitosene-1--dna adducts following dmc treatment or their persistence in the nuclear genome might account for the reduction in nuclear size that correlates with the increased cytotoxicity observed with this mitomycin derivative . dmc treatment of cells caused a reduction in nuclear size which is consistent with a number of types of cell death including mitotic catastrophe and apoptosis ( 46 ) , while swelling is associated with necrosis ( 43 ) . our data demonstrate that chk1 depletion can increase the extent of mc induced cell death and is correlated with dmc induced cell death ; but our data do not indicate if the mechanism is through mitotic catastrophe or a caspase 2-dependent cell death . while we do not have direct evidence for this , our confocal microscopy images of dapi stained nuclei ( figure 2 ) show brightly stained condensed nuclear architecture after the cells were treated with dmc for 24 h. it is known that icls disrupt dna replication and transcription ( 32 ) . it is shown here that increased production of mitosene-1--dna adducts by dmc correlates with a signaling mechanism that results in the down - regulation of chk1 by the ubiquitin proteolysis pathway . sporadically , we see that 24 h treatment of cells with mc results in increased chk1 protein levels ; however , we never see a reduction of chk1 at the 24 h time point ( 33 ) . we previously showed that increasing the time of mc treatment to 72 h caused a reduction in the chk1 level ; here , we show that increasing the mc concentration by 10-fold ( to 100 m ) causes a reduction in chk1 . when chk1 was down - regulated by sirna , similar cytotoxicity of equimolar mc and dmc was achieved . reported that disruption of the chk1 g2 checkpoint using ucn-01 , a chk1 inhibitor , increased the cytotoxicity of mc , and this effect was independent of the wild - type p53 ( 52 ) . however , overexpressing chk1 caused only a slight decrease in the cytotoxicity of the two mitomycins at 24 h post - treatment . the activity of the exogenous chk1 is limited , and therefore , the slight increase in viability detected is most likely due to this fact ( 44 ) . we observed that when the proteasome activity was inhibited by mg115 , chk1 protein levels were recovered even when cells were treated with dmc . our data support the hypothesis that dmc down - regulates chk1 by causing increased ubiquitination and subsequent degradation by the proteasome . importantly , we saw that this mechanism of chk1 down - regulation happened in the absence of increased proteasome activation . we did not observe any increase in proteasome activity in dmc treated samples but observed a decrease in total ubiquitinated protein products upon treatment with dmc . this supports the hypothesis that dmc dna adducts signal for altered protein post - translational modifications that cause rapid degradation of the tagged polypeptides . this was substantiated by preliminary protein microarray data showing that dmc treatment caused a dramatic reduction in the levels of many proteins on the sigma panorama cell signaling antibody array ( data not shown ) . when we examined the p53 transcriptional activity , we observed that while both mc and dmc could stabilize p53 , the p53 in 24 h dmc treated cells did not correlate with increased levels of the mdm2 gene transcript and resulted in lower p21 transcription than that seen in mc treated cells . the reduced p53 transcriptional activity following dmc treatment may be due to the fact that the persistent dna adducts of dmc , over time , negatively influence the transcription machinery . this suggests that even in the presence of wild - type p53 protein , dmc might activate a parallel p53-independent pathway because the p53 target genes are not as robustly turned on by dmc in spite of stabilization of the p53 protein . the parallel death pathways of p53 activation and p53-independent cell death by dmc will require further investigation . we observed that dmc induces p53-independent cell death in cancer cells in a manner that correlated with ubiquitin mediated degradation of chk1 in the presence of persistent dna damage . we see that chk1 degradation is correlated with the predominant formation of mitosene-1- guanine adducts of dmc , in contrast to the mitosene-1- adducts formed with mc . this alteration of the chirality of the drug linkage to dna undoubtedly changes the alignment of dmc adducts , and this may be related to the basis of the different dna damage signaling mechanisms ( see model in figure 6 ) .

the pancreas comprises an exocrine component ( ductal and acinar cells ) and an endocrine component ( cells , cells , cells , pancreatic polypeptide - positive [ pp ] cells , and cells ) . the endocrine cells are organized in defined islet structures embedded in the acinar compartment , which function as key regulators of carbohydrate metabolism ( edlund , 2002 ) . the autoimmune disease type 1 diabetes irreversibly destroys insulin - secreting cells in pancreatic islets , resulting in a lack of insulin production and hyperglycemia ( atkinson et al . , 2011 ) . treatment is most commonly with insulin injections , but the degree of glycemic control with this approach does not compare to functional pancreatic cells . regenerative cell treatments in diabetic patients could allow for the long - term restoration of normal glycemic control and thus represent a potentially curative therapy ( yi et al . , 2013 ) . the generation of new pancreatic cells is being pursued on several fronts in vitro , including differentiation of induced pluripotent stem cells ( ipscs ) and reprogramming of other pancreatic cell types ( pagliuca and melton , 2013 ) . regenerating pancreatic cells in situ is an attractive alternative to these approaches , driven by evidence of spontaneous cell neogenesis in the adult pancreas ( bonner - weir et al . , 2004 ; dor et al . , 2004 ; lysy et al . , 2012 ; pagliuca and melton , 2013 ; teta et al . , cell regeneration during adulthood is very limited but can be achieved experimentally using pancreatic duct ligation in mice ( xu et al . , 2008 ) and inducible depletion of acinar and islet cells with diphtheria toxin showed that duct cells can give rise to both acinar and endocrine cells ( criscimanna et al . , 2011 ) . thus , ductal cells in the adult pancreas show a latent propensity for cell generation . adenoviral overexpression of the three transcription factors neurogenin-3 ( ngn3 ) , maf1a , and pdx1 is sufficient to convert adult acinar cells into cells ( zhou et al . , 2008 ) , and overexpression of pax4 converts glucagon - producing cells into cells ( collombat et al . , 2009 ) . however , the capacity for cell neogenesis in the normal adult pancreas , and the regulatory events surrounding it , remain largely unknown . ngn3 is the earliest factor that specifically regulates the development of the endocrine compartment in the embryonic pancreas ( habener et al . , 2005 ) . ngn3 mice completely lack endocrine islet development ( gradwohl et al . , 2000 ) , and transgenic overexpression of ngn3 activates an islet differentiation program in the embryo and in cultured pancreatic ductal cell lines ( heremans et al . , 2002 ; schwitzgebel et al . , 2000 ) . in the adult pancreas , ngn3 expression is very limited , but levels rise during cell neogenesis induced by pancreatic duct ligation , where ngn3 is required for cell replenishment ( van de casteele et al . , 2013 ; xu et al . , 2008 ) . moreover , expansion of ngn3 + cells bordering the ducts contributes to the cell expansion observed when overexpressing pax4 ( al - hasani et al . , 2013 ) , indicating that manipulation of ngn3 levels and/or activity may be beneficial for regeneration therapies . ngn3 is a highly unstable protein ( roark et al . , 2012 ) , and the level and timing of its expression must be precisely controlled to ensure the correct production of cells , but the details of its posttranslational regulation remain elusive . fbw7 ( f - box and wd-40 domain protein 7 ) is the substrate recognition component of an evolutionarily conserved scf ( complex of skp1 , cul1 , and f - box protein)-type ubiquitin ligase . scf(fbw7 ) degrades proteins that function in cellular growth and division pathways , including c - myc , cyclin e , notch , and c - jun ( welcker and clurman , 2008 ) . emerging evidence shows that fbw7 controls stem cell self - renewal , cell fate decisions , survival , and multipotency in numerous tissues , including the hematopoietic ( iriuchishima et al . , 2011 ) and nervous systems ( hoeck et al . , 2010 ; matsumoto et al . , 2011 ) , liver ( onoyama et al . , 2011 ) , and intestine ( sancho et al . , 2010 ) . this suggests that fbw7 has a crucial function in fundamental cell differentiation processes . here , we show that fbw7 contributes to the regulation of ngn3 stability , and loss of fbw7 induces a direct ductal - to- cell differentiation in the adult pancreas . our study not only reveals a role for fbw7 in pancreatic cell fate determination and identifies ngn3 as a target of fbw7 but also demonstrates that ductal cells can be induced to alter their identity in the adult pancreas in the absence of injury to the organ with a single genetic change . given the role of fbw7 in controlling cell fate decisions in other organs , we asked whether fbw7 also functions in cell type specification in the pancreas . we deleted fbw7 in embryonic pancreatic progenitor cells using a cre recombinase under the control of the pdx1 promoter ( pdx1-cre ; fbw7 mice ) . although pancreatic organ size and gross morphology appeared normal , histological analysis revealed increased ductal cell proliferation and an expansion of the ductal compartment ( figures s1a and s1b available online ) , consistent with the increase in proliferation upon fbw7 deletion observed in other organs ( hoeck et al . , 2010 ; matsumoto et al . , 2011 ; onoyama et al . , 2007 , 2011 ; unexpectedly , scattered cells in the pdx1-cre ; fbw7 ducts showed an enlarged cytoplasm and smaller rounded nuclei when compared with surrounding ductal cells , and more closely resembled islet cells ( figures 1a1d ) . insulin expression , which is normally restricted to islets in control mice ( figure 1e ) , was detected in these aberrant ductal cells ( figure 1f ) . the majority of insulin - positive cells in pdx1-cre ; fbw7 ducts were devoid of the ductal cell marker cytokeratin-19 ( ck19 ) , but costaining of ck19 and insulin was sometimes observed ( figure 1f ) , suggesting an intermediate transition state between ductal and insulin - positive cells . no insulin costaining with the acinar cell marker amylase was observed ( figures s1c and s1d ) . thus , the absence of fbw7 appears to trigger abnormal differentiation of a subset of ductal cells , biasing them toward an endocrine fate . in addition to ectopic insulin - positive cells ( in 17% of ducts ) , pdx1-cre ; fbw7 ducts also contained cells expressing the cell marker glucagon , albeit less frequently ( 3% of ducts ) ( figures s1e and s1f ) . we also observed glucagon / insulin double - positive cells in pdx1-cre ; fbw7 ducts ( figures s1g s1i ) , similar to progenitor cells described in human embryonic pancreas ( piper et al . , 2004 ) . thus , deletion of fbw7 in the pancreas promotes the occurrence of cells coexpressing markers of different pancreatic cell lineages , a cellular phenotype that is not normally observed in the adult pancreas . one of the features of functional cells is the expression of the glucose transporter glut2 ( slc2a2 ) . in control animals , glut2 was coexpressed with insulin in islet cells by double insulin / glut2 immunofluorescence ( if ) ( figure 1 g ) . glut2 was also coexpressed with insulin in the pdx1-cre ; fbw7 aberrant ductal cells ( figures 1h and 1i ) . because pdx1-expressing progenitors give rise to all the pancreatic lineages ( oliver - krasinski and stoffers , 2008 ) , the emergence of insulin - positive cells in pdx1-cre ; fbw7 ducts could be due to a developmental defect . to test whether fbw7 deletion can induce cell neogenesis in adult mice , and to clarify the cells that give rise to ectopic cells , we combined inducible fbw7 deletion using the r26-creert line with lineage tracing using r26-lsl - yfp . ry control mice express a tamoxifen - inducible form of cre recombinase from the ubiquitous rosa26 ( r26 ) promoter , leading to the permanent expression of yellow fluorescent protein ( yfp ) in recombined cells . in the fbw7 background ( rfy line , figure 2a ) , tamoxifen treatment results in recombination of the floxed fbw7 alleles in cre - expressing cells concomitantly with activation of yfp expression ( figure 2b ) . intraperitoneal injection of tamoxifen induced recombination in all pancreatic cell types but with different efficiencies . almost all acinar cells showed yfp positivity ( 91% ) , while recombination occurred much less frequently in islet ( 10% ) and ductal cells ( 5% ) ( figures 2c and 2d ) . despite the high percentage of recombination in the acinar compartment ( figure 2d ) , no insulin positivity was observed in cells with acinar cell morphology , and insulin / amylase double - positive cells could not be detected in the rfy pancreas ( figures 2e2n ) . likewise , direct intrapancreatic injection of 4-oh - tamoxifen into the pancreatic tail resulted in exclusive recombination in acinar cells , and here , no cell neogenesis was observed ( figures s2a s2d ) . in contrast , despite the low percentage of recombination in ducts ( 5% ) , rfy mice gave rise to insulin / green fluorescent protein ( gfp ) double - positive cells in ducts as early as 13 days postintraperitoneal injection of tamoxifen ( figures 2e2n ) as well as at later time points ( figures s2e and s2f ) . therefore , fbw7 deletion in ductal cells , but not in acinar cells , induces the acquisition of cell identity . fbw7 targets many proteins involved in proliferation and differentiation for proteasomal degradation , such as n - terminally phosphorylated c - jun ( p - c - jun ) , notch intracellular domain 1 ( nicd1 ) , phosphorylated c - myc , and phosphorylated cyclin e ( welcker and clurman , 2008 ) . it has been shown that scf(fbw7)-mediated substrate degradation is tissue specific ( hoeck et al . , 2010 ; ishikawa et al . , 2008 ; nakayama and nakayama , 2006 ; onoyama et al . , 2007 , 2011 ; sancho et al . , 2010 ; thompson et al . , 2008 ; western blotting of lysates from whole pdx1-cre ; fbw7 pancreas , in which fbw7 is inactivated in all pancreatic cell types , showed increased p - c - jun and nicd1 protein levels when compared to fbw7 controls , while phosphorylated c - myc and cyclin e levels were not substantially affected ( figure 3a ; quantifications in figures s3a and s3b ) . in cells , nicd1 and p - c - jun were barely detectable , either in control ry or in fbw7-deleted rfy pancreas ( figure s3c ) . in the acinar compartment , the loss of fbw7 did not increase p - c - jun or nicd1 in rfy compared with ry mice ( figure s3d ) . in order to analyze fbw7 function in ducts , we examined sections from pdx1-cre ; fbw7 animals . p - c - jun fluorescence intensity was increased in pdx1-cre ; fbw7 compared with control ducts ( figures 3b and 3c ) , suggesting that scf(fbw7 ) primarily acts in pancreatic ductal cells . of the known substrates increased by fbw7 loss in the pancreas , none are thought to be involved in cell neogenesis . while notch has been reported to be involved in embryonic pancreatic differentiation , it has been proposed to inhibit cell neogenesis rather than promoting it ( esni et al . , 2004 ; we therefore examined the possibility that fbw7 in the pancreas may control the levels of one or more other substrates . transcription factors involved in embryonic cell development include pdx1 , ngn3 , hnf3 , and hnf6 ( zaret , 2008 ) . pdx1 , hnf3 , and hnf6 protein levels were unaltered in pdx1-cre ; fbw7 compared with fbw7 pancreas , but the protein levels of ngn3 were strongly increased ( figure 3d ; quantifications in figures s3e and s3f ) . ngn3 messenger rna ( mrna ) levels were also increased ( figure 3e ) , in agreement with previous reports of positive autoregulatory loops controlling ngn3 expression ( ejarque et al . the increase in ngn3 after fbw7 loss also correlated with higher mrna levels of the ngn3 transcriptional target genes insm1 , heyl , ctgf , and nkx2 - 2 ( swales et al . , 2012 ) when analyzed by quantitative pcr ( qpcr ) ( figure 3e ) , and increased protein levels of the ngn3 transcriptional targets neurod1 and insm1 ( figures 3d , 3f , 3 g , s3e , and s3f ) . ngn3 is a key regulator of endocrine differentiation , making it an excellent candidate for inducing cell neogenesis induced by fbw7 loss . to investigate the mechanism by which fbw7 affects ngn3 , we first analyzed the stability of ngn3 protein using cycloheximide to inhibit protein synthesis . ngn3 half - life was increased more than 2-fold after fbw7 silencing , suggesting that fbw7 acts to destabilize ngn3 protein ( figure 4a ) . ngn3-hemagglutinin ( ngn3-ha ) coimmunoprecipitated flag - tagged fbw7 isoform- and , to a lesser extent , isoform- ( figure 4b , left panel ; and vice versa , as shown in the right panel ) , and endogenous fbw7 interacted with ngn3-ha ( figure 4c ) . ngn3 is a heavily ubiquitinated protein ( roark et al . , 2012 ) , but ngn3 ubiquitination was strongly reduced in fbw7 hct116 cells when compared to congenic fbw7 cells ( figure 4d ) . in vitro , wild - type ( wt ) fbw7flag protein complexes promoted efficient ubiquitination of recombinant ngn3 , but the inactive mutant fbw7-fbox flag did not ( welcker et al . , 2004 ) , these data suggest that ngn3 is a substrate of the scf(fbw7 ) ubiquitin ligase . most fbw7 substrates contain a phosphodegron motif that serves as the recognition motif for fbw7 interaction ( welcker and clurman , 2008 ) . multiple higher molecular weight bands of ngn3-ha detected by immunoblot collapsed after calf intestinal phosphatase ( cip ) treatment , suggesting that they represent phosphorylated forms . silencing of fbw7 increased the levels of these higher molecular weight forms ( figure 4f ) . gsk3 is the kinase responsible for modifying the phosphodegron motifs of c - myc and notch1 ( welcker and clurman , 2008 ; welcker et al . in silico analysis revealed a gsk3 consensus site at the ngn3 c terminus ( ser183ser187 ) . gsk3 inhibitor treatment increased ngn3 protein levels ( figure 4 g ) , suggesting that gsk3 regulates the stability of ngn3 . we generated constructs of ngn3 with ser183 and/or ser187 mutated to alanine to assess the role of the putative phosphodegron motif ( figure 4h ) . mutation of the predicted gsk3 phosphorylation site ser183 altered the electrophoretic mobility of ngn3 protein , while mutation of ser187 had less effect ( figure 4i , left panel ) . while wt ngn3 could efficiently interact with fbw7 , the interaction was severely impaired when ser183 was mutated ( ngn3-aa and ngn3-as ; figure 4i , right panel ) . accordingly , while the mrna levels from all four ngn3 constructs were comparable , the stability of ngn3-aa and ngn3-as was greatly increased ( figures s4b and s4j ) . these data suggest that fbw7 directly controls ngn3 stability by regulating its ubiquitination and proteasomal degradation and that gsk3-mediated phosphorylation of ser183 might regulate this process . since ngn3 has been shown to be involved in cell neogenesis in the adult pancreas ( al - hasani et al . , 2013 ; baeyens et al . , 2006 ; xu et al . , 2008 ) , accumulation of ngn3 protein is likely to contribute to adult cell neogenesis induced by fbw7 inactivation . to determine whether accumulation of ngn3 protein is sufficient to induce cell neogenesis , we generated a conditional inducible transgenic mouse line that expresses the phospho mutant , stable form of ngn3 ( ngn3-aa ) together with gfp after cre recombination ( pdx1-cre ; rosa26-loxstoplox - ngn3-aa - ires - gfp or pdx1-cre ; r26-lsl - ngn3-aa ; figure 5a ) . pdx - cre induced recombination in mouse pancreas but not in liver or tail ( figure 5b ) . ngn3 protein , which is undetectable in control adult pancreas , was detected in pdx1-cre ; r26-lsl - ngn3-aa pancreas ( figure 5c ) but not in liver . pdx1-cre ; r26-lsl - ngn3-aa pancreas showed increased ngn3 and ins2 mrna levels when compared to unrecombined r26-lsl - ngn3-aa mice ( figure 5d ) . transgenic ngn3-aa expression resulted in increased cell area as analyzed by immunostaining for insulin ( figures 5e and 5f ) . these data suggested that the overexpression of a stable form of ngn3 ( ngn3-aa ) from embryonic pancreas development onward results in an increase in cells . in order to test whether overexpression of ngn3-aa in the adult pancreatic ducts was sufficient to induce cell reprogramming , we crossed r26-lsl - ngn3-aa mice to ck19-creert mice , in which the expression of tamoxifen - inducible cre - ert protein is driven by the promoter of the ductal marker cytokeratin 19 ( means et al . , 2008 ) thirteen days post - tamoxifen injection ( figure 5h ) , gfp expression could be detected specifically in ductal cells of ck19-creert ; r26-lsl - ngn3-aa mice , while it was absent in r26-lsl - ngn3-aa ducts ( figure 5i ) . furthermore , we detected a significant increase in insulin - positive ductal cells in tamoxifen - injected ck19-creert ; r26-lsl - ngn3-aa pancreas ( figures 5j and 5k ) , suggesting that ngn3-aa overexpression in the adult pancreatic duct is sufficient to induce ductal - to- cell conversion . the aforementioned data suggest that fbw7 may control adult cell neogenesis by regulating ngn3 protein stability . to test whether cells arise as a direct consequence of fbw7 loss in ductal cells , we asked whether loss of fbw7 , specifically in the adult ductal compartment , is sufficient to achieve cell conversion . to this end , we generated ck19-creert ; fbw7 ; r26-lsl - yfp ( cy ) control and ck19-creert ; fbw7 ; r26-lsl - yfp ( cfy ) inducible fbw7 deletion mice ( figures 6a and 6b ) . the efficiency of recombination 2 weeks after tamoxifen injection was between 40% and 50% in both cy and cfy ducts , as reported previously ( means et al . , 2008 ) . complete recombination was confirmed by pcr analysis of genomic dna isolated from yfp+ cfy cells ( figure 6c ) . qpcr analysis demonstrated that fbw7 mrna was highly expressed in cy ductal cells but undetectable in cfy ductal cells and mature pancreatic -cells sorted from mip - gfp mice ( in which gfp expression is driven by the insulin promoter ) ( figure 6d ) . these data suggest that fbw7 mrna is enriched in ductal cells , in agreement with substrate stabilization predominantly in this cell type after fbw7 loss ( figure 3b ) . fbw7 inactivation in ductal cells did not alter the number of ducts ( figure 6e ) or islets ( figure 6f ) . however , a significant number of insulin - positive cells ( almost 0.5% , i.e. 1% of the fbw7 knockout cells if considering 50% recombination efficiency ) was observed in the ducts of cfy mice , while they rarely arose in cy mice ( figure 6 g ) . about 12% of the cfy ducts contained induced cells , typically between one and three cells per duct . it is interesting that 3.8% of cfy ducts contained glucagon - positive cells and 5.5% contained somatostatin - positive cells , while pp or amylase - positive ductal cells were never detected ( figures s4c and s4d ) . thus , deletion of fbw7 in adult pancreatic ductal cells induces conversion of some ductal cells to or cells or , most frequently , to cells . inactivation of fbw7 could trigger resident ductal progenitor cell proliferation followed by redifferentiation or induce direct transdifferentiation . to distinguish between these possibilities , fbw7 inactivation and lineage tracing was combined with long - term bromodeoxyuridine ( brdu ) labeling . brdu was incorporated in scattered cells in the pancreatic cy ducts and increased in cfy ducts ( figures 6h , 6i , and s4e ) . however , less than 1% of insulin - positive cfy duct cells were labeled after 2 weeks of continuous brdu exposure , beginning directly before fbw7 deletion ( figures 6j , s4f , and s4 g ) . thus fbw7 deletion in the adult pancreatic ducts induces direct conversion of a subset of exocrine ductal cells into endocrine cells , without a requirement for cell proliferation . to explore the functionality of the cells formed after fbw7 deletion , we performed mrna expression profiling of gfp+ sorted cells from tamoxifen - injected cy and cfy mice and compared them to gfp+ cells from mip - gfp mice as a positive control for cells ( figure 7a ) . cfy gfp+ ductal cells showed a modest increase in expression of numerous cell specific genes , consistent with a small subset of ductal cells undergoing cell conversion . in agreement with the increase in ngn3 stability in fbw7-deleted cells , cfy gfp+ ductal cells also showed an increase in the expression of reported ngn3 target genes ( chga , insm1 , dll3 , syp , chn1 , heyl , atp2a3 , and pcsk2 ; swales et al . , 2012 ) qpcr analysis confirmed increased mrna expression of the cell marker genes ins2 , gck , pdx1 , and nkx6.1 in cfy gfp+ ductal cells compared with cy gfp+ cells ( figure 7c ) . as well as showing gene expression characteristics of islet cells , insulin - positive cells in the ducts of cfy mice costained with the functional cell markers c - ppt , glut2 , mafa1 , nkx6.1 , pax6 , pc1/3 , pdx1 , urocortin 3 ( ucn3 ) , and isl1 , showing comparable staining to islet cells ( figures 7d , 7e , and s5 ) . insulin - positive cfy ductal cells were negative for the ductal markers sox9 and dba , while other cfy ductal cells retained expression of these markers ( figures 7e and s5 ) . an important hallmark of cell function is the ability to release insulin after glucose stimulation . to test this , we subjected gfp+ cells sorted from cy and cfy mouse pancreas to in vitro glucose challenge ( figures 7f and 7 g ) . while cy gfp+ cells did not respond to glucose , cfy gfp+ cells showed a substantial release of insulin ( figure 7 g ) . each cfy well of 30,000 cells contained approximately 300 converted cells ( based on a 1% conversion frequency ) , which secreted 214 pg ( 1.07 ng / ml ) of insulin , i.e. , 0.71 pg per cell . by comparison , 5,000 islet cells sorted from a mip - gfp pancreas responded to glucose by releasing 3,378 pg ( 16.89 ng / ml ) of insulin , i.e. , 0.67 pg per cell ( figure 7h ) . based on this result , the response to glucose challenge in the converted cells is comparable to that of islet cells ( figure 7i ) . thus , the cells converted after fbw7 loss in the adult pancreatic ducts show both the characteristic marker expression and functionality of mature cells . ectopic expression of combinations of transcription factors can induce changes of cellular fate in adult pancreatic tissue ( zhou et al . , 2008 ) ; however , examples of reprogramming in vivo by loss of a single molecule are rare . in this study we show that fbw7 maintains adult ductal cell fate , as fbw7 inactivation results in transdifferentiation of ductal cells into and cells and , predominantly , cells . the newly formed cells resemble islet cells with regard to cellular morphology , marker gene expression , and insulin secretion in response to glucose challenge . our findings indicate an unexpected plasticity of ductal cells , in which loss of a single gene ( fbw7 ) renders the duct cells multipotent , able to remain exocrine or transdifferentiate into endocrine , , or cell types . fbw7 is a key regulator of stem cell function , as fbw7 inactivation results in increased proliferation and impaired differentiation of hematopoietic , liver , intestinal , and neural progenitor cells ( hoeck et al . , 2010 ; , 2011 ; matsumoto et al . , 2011 ; onoyama et al . , 2011 ; the potent tumor suppressor function of fbw7 is likely to be a direct consequence of deregulated stem cell proliferation and differentiation ( wang et al . , 2012 ) . however , the role of fbw7 in the pancreatic ducts is distinct from fbw7 function in other organ systems . ductal to endocrine cell transdifferentiation after fbw7 loss occurs in the absence of proliferation , suggesting that the subset of cells that respond to fbw7 deletion in this way does not behave as adult stem cells in other organ systems , requiring cell division before differentiation . rather , fbw7 seems to function in the adult pancreas to constantly maintain cell fate in a subset of ductal cells . we found that the expression and activity of fbw7 in the adult pancreas is enriched in the ductal compartment . adult pancreatic ducts have been suggested to harbor cell progenitors , which are reactivated after challenge ( bonner - weir et al . , 2008 ) . pancreatic duct ligation ( pdl ) , combined with lineage tracing of the ductal epithelium , demonstrated that a quarter of new cells formed in response to injury were derived from ductal cells ( inada et al . , 2008 ) . in contrast , alloxan treatment was recently shown to induce transdifferentiation of acinar cells into cells ( baeyens et al . , 2014 ) . we found that fbw7 expression was quickly and dramatically downregulated 24 hr after pdl , but alloxan treatment induced no change in fbw7 expression ( figures s6a and s6b ) . these data suggest that fbw7 transcriptional downregulation may contribute to duct - derived cell neogenesis in response to pancreatic injury ( figure s6c ) . although it is conceivable that cells produced elsewhere could migrate to the ducts , the location of induced cells embedded within the ducts suggests that they originated in this compartment . several lines of evidence support this interpretation : first , transition cells coexpressing ductal cell ( ck19 ) and cell ( ins ) markers are observed after fbw7 loss ; second , genetic models in which recombination is inefficient in ducts ( such as rfy mice injected intrapancreatically with oh - tamoxifen ) do not induce cell neogenesis ; and third , duct - specific fbw7 loss combined with lineage tracing ( our cfy model ) induces the occurrence of ductal yfp - labeled insulin - positive cells . insulin - positive cells are also observed in ducts of normal unchallenged mice , albeit rarely ( teta et al . , 2005 ) , a finding we confirmed in this study . it is not known whether this spontaneous transdifferentiation process is similar to the reprogramming induced by fbw7 inactivation . however , bihormonal insulin / glucagon double - positive cells presumptive and cell precursors during embryonic pancreas development ( de krijger et al . , 1992 ) that we also observed in fbw7 deleted ducts have not been described in unchallenged adult wt pancreas . thus , cell reprogramming induced by fbw7 inactivation appears to be a distinct process from the spontaneous appearance of insulin - positive cells in wt pancreatic ducts and may represent a reawakening of a multipotent state . a frequent stumbling block in previous models inducing cell reprogramming has been the functionality of the newly formed cells . it is important to note that fbw7-mutant induced cells secrete comparable amounts of insulin after glucose challenge as bona fide cells isolated from mip - gfp mice . therefore , fbw7 loss appears to trigger the conversion of adult pancreatic ductal cells into apparently functional cells . the activity of fbw7 toward different substrates is tissue specific , and our results show that , in the pancreas , c - jun , notch , and ngn3 levels are increased after fbw7 deletion . while c - jun has no reported function in pancreatic cell fate decisions , the notch signaling pathway is thought to inhibit pancreatic endocrine development ( apelqvist et al . , 1999 ; in contrast , we find that endocrine differentiation in the ducts after fbw7 deletion is accompanied by an increase in nicd1 levels . this could imply that notch has different roles in embryonic and adult pancreatic cell differentiation , but it is also possible that increased notch signaling is not required for cell neogenesis after fbw7 loss , and cell neogenesis is induced despite an overall increase in notch levels . the stabilization of ngn3 after fbw7 loss is consistent with a strong proendocrine signal . ngn3 has been previously reported to be required for pdl- and pax4 overexpression - induced cell neogenesis ( al - hasani et al . , 2013 ; xu et al . , 2008 ) , and our data show that ngn3 stabilization in the ducts is sufficient to induce cell neogenesis ( figure 5 ) . despite its essential role in endocrine differentiation , and the reported ngn3 instability at the protein level , the regulatory mechanisms that control the abundance of ngn3 are not fully understood . in this study , we show that ngn3 is a substrate for scf(fbw7 ) . ngn3 behaves as a canonical fbw7 substrate , containing a gsk3 consensus phosphorylation site that , when mutated , increases the stability of the protein . our data indicate that ngn3 stabilization after fbw7 loss contributes to a transdifferentiation program , inducing ductal cells to differentiate into cells . induction of adult cell neogenesis is desirable for diabetes treatment , and modulation of the fbw7-ngn3 axis could potentially be exploited as a therapeutic approach toward generation of new cells for cell replacement therapies . r26-lsl - yfp ( srinivas et al . , 2001 ) , ck19-creert ( means et al . , 2008 ) , r26-creert ( ventura et al . , 2007 ) , mip - gfp ( hara et al . , 2003 ) , and fbw7 ( jandke et al . , 2011 ) mouse lines have been previously described . the r26-lsl - ngn3-aa mouse was generated using mouse ngn3-aa complementary dna ( cdna ) to create a conditional rosa26-ngn3-aa - ires - egfp - pa targeting vector as described elsewhere ( nyabi et al . , 2009 ) , followed by selection of embryonic stem cell clones targeted with linearized vector and generation of chimeric swiss diploid embryos . all animal experiments were approved by the cruk london research institute animal ethics committee and conformed with uk home office regulations under the animals ( scientific procedures ) act 1986 including amendment regulations 2012 . hct116-fbw7 and hct116-fbw7 cells , and fbw7flag and fbw7flag constructs , have been described elsewhere ( grim et al . , 2008 ) . full - length ngn3 cdna from mouse embryonic pancreas was obtained by pcr and cloned into pcdna3 to generate the pcdna3-ngn3 plasmid . mutation of ngn3 ser183/ser187 to alanine was achieved by conventional pcr site - directed mutagenesis . p - rs - sh - control and p - rs - sh - fbw7 constructs were generated by cloning short hairpin - containing oligos into the prs vector ( addgene ) . for all experiments , adult ( 69 weeks except where indicated ) age- and strain - matched animals were used . mice were either injected intraperitoneally with 100 g / g body weight of tamoxifen dissolved in peanut oil ( at least three mice per genotype ) or intrapancreatically injected with 20 l50 m 4-oh - tamoxifen ( two mice per genotype ) as indicated . analyses were performed 5/13 days ( short term ) or 60/82 days ( long term ) postinjection . where indicated , brdu ( 0.8 mg / ml ) was given in drinking water 1 day before the first tamoxifen injection and kept until the end of the experiment . the pancreas was excised , processed , and stained as described in the supplemental experimental procedures . rhodamine - dba ( sigma ) was used to detect ductal cells by confocal microscopy . if and immunohistochemistry staining was performed as described elsewhere ( sancho et al . , 2010 ) . quantification of the insulin - positive area in ngn3 conditional transgenic mouse pancreas was performed on nanozoomer 2.0-ht ( hamamatsu ) scanned slides using adobeps - cs5 . pancreas lysates were homogenized in ripa lysis buffer supplemented with protease inhibitor ( sigma ) . immunoblots were carried out as described elsewhere ( nateri et al . , 2005 ) . human embryonic kidney 293 t ( hek293 t ) cells coexpressing ha - ngn3 and fbw7-flag were treated for 5 hr with proteasome inhibitor mg-132 ( 25 m ; calbiochem ) , lysed with 0.2% np40 buffer , and incubated with anti - flag or anti - ha agarose beads ( sigma ) . for the endogenous fbw7-ngn3 interaction assay endogenous fbw7 in inputs and immunoprecipitation ( ip ) samples was detected using anti - fbw7 antibody ( bethyl laboratories ) . for in vivo ubiquitination assays , his - ub was affinity purified with nickel - nitrilotriacetic acid ( nta)-agarose beads , as described elsewhere ( davies et al . , 2010 ) . in vitro ubiquitination assays with fbw7 and fbw7-fbox - flag immunoprecipitated complexes were performed as described elsewhere ( popov et al . , 2007 ) . single pancreatic cell suspensions were obtained by 30 min digestion in 1.6 mg / ml collagenase type iv ( whorttington ) , followed by filtration on a 70 m nylon mesh . cells from six age - matched ( 6- to 8-week - old ) mice per genotype were sorted for gfp expression for each independent experiment . genomic dna from gfp+/gfp cells sorted from six tamoxifen - injected cfy and cy mice was isolated by digestion in directpcr lysis reagent ( viagen ) . pcr primers used to detect the efficiency of recombination of fbw7 and r26 alleles are given in the supplemental experimental procedures . rna was isolated from sorted gfp+ cells from tamoxifen - injected cy and cfy mice ( six pooled pancreas per genotype ) or mip - gfp mice ( three pooled pancreas ) using a rneasy micro kit ( qiagen ) . rna microarray hybridizations were performed by the cancer research uk manchester institute microarray service using the genechip mouse gene 1.0 st array ( affymetrix ) after genome amplification of the rna . for qpcr analysis of sorted cells , rna was isolated as described for the microarray , and cdna amplification was performed using the quantitect whole transcriptome amplification kit ( qiagen ) . for qpcr analysis in pdx1-cre ; fbw7 mice , rna was isolated using the rneasy mini kit ( qiagen ) , and cdna was generated using the transcriptor first strand cdna synthesis kit ( roche ) . diluted cdnas were used for qpcr sybr - green detection of target genes , using primer sequences given in the supplemental experimental procedures . determination of insulin release after glucose challenge was performed as described elsewhere ( banga et al . , 2012 ) , with minor modifications . briefly , 30,000 gfp+ sorted cy or cfy cells ( from six pooled pancreas per genotype ) or 5,000 gfp+ cells from mip - gfp pancreas were plated per well in dulbecco s modified eagle s medium without serum / glucose / phenol red . cells were starved for 2 hr , the medium was changed , and 20 mm glucose was added ( final volume , 200 l ) . insulin concentration was determined in supernatants using the mouse insulin elisa kit ( crystal chem ) . p 0.05 , p 0.01 , p 0.001 were considered statistically significant .

summarythe adult pancreas is capable of limited regeneration after injury but has no defined stem cell population . the cell types and molecular signals that govern the production of new pancreatic tissue are not well understood . here , we show that inactivation of the scf - type e3 ubiquitin ligase substrate recognition component fbw7 induces pancreatic ductal cells to reprogram into , , and cells . loss of fbw7 stabilized the transcription factor ngn3 , a key regulator of endocrine cell differentiation . the induced cells resemble islet cells in morphology and histology , express genes essential for cell function , and release insulin after glucose challenge . thus , loss of fbw7 appears to reawaken an endocrine developmental differentiation program in adult pancreatic ductal cells . our study highlights the plasticity of seemingly differentiated adult cells , identifies fbw7 as a master regulator of cell fate decisions in the pancreas , and reveals adult pancreatic duct cells as a latent multipotent cell type .

Introduction Results Discussion Experimental Procedures

the pancreas comprises an exocrine component ( ductal and acinar cells ) and an endocrine component ( cells , cells , cells , pancreatic polypeptide - positive [ pp ] cells , and cells ) . the generation of new pancreatic cells is being pursued on several fronts in vitro , including differentiation of induced pluripotent stem cells ( ipscs ) and reprogramming of other pancreatic cell types ( pagliuca and melton , 2013 ) . regenerating pancreatic cells in situ is an attractive alternative to these approaches , driven by evidence of spontaneous cell neogenesis in the adult pancreas ( bonner - weir et al . , 2008 ) and inducible depletion of acinar and islet cells with diphtheria toxin showed that duct cells can give rise to both acinar and endocrine cells ( criscimanna et al . thus , ductal cells in the adult pancreas show a latent propensity for cell generation . adenoviral overexpression of the three transcription factors neurogenin-3 ( ngn3 ) , maf1a , and pdx1 is sufficient to convert adult acinar cells into cells ( zhou et al . however , the capacity for cell neogenesis in the normal adult pancreas , and the regulatory events surrounding it , remain largely unknown . ngn3 is the earliest factor that specifically regulates the development of the endocrine compartment in the embryonic pancreas ( habener et al . , 2000 ) , and transgenic overexpression of ngn3 activates an islet differentiation program in the embryo and in cultured pancreatic ductal cell lines ( heremans et al . in the adult pancreas , ngn3 expression is very limited , but levels rise during cell neogenesis induced by pancreatic duct ligation , where ngn3 is required for cell replenishment ( van de casteele et al . , 2012 ) , and the level and timing of its expression must be precisely controlled to ensure the correct production of cells , but the details of its posttranslational regulation remain elusive . fbw7 ( f - box and wd-40 domain protein 7 ) is the substrate recognition component of an evolutionarily conserved scf ( complex of skp1 , cul1 , and f - box protein)-type ubiquitin ligase . emerging evidence shows that fbw7 controls stem cell self - renewal , cell fate decisions , survival , and multipotency in numerous tissues , including the hematopoietic ( iriuchishima et al . here , we show that fbw7 contributes to the regulation of ngn3 stability , and loss of fbw7 induces a direct ductal - to- cell differentiation in the adult pancreas . our study not only reveals a role for fbw7 in pancreatic cell fate determination and identifies ngn3 as a target of fbw7 but also demonstrates that ductal cells can be induced to alter their identity in the adult pancreas in the absence of injury to the organ with a single genetic change . given the role of fbw7 in controlling cell fate decisions in other organs , we asked whether fbw7 also functions in cell type specification in the pancreas . , 2007 , 2011 ; unexpectedly , scattered cells in the pdx1-cre ; fbw7 ducts showed an enlarged cytoplasm and smaller rounded nuclei when compared with surrounding ductal cells , and more closely resembled islet cells ( figures 1a1d ) . the majority of insulin - positive cells in pdx1-cre ; fbw7 ducts were devoid of the ductal cell marker cytokeratin-19 ( ck19 ) , but costaining of ck19 and insulin was sometimes observed ( figure 1f ) , suggesting an intermediate transition state between ductal and insulin - positive cells . thus , the absence of fbw7 appears to trigger abnormal differentiation of a subset of ductal cells , biasing them toward an endocrine fate . thus , deletion of fbw7 in the pancreas promotes the occurrence of cells coexpressing markers of different pancreatic cell lineages , a cellular phenotype that is not normally observed in the adult pancreas . glut2 was also coexpressed with insulin in the pdx1-cre ; fbw7 aberrant ductal cells ( figures 1h and 1i ) . to test whether fbw7 deletion can induce cell neogenesis in adult mice , and to clarify the cells that give rise to ectopic cells , we combined inducible fbw7 deletion using the r26-creert line with lineage tracing using r26-lsl - yfp . in the fbw7 background ( rfy line , figure 2a ) , tamoxifen treatment results in recombination of the floxed fbw7 alleles in cre - expressing cells concomitantly with activation of yfp expression ( figure 2b ) . despite the high percentage of recombination in the acinar compartment ( figure 2d ) , no insulin positivity was observed in cells with acinar cell morphology , and insulin / amylase double - positive cells could not be detected in the rfy pancreas ( figures 2e2n ) . likewise , direct intrapancreatic injection of 4-oh - tamoxifen into the pancreatic tail resulted in exclusive recombination in acinar cells , and here , no cell neogenesis was observed ( figures s2a s2d ) . therefore , fbw7 deletion in ductal cells , but not in acinar cells , induces the acquisition of cell identity . , 2008 ; western blotting of lysates from whole pdx1-cre ; fbw7 pancreas , in which fbw7 is inactivated in all pancreatic cell types , showed increased p - c - jun and nicd1 protein levels when compared to fbw7 controls , while phosphorylated c - myc and cyclin e levels were not substantially affected ( figure 3a ; quantifications in figures s3a and s3b ) . in the acinar compartment , the loss of fbw7 did not increase p - c - jun or nicd1 in rfy compared with ry mice ( figure s3d ) . p - c - jun fluorescence intensity was increased in pdx1-cre ; fbw7 compared with control ducts ( figures 3b and 3c ) , suggesting that scf(fbw7 ) primarily acts in pancreatic ductal cells . of the known substrates increased by fbw7 loss in the pancreas , none are thought to be involved in cell neogenesis . , 2004 ; we therefore examined the possibility that fbw7 in the pancreas may control the levels of one or more other substrates . transcription factors involved in embryonic cell development include pdx1 , ngn3 , hnf3 , and hnf6 ( zaret , 2008 ) . pdx1 , hnf3 , and hnf6 protein levels were unaltered in pdx1-cre ; fbw7 compared with fbw7 pancreas , but the protein levels of ngn3 were strongly increased ( figure 3d ; quantifications in figures s3e and s3f ) . the increase in ngn3 after fbw7 loss also correlated with higher mrna levels of the ngn3 transcriptional target genes insm1 , heyl , ctgf , and nkx2 - 2 ( swales et al . , 2012 ) when analyzed by quantitative pcr ( qpcr ) ( figure 3e ) , and increased protein levels of the ngn3 transcriptional targets neurod1 and insm1 ( figures 3d , 3f , 3 g , s3e , and s3f ) . ngn3 is a key regulator of endocrine differentiation , making it an excellent candidate for inducing cell neogenesis induced by fbw7 loss . to investigate the mechanism by which fbw7 affects ngn3 , we first analyzed the stability of ngn3 protein using cycloheximide to inhibit protein synthesis . ngn3-hemagglutinin ( ngn3-ha ) coimmunoprecipitated flag - tagged fbw7 isoform- and , to a lesser extent , isoform- ( figure 4b , left panel ; and vice versa , as shown in the right panel ) , and endogenous fbw7 interacted with ngn3-ha ( figure 4c ) . in vitro , wild - type ( wt ) fbw7flag protein complexes promoted efficient ubiquitination of recombinant ngn3 , but the inactive mutant fbw7-fbox flag did not ( welcker et al . , 2004 ) , these data suggest that ngn3 is a substrate of the scf(fbw7 ) ubiquitin ligase . since ngn3 has been shown to be involved in cell neogenesis in the adult pancreas ( al - hasani et al . ngn3 protein , which is undetectable in control adult pancreas , was detected in pdx1-cre ; r26-lsl - ngn3-aa pancreas ( figure 5c ) but not in liver . in order to test whether overexpression of ngn3-aa in the adult pancreatic ducts was sufficient to induce cell reprogramming , we crossed r26-lsl - ngn3-aa mice to ck19-creert mice , in which the expression of tamoxifen - inducible cre - ert protein is driven by the promoter of the ductal marker cytokeratin 19 ( means et al . furthermore , we detected a significant increase in insulin - positive ductal cells in tamoxifen - injected ck19-creert ; r26-lsl - ngn3-aa pancreas ( figures 5j and 5k ) , suggesting that ngn3-aa overexpression in the adult pancreatic duct is sufficient to induce ductal - to- cell conversion . to test whether cells arise as a direct consequence of fbw7 loss in ductal cells , we asked whether loss of fbw7 , specifically in the adult ductal compartment , is sufficient to achieve cell conversion . these data suggest that fbw7 mrna is enriched in ductal cells , in agreement with substrate stabilization predominantly in this cell type after fbw7 loss ( figure 3b ) . 1% of the fbw7 knockout cells if considering 50% recombination efficiency ) was observed in the ducts of cfy mice , while they rarely arose in cy mice ( figure 6 g ) . about 12% of the cfy ducts contained induced cells , typically between one and three cells per duct . it is interesting that 3.8% of cfy ducts contained glucagon - positive cells and 5.5% contained somatostatin - positive cells , while pp or amylase - positive ductal cells were never detected ( figures s4c and s4d ) . thus , deletion of fbw7 in adult pancreatic ductal cells induces conversion of some ductal cells to or cells or , most frequently , to cells . inactivation of fbw7 could trigger resident ductal progenitor cell proliferation followed by redifferentiation or induce direct transdifferentiation . brdu was incorporated in scattered cells in the pancreatic cy ducts and increased in cfy ducts ( figures 6h , 6i , and s4e ) . however , less than 1% of insulin - positive cfy duct cells were labeled after 2 weeks of continuous brdu exposure , beginning directly before fbw7 deletion ( figures 6j , s4f , and s4 g ) . thus fbw7 deletion in the adult pancreatic ducts induces direct conversion of a subset of exocrine ductal cells into endocrine cells , without a requirement for cell proliferation . to explore the functionality of the cells formed after fbw7 deletion , we performed mrna expression profiling of gfp+ sorted cells from tamoxifen - injected cy and cfy mice and compared them to gfp+ cells from mip - gfp mice as a positive control for cells ( figure 7a ) . in agreement with the increase in ngn3 stability in fbw7-deleted cells , cfy gfp+ ductal cells also showed an increase in the expression of reported ngn3 target genes ( chga , insm1 , dll3 , syp , chn1 , heyl , atp2a3 , and pcsk2 ; swales et al . , 2012 ) qpcr analysis confirmed increased mrna expression of the cell marker genes ins2 , gck , pdx1 , and nkx6.1 in cfy gfp+ ductal cells compared with cy gfp+ cells ( figure 7c ) . as well as showing gene expression characteristics of islet cells , insulin - positive cells in the ducts of cfy mice costained with the functional cell markers c - ppt , glut2 , mafa1 , nkx6.1 , pax6 , pc1/3 , pdx1 , urocortin 3 ( ucn3 ) , and isl1 , showing comparable staining to islet cells ( figures 7d , 7e , and s5 ) . an important hallmark of cell function is the ability to release insulin after glucose stimulation . to test this , we subjected gfp+ cells sorted from cy and cfy mouse pancreas to in vitro glucose challenge ( figures 7f and 7 g ) . based on this result , the response to glucose challenge in the converted cells is comparable to that of islet cells ( figure 7i ) . thus , the cells converted after fbw7 loss in the adult pancreatic ducts show both the characteristic marker expression and functionality of mature cells . ectopic expression of combinations of transcription factors can induce changes of cellular fate in adult pancreatic tissue ( zhou et al . in this study we show that fbw7 maintains adult ductal cell fate , as fbw7 inactivation results in transdifferentiation of ductal cells into and cells and , predominantly , cells . the newly formed cells resemble islet cells with regard to cellular morphology , marker gene expression , and insulin secretion in response to glucose challenge . our findings indicate an unexpected plasticity of ductal cells , in which loss of a single gene ( fbw7 ) renders the duct cells multipotent , able to remain exocrine or transdifferentiate into endocrine , , or cell types . fbw7 is a key regulator of stem cell function , as fbw7 inactivation results in increased proliferation and impaired differentiation of hematopoietic , liver , intestinal , and neural progenitor cells ( hoeck et al . , 2011 ; the potent tumor suppressor function of fbw7 is likely to be a direct consequence of deregulated stem cell proliferation and differentiation ( wang et al . however , the role of fbw7 in the pancreatic ducts is distinct from fbw7 function in other organ systems . ductal to endocrine cell transdifferentiation after fbw7 loss occurs in the absence of proliferation , suggesting that the subset of cells that respond to fbw7 deletion in this way does not behave as adult stem cells in other organ systems , requiring cell division before differentiation . rather , fbw7 seems to function in the adult pancreas to constantly maintain cell fate in a subset of ductal cells . we found that the expression and activity of fbw7 in the adult pancreas is enriched in the ductal compartment . pancreatic duct ligation ( pdl ) , combined with lineage tracing of the ductal epithelium , demonstrated that a quarter of new cells formed in response to injury were derived from ductal cells ( inada et al . thus , cell reprogramming induced by fbw7 inactivation appears to be a distinct process from the spontaneous appearance of insulin - positive cells in wt pancreatic ducts and may represent a reawakening of a multipotent state . it is important to note that fbw7-mutant induced cells secrete comparable amounts of insulin after glucose challenge as bona fide cells isolated from mip - gfp mice . therefore , fbw7 loss appears to trigger the conversion of adult pancreatic ductal cells into apparently functional cells . the activity of fbw7 toward different substrates is tissue specific , and our results show that , in the pancreas , c - jun , notch , and ngn3 levels are increased after fbw7 deletion . while c - jun has no reported function in pancreatic cell fate decisions , the notch signaling pathway is thought to inhibit pancreatic endocrine development ( apelqvist et al . , 1999 ; in contrast , we find that endocrine differentiation in the ducts after fbw7 deletion is accompanied by an increase in nicd1 levels . this could imply that notch has different roles in embryonic and adult pancreatic cell differentiation , but it is also possible that increased notch signaling is not required for cell neogenesis after fbw7 loss , and cell neogenesis is induced despite an overall increase in notch levels . , 2008 ) , and our data show that ngn3 stabilization in the ducts is sufficient to induce cell neogenesis ( figure 5 ) . despite its essential role in endocrine differentiation , and the reported ngn3 instability at the protein level , the regulatory mechanisms that control the abundance of ngn3 are not fully understood . in this study , we show that ngn3 is a substrate for scf(fbw7 ) . ngn3 behaves as a canonical fbw7 substrate , containing a gsk3 consensus phosphorylation site that , when mutated , increases the stability of the protein . our data indicate that ngn3 stabilization after fbw7 loss contributes to a transdifferentiation program , inducing ductal cells to differentiate into cells . induction of adult cell neogenesis is desirable for diabetes treatment , and modulation of the fbw7-ngn3 axis could potentially be exploited as a therapeutic approach toward generation of new cells for cell replacement therapies . hct116-fbw7 and hct116-fbw7 cells , and fbw7flag and fbw7flag constructs , have been described elsewhere ( grim et al . the pancreas was excised , processed , and stained as described in the supplemental experimental procedures . pcr primers used to detect the efficiency of recombination of fbw7 and r26 alleles are given in the supplemental experimental procedures . for qpcr analysis of sorted cells , rna was isolated as described for the microarray , and cdna amplification was performed using the quantitect whole transcriptome amplification kit ( qiagen ) . determination of insulin release after glucose challenge was performed as described elsewhere ( banga et al .

a blood specimen was collected from live patients with suspected evd to establish the diagnosis . the who case definition for a suspected case was as follows : any person , alive or dead , suffering or having suffered from a sudden onset of high fever and having had contact with : a suspected , probable or confirmed ebola or marburg case ; a dead or sick animal ( for ebola ) ; a mine ( for marburg ) ; or any person with sudden onset of high fever and at least three of the following symptoms : headaches , lethargy , anorexia / loss of appetite , aching muscles or joints , stomach pain , difficulty swallowing , vomiting , difficulty breathing , diarrhea , hiccups ; or any person with inexplicable bleeding ; or any sudden , inexplicable death [ 6p2 ] . the vast majority of suspected evd cases were managed at the etus in guckdou and macenta , both of which were operated by mdecins sans frontires . the emlab unit also tested suspected evd cases being managed in liberia and sierra leone . patients with suspected evd were retested 12 days later if the result of their first test , performed on a sample collected within the first 3 days after onset of symptoms , was negative . during this time , they were kept in the area of the etu reserved for individuals with suspected evd . another blood sample was collected for analysis from patients who were scheduled for discharge from the etu . in rare cases , specimens of other body fluids , such as urine , were collected from live patients with evd and tested . in addition , emlab tested oral swab specimens from patients who were found dead in their communities ( hereafter , community deaths ) . viral rna was extracted from 50 l of whole blood collected in ethylenediaminetetraacetic acid ( edta)lined tubes ( hereafter , edta whole blood ) or 140 l of cell - free fluid ( plasma , urine , amniotic fluid , or saliva ) , using the qiaamp viral rna mini kit ( qiagen ) . comparison in the field of cycle threshold ( ct ) values , using rt - pcr analysis of 50 l of edta whole blood or 140 l of plasma , revealed equivalent results ( mean ct difference [ sd ] between whole blood and plasma,0.08 2.46 ; n = 12 ) . material on dry swabs was released in 200 l of water by agitation , of which 50 l was processed . after addition of avl buffer and incubation , the tubes were decontaminated and moved out of the glove box for rna extraction . ebov rna was detected using the realstar filovirus screen rt - pcr kit 1.0 ( altona diagnostics ) on a smartcycler ( cepheid ) . the internal control template of the kit was added to the sample before rna extraction , and only results with a valid run control were communicated . whole blood was evaluated for the presence of plasmodium falciparum , plasmodium vivax , plasmodium malariae , and plasmodium ovale antigens by using the binaxnow malaria ( alere ) rapid diagnostic test ( rdt ) in the glove box according to the manufacturers instructions . this test has an analytical sensitivity of 99.5% for a parasitemia level of > 1000 parasites/l blood for p. falciparum , the predominant plasmodium species in guinea . this threshold provides a reasonable compromise between sensitivity and specificity in detecting true severe malaria , rather than severe disease with incidental parasitemia , in areas with moderate - to - high transmission [ 10 , 11 ] . demographic data for patients were provided by mdecins sans frontires , the red cross , the who , national authorities , contact tracing teams , and other partners in field on the laboratory request forms accompanying the sample . name , age , sex , residence , etu patient identifier , sample identifier , sample type , collection date , date of symptom onset , ebov rt - pcr result with corresponding ct value , and malaria rdt results were captured in the emlab database ( excel , microsoft ) and reported on a daily basis to the who and national authorities . the operational emlab sample database was the basis for further analysis to facilitate allocation of various samples to individual patients , validate the demographic information , and document outcome , the emlab database was merged manually with the guinean evd patient database maintained at the who country office in conakry . patient name and sample identifier recorded in both databases were used as primary identifiers for merging ; additional variables were used to verify the match . inconsistencies between the 2 databases and between sample entries for the same patient were resolved , and the data were cleaned as much as possible , using stata 14 ( statacorp ) . on the basis of specific criteria , patients were classified into 3 main categories for analysis : ( 1 ) suspected cases of evd not confirmed by pcr testing ( noncases ) , ( 2 ) patients with pcr - confirmed evd ( evd cases ) , and ( 3 ) community deaths . database entries for patients who could not be assigned to one of these categories because of missing or conflicting data ( n = 200 samples ) and cases managed in liberia and sierra leone ( n = 1083 samples lacking any epidemiological data ) were excluded from the analysis . the final database comprised 2741 patients with 4719 samples collected between 17 march 2014 and 29 march 2015 . in hospitalized patients , only samples collected at admission associations of independent variables with the dichotomous outcome ( survival or death ) were displayed with crude ( unadjusted ) odds ratios ( ors ) . the final model , an interaction term ( the product of 2 interacting categorical variables ) was included to assess outcome associations of one independent variable within levels of another independent variable . to describe the interaction effect , the corresponding effect estimates of the interaction term , used to derive the ors , are provided as well . the national committee of ethics in medical research of guinea and the ethics committee of the medical association of hamburg approved the use of diagnostic leftover samples and corresponding patient data for this study ( permits 11/cners/14 and pv4910 ) . a blood specimen was collected from live patients with suspected evd to establish the diagnosis . the who case definition for a suspected case was as follows : any person , alive or dead , suffering or having suffered from a sudden onset of high fever and having had contact with : a suspected , probable or confirmed ebola or marburg case ; a dead or sick animal ( for ebola ) ; a mine ( for marburg ) ; or any person with sudden onset of high fever and at least three of the following symptoms : headaches , lethargy , anorexia / loss of appetite , aching muscles or joints , stomach pain , difficulty swallowing , vomiting , difficulty breathing , diarrhea , hiccups ; or any person with inexplicable bleeding ; or any sudden , inexplicable death [ 6p2 ] . the vast majority of suspected evd cases were managed at the etus in guckdou and macenta , both of which were operated by mdecins sans frontires . the emlab unit also tested suspected evd cases being managed in liberia and sierra leone . patients with suspected evd were retested 12 days later if the result of their first test , performed on a sample collected within the first 3 days after onset of symptoms , was negative . during this time , they were kept in the area of the etu reserved for individuals with suspected evd . another blood sample was collected for analysis from patients who were scheduled for discharge from the etu . in rare cases , specimens of other body fluids , such as urine , were collected from live patients with evd and tested . in addition , emlab tested oral swab specimens from patients who were found dead in their communities ( hereafter , community deaths ) . viral rna was extracted from 50 l of whole blood collected in ethylenediaminetetraacetic acid ( edta)lined tubes ( hereafter , edta whole blood ) or 140 l of cell - free fluid ( plasma , urine , amniotic fluid , or saliva ) , using the qiaamp viral rna mini kit ( qiagen ) . comparison in the field of cycle threshold ( ct ) values , using rt - pcr analysis of 50 l of edta whole blood or 140 l of plasma , revealed equivalent results ( mean ct difference [ sd ] between whole blood and plasma,0.08 2.46 ; n = 12 ) . material on dry swabs was released in 200 l of water by agitation , of which 50 l was processed . after addition of avl buffer and incubation , the tubes were decontaminated and moved out of the glove box for rna extraction . ebov rna was detected using the realstar filovirus screen rt - pcr kit 1.0 ( altona diagnostics ) on a smartcycler ( cepheid ) . the internal control template of the kit was added to the sample before rna extraction , and only results with a valid run control were communicated . whole blood was evaluated for the presence of plasmodium falciparum , plasmodium vivax , plasmodium malariae , and plasmodium ovale antigens by using the binaxnow malaria ( alere ) rapid diagnostic test ( rdt ) in the glove box according to the manufacturers instructions . this test has an analytical sensitivity of 99.5% for a parasitemia level of > 1000 parasites/l blood for p. falciparum , the predominant plasmodium species in guinea . this threshold provides a reasonable compromise between sensitivity and specificity in detecting true severe malaria , rather than severe disease with incidental parasitemia , in areas with moderate - to - high transmission [ 10 , 11 ] . demographic data for patients were provided by mdecins sans frontires , the red cross , the who , national authorities , contact tracing teams , and other partners in field on the laboratory request forms accompanying the sample . name , age , sex , residence , etu patient identifier , sample identifier , sample type , collection date , date of symptom onset , ebov rt - pcr result with corresponding ct value , and malaria rdt results were captured in the emlab database ( excel , microsoft ) and reported on a daily basis to the who and national authorities . the operational emlab sample database was the basis for further analysis . to facilitate allocation of various samples to individual patients , validate the demographic information , and document outcome , the emlab database was merged manually with the guinean evd patient database maintained at the who country office in conakry . patient name and sample identifier recorded in both databases were used as primary identifiers for merging ; additional variables were used to verify the match . inconsistencies between the 2 databases and between sample entries for the same patient were resolved , and the data were cleaned as much as possible , using stata 14 ( statacorp ) . on the basis of specific criteria , patients were classified into 3 main categories for analysis : ( 1 ) suspected cases of evd not confirmed by pcr testing ( noncases ) , ( 2 ) patients with pcr - confirmed evd ( evd cases ) , and ( 3 ) community deaths . database entries for patients who could not be assigned to one of these categories because of missing or conflicting data ( n = 200 samples ) and cases managed in liberia and sierra leone ( n = 1083 samples lacking any epidemiological data ) were excluded from the analysis . the final database comprised 2741 patients with 4719 samples collected between 17 march 2014 and 29 march 2015 . in hospitalized patients , only samples collected at admission associations of independent variables with the dichotomous outcome ( survival or death ) were displayed with crude ( unadjusted ) odds ratios ( ors ) . the final model , an interaction term ( the product of 2 interacting categorical variables ) was included to assess outcome associations of one independent variable within levels of another independent variable . to describe the interaction effect , ors were calculated for each level of the second independent variable . the corresponding effect estimates of the interaction term , used to derive the ors , are provided as well . the national committee of ethics in medical research of guinea and the ethics committee of the medical association of hamburg approved the use of diagnostic leftover samples and corresponding patient data for this study ( permits 11/cners/14 and pv4910 ) . the cleaned emlab database contained 2178 cases of suspected evd ( 79% ) who attended a hospital / etu and 563 community deaths ( 21% ) . evd was confirmed by pcr in 1231 suspected cases ( 57% ) and 281 community deaths ( 50% ) . most patients originated from the regions of n'zrkor ( 1955 [ 77% ] ) , kankan ( 374 [ 15% ] ) , and faranah ( 196 [ 8% ] ) . table 1.characteristics of individuals included in the analysischaracteristicevd suspected cases in hospitalcommunity deathsoverallebov rt - pcr positiveebov rt - pcr negativeoverallebov rt - pcr positiveebov rt - pcr negativeindividuals2178/2178 ( 100)1231/2178 ( 57)947/2178 ( 43)563281/563 ( 50)282/563 ( 50)female sex1135/2157 ( 53)645/1228 ( 53)490/929 ( 53)260/545 ( 48)136/271 ( 50)124/274 ( 45)age , y , median ( iqr)30 ( 1844)30 ( 1945)30 ( 1842)37 ( 2555)35 ( 2353)40 ( 2556)malaria rdt positive541/1937 ( 28)261/1091 ( 24)280/846 ( 33)not testednot testednot testedfatal outcome769/2049 ( 38)719/1205 ( 60)50/844 ( 6)563 ( 100)281 ( 100)282 ( 100)data are proportion of individuals with the characteristic / no . evaluated ( % ) , unless otherwise indicated.abbreviations : ebov , ebola virus ; evd , ebola virus disease ; iqr , interquartile range ; rdt , rapid diagnostic test ; rt - pcr , reverse transcription polymerase chain reaction . . characteristics of individuals included in the analysis data are proportion of individuals with the characteristic / no . abbreviations : ebov , ebola virus ; evd , ebola virus disease ; iqr , interquartile range ; rdt , rapid diagnostic test ; rt - pcr , reverse transcription polymerase chain reaction . the weekly incidence of ebov rt - pcr positive cases in the hospital and community shows that the outbreak in guckdou progressed in 2 major waves ( march july and august january ; figure 1a ) . however , specifically the community data suggest that the 2 major waves actually consisted of 5 subwaves : march april , may the median of the weekly evd confirmation rate among hospital attendees was 52% ( iqr , 31%66% ) . the median weekly cfr for confirmed cases of evd was 66% ( iqr , 54%79% ) , with a decreasing trend during the outbreak period ( figure 1a ) . among community deaths , the median ct for patients with evd on admission to the hospital showed no trend over time ( figure 1b ) . the coinfection rate with malaria parasites among hospitalized patients with evd also remained at a similar level during the epidemic , with the notable exception of a drop in january 2015 ( figure 1b ) . figure 1.frequency of patients tested by ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) , case - fatality ratios ( cfrs ) , cycle threshold ( ct ) values , and malaria parasite coinfection rate over time . a , ebov rt - pcr results are shown for 2178 patients attending an ebola virus disease ( evd ) treatment unit ( etu ; upper panel ) and 563 patients who died in their communities ( lower panel ) , by week of the deployment period . for patients with evd who were treated at an etu , the cfr is shown in the upper panel . b , ct values on admission and malaria parasite coinfection rate for patients with evd who were treated at an etu . abbreviation : rdt , rapid diagnostic test . frequency of patients tested by ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) , case - fatality ratios ( cfrs ) , cycle threshold ( ct ) values , and malaria parasite coinfection rate over time . a , ebov rt - pcr results are shown for 2178 patients attending an ebola virus disease ( evd ) treatment unit ( etu ; upper panel ) and 563 patients who died in their communities ( lower panel ) , by week of the deployment period . for patients with evd who were treated at an etu , the cfr is shown in the upper panel . b , ct values on admission and malaria parasite coinfection rate for patients with evd who were treated at an etu . abbreviation : rdt , rapid diagnostic test . figure 2 shows the age distributions among hospitalized patients with evd and community deaths . essentially , both distributions show 3 peaks young children , young adults aged 1550 years , and elderly persons although this structure was more pronounced among community deaths . evd confirmation rates in the hospital were comparable among age groups , with a median of 58% ( iqr , 54%62% ) . in the communities , the evd confirmation rate varied more among the age groups ( median , 50% ; iqr , 38%59% ) but had an overall decreasing trend toward higher age . figure 2.age distribution for patients tested by ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) . results are shown for 2153 patients attending an ebola virus disease ( evd ) treatment unit ( a ) and 521 patients who died in their communities ( b ) , by age category . age distribution for patients tested by ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) . results are shown for 2153 patients attending an ebola virus disease ( evd ) treatment unit ( a ) and 521 patients who died in their communities ( b ) , by age category . a malaria rdt was performed for 1937 hospital attendees ( 89% ) , of whom 541 ( 28% ) tested positive . malaria rdt positive patients had a median age of 20 years ( iqr , 735 years ) and thus were younger than malaria rdt negative patients ( median age , 33 years ; iqr , 2445 years ) . the highest malaria prevalence was observed among children aged < 15 years , of whom 220 ( 59% ) had a positive test result . the proportion of malaria rdt positive patients decreased relative to that of evd - positive patients toward the higher age groups ( figure 3a ) . in total , 261 ( 24% ) the cfr for evd showed an age - related effect with 2 maxima and a minimum ( figure 3a ) . maximum cfrs were observed in young children aged < 5 years ( 80% ) and elderly patients aged > 74 years ( 90% ) . the lowest cfr was observed in 1519-year - old young adults ( 39% ) . malaria parasite coinfection increased the cfr in 514-year - old children by > 20% ( figure 3b ) . figure 3.proportion of patients with ebola virus disease ( evd ) and/or malaria , as well as case - fatality ratios ( cfrs ) for evd , according to age and malaria parasite coinfection status . a , the relative frequencies of hospitalized patients with positive results of ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) analysis and/or malaria rapid diagnostic tests ( rdts ) the number of fatalities and total number of patients per age group are shown below the graph . the data set used to generate the graph ( for 1047 patients ) corresponds to the data set used to calculate the regression models in tables 2 and 3 . proportion of patients with ebola virus disease ( evd ) and/or malaria , as well as case - fatality ratios ( cfrs ) for evd , according to age and malaria parasite coinfection status . a , the relative frequencies of hospitalized patients with positive results of ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) analysis and/or malaria rapid diagnostic tests ( rdts ) are shown . the number of fatalities and total number of patients per age group are shown below the graph . the data set used to generate the graph ( for 1047 patients ) corresponds to the data set used to calculate the regression models in tables 2 and 3 . figure 4 shows the distributions of ct values for the first blood sample collected from hospitalized patients who died of or survived evd and for the throat swab collected from community deaths . patients who died in the hospital had a lower median ct on admission , indicating a higher virus load , than survivors ( 18.1 vs 23.2 ) . the median ct for community deaths ( 21.5 ) was 3.4 ct units higher than for patients who died while hospitalized , which may be related to the different clinical material tested . given the difference in ct between people who survived and those who died of evd , we have plotted the cfr versus ct categories to evaluate the relationship between virus load and outcome in more detail ( figure 5 ) . the data show a clear inverse correlation between ct and cfr , indicating that the ct on admission has a strong prognostic value . figure 4.distribution of cycle threshold ( ct ) values on admission to hospital for patients who died of or survived ebola virus disease ( evd ) and for individuals who died of evd in the community . arrows and horizontal bars above the histograms indicate medians and interquartile ranges ( iqrs ) , respectively . figure 5.case-fatality ratios ( cfrs ) among hospitalized patients with ebola virus disease ( evd ) , according to cycle threshold ( ct ) category . distribution of cycle threshold ( ct ) values on admission to hospital for patients who died of or survived ebola virus disease ( evd ) and for individuals who died of evd in the community . arrows and horizontal bars above the histograms indicate medians and interquartile ranges ( iqrs ) , respectively . case - fatality ratios ( cfrs ) among hospitalized patients with ebola virus disease ( evd ) , according to cycle threshold ( ct ) category . the ct values for the first ebola virus reverse transcription polymerase chain reaction positive blood sample from 2527 patients were included in the analysis . the analysis of individual factors indicated that age , malaria parasite coinfection , and virus load may be outcome determinants . variables were ct of the first ebov - positive blood sample , age category ( ie , 04 , 514 , 1545 , and > 45 years ) , and malaria rdt result , stratified within the established age groups . complete data sets for 1047 evd patients were available for analysis . in the crude analysis , patients with evd who had lower ct values on admission and an age of 4 years or 45 years had a higher chance of death ( table 2 ) . malaria had no effect on outcome in the crude analysis . however , as 514-year - old children had the highest malaria parasite coinfection rate and an increased cfr if coinfected with malaria parasites ( figure 3b ) , we assumed an effect of malaria on outcome in this specific age group , which is obliterated in the crude analysis . therefore , an interaction term ( the combination of age category and malaria rdt positivity ) was included in the full regression to model an interaction of age and malaria . in agreement with the crude analysis , the full model revealed a higher chance of fatal outcome in particular within the age categories 4 years and 45 years , irrespective of malaria parasite coinfection ( table 3 ) . consistent with the data shown in figure 3b , an effect of malaria rdt positivity was only seen in children 514 years of age , who had a higher chance of dying if coinfected with malaria parasites . there was no evidence of an impact of malaria parasite coinfection on outcome in the other age groups . the ct was not confounded by these variables and showed a similar effect estimate as in the crude analysis . table 2.crude ( unadjusted ) logistic regression analysis of the association between a fatal outcome and both age and malaria rapid diagnostic test ( rdt ) result among 1047 patients with ebola virus diseasevariablefatal cases / total cases ( % ) crude model , or for fatal outcome ( 95% ci)p valuect of ebov rt - pcr ( increasing , continuous)602/1047 ( 57.5)0.7 ( .7.7)<.001age category , y 0442/55 ( 76.4)2.9 ( 1.45.9).004 51465/123 ( 52.8)1 ( reference ) 1544322/603 ( 53.4)1.0 ( .71.5).91 45173/266 ( 65.0)1.6 ( 1.12.6).02malaria rdt result negative452/798 ( 56.6)1 ( reference ) positive150/249 ( 60.2)1.2 ( .91.5).32abbreviations : ci , confidence interval ; ct , cycle threshold ; ebov , ebola virus ; or , odds ratio ; rt - pcr , reverse transcription polymerase chain reaction . table 3.multivariate logistic regression analysis of the association between age and fatal outcome , by malaria rapid diagnostic test ( rdt ) result , and the effect of malaria per age group ( interaction ) among 1047 patients with ebola virus diseasevariablemalaria rdt negativemalaria rdt positiveinteractionfull model , or for fatal outcome ( 95% ci)p valuefull model , or for fatal outcome ( 95% ci)p valuefull model , or for fatal outcome ( 95% ci)p valuect of ebov rt - pcr ( increasing , continuous)0.7 ( .7.7)<.0010.7 ( .7.7)<.0010.7 ( .7.7)<.001age category , y 0414.3 ( 3.558.5)<.00112.3 ( 3.247.7)<.0010.9 ( .24.8).86 5141 ( reference)4.2 ( 1.710.1).0024.2 ( 1.710.1).002 15443.0 ( 1.55.9).0022.6 ( 1.25.9).020.9 ( .51.5).63 455.0 ( 2.410.5)<.0013.9 ( 1.510.2).0060.8 ( .31.7).52abbreviations : ci , confidence interval ; ct , cycle threshold ; ebov , ebola virus ; or , odds ratio ; rt - pcr , reverse transcription polymerase chain reaction . estimates of the corresponding interaction terms are as follows : age 04 years : or , 0.2 ( 95% ci , .11.4 ; p = .11 ) ; age 514 years : or , 1 ( reference ) ; age 1544 years : or , 0.2 ( 95% ci , .1.6 ; p = .003 ) ; and age 45 years : or , 0.2 ( 95% ci , .1.6 ; p = .006 ) . crude ( unadjusted ) logistic regression analysis of the association between a fatal outcome and both age and malaria rapid diagnostic test ( rdt ) result among 1047 patients with ebola virus disease abbreviations : ci , confidence interval ; ct , cycle threshold ; ebov , ebola virus ; or , odds ratio ; rt - pcr , reverse transcription polymerase chain reaction . multivariate logistic regression analysis of the association between age and fatal outcome , by malaria rapid diagnostic test ( rdt ) result , and the effect of malaria per age group ( interaction ) among 1047 patients with ebola virus disease abbreviations : ci , confidence interval ; ct , cycle threshold ; ebov , ebola virus ; or , odds ratio ; rt - pcr , reverse transcription polymerase chain reaction . estimates of the corresponding interaction terms are as follows : age 04 years : or , 0.2 ( 95% ci , .11.4 ; p = .11 ) ; age 514 years : or , 1 ( reference ) ; age 1544 years : or , 0.2 ( 95% ci , .1.6 ; p = .003 ) ; and age 45 years : or , 0.2 ( 95% ci , .1.6 ; p = .006 ) . during the stay in guckdou , emlab tested specimens from 2741 patients with suspected evd from guinea who either attended a hospital or died in their community . evd was confirmed in 1512 cases , representing 44% of all evd cases reported from the entire country during that period . irrespective of whether patients attended a hospital or died in the community , evd was diagnosed in about 50% of all suspected cases . this high incidence suggests that evd was a major cause of mortality and morbidity in the affected area during the epidemic . nearly 20% of all evd cases died in the community and were diagnosed on the basis of analysis of swabs . the median ct for swabs was 3.4 ct units higher than the admission ct for blood from fatal hospital cases , which roughly corresponds to a 1 log unit difference in virus load . nevertheless , the ct distribution curve for swabs lies well within the detection range of the ebov rt - pcr assay . as the ct values appear to be largely normally distributed , the observed curve suggests that the vast majority of throat swabs contain a virus load that can easily be detected in that assay . thus , a throat swab is a suitable clinical specimen for postmortem evd diagnostic testing . in addition , the epidemic curve for evd community deaths corresponds quite well to the epidemic curve for people hospitalized with evd . both imply that testing of community deaths is a reliable and sensitive method for surveillance . indeed , it has been successfully used in the affected countries in the postoutbreak phase . the cfr remained largely constant or slightly decreased during the epidemic , until december 2014 , when it dropped considerable . the reason for this drop is not clear but may be related to the higher median ct value ( 23 ) and lower malaria parasite coinfection rate ( 3% ) , compared with previous months , and to the initiation of the jiki trial in guckdou during this period , which showed a trend toward efficacy of favipiravir in patients with a ct of 20 . the emlab data have not been collected for scientific purposes , and therefore our results should be interpreted with caution . patients attending the treatment centers are not a random sample from the hospital 's catchment area . attendance at the etu may be influenced by campaigns , reputation of the center , perceived individual disease severity , willingness to be tested , distance to the center , or availability of alternative treatment options . virus load in the field , usually represented by the ct is closely correlated with outcome , as has been observed in previous outbreaks as well as in the west african outbreak [ 14 , 1624 ] . we found a difference between the median ct values of evd fatalities and survivors of 5.1 ct units , roughly corresponding to a difference in virus rna concentration of 1.5 log units . moreover , the ct on admission has strong prognostic value , providing a quantitative estimate of outcome . patients with evd who have a ct of < 17 have a cfr of 95% , and those with a ct of > 26 have a cfr of 15% . between these 2 extremes , the ct is nearly perfectly ( negatively ) correlated with the cfr . malaria parasite coinfections occur in a significant fraction of patients with evd and seem to codetermine the outcome as expected , we found the highest incidence of malaria in children < 15 years of age . consistent with this finding , the coinfection rate of ebola virus and malaria parasites was highest in this age group . however , the interaction between malaria and evd and their effect on outcome seems to be complex . the cfr has a first maximum in children aged < 5 years , followed by a minimum among individuals aged 1019 years and a second maximum among patients aged > 74 years . similar distributions have been observed in other studies from the west african outbreak [ 20 , 23 , 26 ] . it may be that both an immature immune system in conjunction with malaria parasite coinfection leads to the increased cfr in young children , while the high cfr in elderly individuals may be due to comorbidities and a generally reduced health and immune status . the shape of the evd cfr curve by age resembles the u or w shape of the mortality and cfr curves for severe influenza , suggesting that similar host determinants might underlie both distributions . the uneven malaria distribution among the patients with evd and the age dependency of the effect of malaria parasite coinfection has been taken into account by our full regression model . it revealed that both young age ( 4 years ) and malaria parasite coinfection in children aged 514 years are independent risk factors for a fatal outcome . the lack of significant contribution of malaria parasite coinfection in most age groups may be the result of treatment with antimalarials in the etu ( irrespective of age , all patients received artemisinin - based combination therapy ) .

background . a unit of the european mobile laboratory ( emlab ) consortium was deployed to the ebola virus disease ( evd ) treatment unit in guckdou , guinea , from march 2014 through march 2015.methods . the unit diagnosed evd and malaria , using the realstar filovirus screen reverse transcription polymerase chain reaction ( rt - pcr ) kit and a malaria rapid diagnostic test , respectively.results . the cleaned emlab database comprised 4719 samples from 2741 cases of suspected evd from guinea . evd was diagnosed in 1231 of 2178 hospitalized patients ( 57% ) and in 281 of 563 who died in the community ( 50% ) . children aged < 15 years had the highest proportion of ebola virus malaria parasite coinfections . the case - fatality ratio was high in patients aged < 5 years ( 80% ) and those aged > 74 years ( 90% ) and low in patients aged 1019 years ( 40% ) . on admission , rt - pcr analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold ( ct ) than analysis of blood specimens from survivors ( 18.1 vs 23.2 ) . individuals who died in the community had a median ct of 21.5 for throat swabs . multivariate logistic regression on 1047 data sets revealed that low ct values , ages of < 5 and 45 years , and , among children aged 514 years , malaria parasite coinfection were independent determinants of a poor evd outcome.conclusions . virus load , age , and malaria parasite coinfection play a role in the outcome of evd .

METHODS Patients and Specimens Diagnostic Assays Data Management and Statistical Analysis Ethics RESULTS DISCUSSION

during this time , they were kept in the area of the etu reserved for individuals with suspected evd . comparison in the field of cycle threshold ( ct ) values , using rt - pcr analysis of 50 l of edta whole blood or 140 l of plasma , revealed equivalent results ( mean ct difference [ sd ] between whole blood and plasma,0.08 2.46 ; n = 12 ) . ebov rna was detected using the realstar filovirus screen rt - pcr kit 1.0 ( altona diagnostics ) on a smartcycler ( cepheid ) . the internal control template of the kit was added to the sample before rna extraction , and only results with a valid run control were communicated . whole blood was evaluated for the presence of plasmodium falciparum , plasmodium vivax , plasmodium malariae , and plasmodium ovale antigens by using the binaxnow malaria ( alere ) rapid diagnostic test ( rdt ) in the glove box according to the manufacturers instructions . name , age , sex , residence , etu patient identifier , sample identifier , sample type , collection date , date of symptom onset , ebov rt - pcr result with corresponding ct value , and malaria rdt results were captured in the emlab database ( excel , microsoft ) and reported on a daily basis to the who and national authorities . on the basis of specific criteria , patients were classified into 3 main categories for analysis : ( 1 ) suspected cases of evd not confirmed by pcr testing ( noncases ) , ( 2 ) patients with pcr - confirmed evd ( evd cases ) , and ( 3 ) community deaths . the final database comprised 2741 patients with 4719 samples collected between 17 march 2014 and 29 march 2015 . comparison in the field of cycle threshold ( ct ) values , using rt - pcr analysis of 50 l of edta whole blood or 140 l of plasma , revealed equivalent results ( mean ct difference [ sd ] between whole blood and plasma,0.08 2.46 ; n = 12 ) . ebov rna was detected using the realstar filovirus screen rt - pcr kit 1.0 ( altona diagnostics ) on a smartcycler ( cepheid ) . the internal control template of the kit was added to the sample before rna extraction , and only results with a valid run control were communicated . whole blood was evaluated for the presence of plasmodium falciparum , plasmodium vivax , plasmodium malariae , and plasmodium ovale antigens by using the binaxnow malaria ( alere ) rapid diagnostic test ( rdt ) in the glove box according to the manufacturers instructions . name , age , sex , residence , etu patient identifier , sample identifier , sample type , collection date , date of symptom onset , ebov rt - pcr result with corresponding ct value , and malaria rdt results were captured in the emlab database ( excel , microsoft ) and reported on a daily basis to the who and national authorities . on the basis of specific criteria , patients were classified into 3 main categories for analysis : ( 1 ) suspected cases of evd not confirmed by pcr testing ( noncases ) , ( 2 ) patients with pcr - confirmed evd ( evd cases ) , and ( 3 ) community deaths . the final database comprised 2741 patients with 4719 samples collected between 17 march 2014 and 29 march 2015 . the cleaned emlab database contained 2178 cases of suspected evd ( 79% ) who attended a hospital / etu and 563 community deaths ( 21% ) . evd was confirmed by pcr in 1231 suspected cases ( 57% ) and 281 community deaths ( 50% ) . table 1.characteristics of individuals included in the analysischaracteristicevd suspected cases in hospitalcommunity deathsoverallebov rt - pcr positiveebov rt - pcr negativeoverallebov rt - pcr positiveebov rt - pcr negativeindividuals2178/2178 ( 100)1231/2178 ( 57)947/2178 ( 43)563281/563 ( 50)282/563 ( 50)female sex1135/2157 ( 53)645/1228 ( 53)490/929 ( 53)260/545 ( 48)136/271 ( 50)124/274 ( 45)age , y , median ( iqr)30 ( 1844)30 ( 1945)30 ( 1842)37 ( 2555)35 ( 2353)40 ( 2556)malaria rdt positive541/1937 ( 28)261/1091 ( 24)280/846 ( 33)not testednot testednot testedfatal outcome769/2049 ( 38)719/1205 ( 60)50/844 ( 6)563 ( 100)281 ( 100)282 ( 100)data are proportion of individuals with the characteristic / no . evaluated ( % ) , unless otherwise indicated.abbreviations : ebov , ebola virus ; evd , ebola virus disease ; iqr , interquartile range ; rdt , rapid diagnostic test ; rt - pcr , reverse transcription polymerase chain reaction . abbreviations : ebov , ebola virus ; evd , ebola virus disease ; iqr , interquartile range ; rdt , rapid diagnostic test ; rt - pcr , reverse transcription polymerase chain reaction . the weekly incidence of ebov rt - pcr positive cases in the hospital and community shows that the outbreak in guckdou progressed in 2 major waves ( march july and august january ; figure 1a ) . the median weekly cfr for confirmed cases of evd was 66% ( iqr , 54%79% ) , with a decreasing trend during the outbreak period ( figure 1a ) . among community deaths , the median ct for patients with evd on admission to the hospital showed no trend over time ( figure 1b ) . figure 1.frequency of patients tested by ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) , case - fatality ratios ( cfrs ) , cycle threshold ( ct ) values , and malaria parasite coinfection rate over time . a , ebov rt - pcr results are shown for 2178 patients attending an ebola virus disease ( evd ) treatment unit ( etu ; upper panel ) and 563 patients who died in their communities ( lower panel ) , by week of the deployment period . b , ct values on admission and malaria parasite coinfection rate for patients with evd who were treated at an etu . frequency of patients tested by ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) , case - fatality ratios ( cfrs ) , cycle threshold ( ct ) values , and malaria parasite coinfection rate over time . a , ebov rt - pcr results are shown for 2178 patients attending an ebola virus disease ( evd ) treatment unit ( etu ; upper panel ) and 563 patients who died in their communities ( lower panel ) , by week of the deployment period . b , ct values on admission and malaria parasite coinfection rate for patients with evd who were treated at an etu . figure 2.age distribution for patients tested by ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) . results are shown for 2153 patients attending an ebola virus disease ( evd ) treatment unit ( a ) and 521 patients who died in their communities ( b ) , by age category . age distribution for patients tested by ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) . results are shown for 2153 patients attending an ebola virus disease ( evd ) treatment unit ( a ) and 521 patients who died in their communities ( b ) , by age category . malaria rdt positive patients had a median age of 20 years ( iqr , 735 years ) and thus were younger than malaria rdt negative patients ( median age , 33 years ; iqr , 2445 years ) . the highest malaria prevalence was observed among children aged < 15 years , of whom 220 ( 59% ) had a positive test result . maximum cfrs were observed in young children aged < 5 years ( 80% ) and elderly patients aged > 74 years ( 90% ) . figure 3.proportion of patients with ebola virus disease ( evd ) and/or malaria , as well as case - fatality ratios ( cfrs ) for evd , according to age and malaria parasite coinfection status . a , the relative frequencies of hospitalized patients with positive results of ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) analysis and/or malaria rapid diagnostic tests ( rdts ) the number of fatalities and total number of patients per age group are shown below the graph . proportion of patients with ebola virus disease ( evd ) and/or malaria , as well as case - fatality ratios ( cfrs ) for evd , according to age and malaria parasite coinfection status . a , the relative frequencies of hospitalized patients with positive results of ebola virus ( ebov ) reverse transcription polymerase chain reaction ( rt - pcr ) analysis and/or malaria rapid diagnostic tests ( rdts ) are shown . figure 4 shows the distributions of ct values for the first blood sample collected from hospitalized patients who died of or survived evd and for the throat swab collected from community deaths . patients who died in the hospital had a lower median ct on admission , indicating a higher virus load , than survivors ( 18.1 vs 23.2 ) . the median ct for community deaths ( 21.5 ) was 3.4 ct units higher than for patients who died while hospitalized , which may be related to the different clinical material tested . given the difference in ct between people who survived and those who died of evd , we have plotted the cfr versus ct categories to evaluate the relationship between virus load and outcome in more detail ( figure 5 ) . figure 4.distribution of cycle threshold ( ct ) values on admission to hospital for patients who died of or survived ebola virus disease ( evd ) and for individuals who died of evd in the community . figure 5.case-fatality ratios ( cfrs ) among hospitalized patients with ebola virus disease ( evd ) , according to cycle threshold ( ct ) category . distribution of cycle threshold ( ct ) values on admission to hospital for patients who died of or survived ebola virus disease ( evd ) and for individuals who died of evd in the community . case - fatality ratios ( cfrs ) among hospitalized patients with ebola virus disease ( evd ) , according to cycle threshold ( ct ) category . the ct values for the first ebola virus reverse transcription polymerase chain reaction positive blood sample from 2527 patients were included in the analysis . the analysis of individual factors indicated that age , malaria parasite coinfection , and virus load may be outcome determinants . variables were ct of the first ebov - positive blood sample , age category ( ie , 04 , 514 , 1545 , and > 45 years ) , and malaria rdt result , stratified within the established age groups . in the crude analysis , patients with evd who had lower ct values on admission and an age of 4 years or 45 years had a higher chance of death ( table 2 ) . however , as 514-year - old children had the highest malaria parasite coinfection rate and an increased cfr if coinfected with malaria parasites ( figure 3b ) , we assumed an effect of malaria on outcome in this specific age group , which is obliterated in the crude analysis . in agreement with the crude analysis , the full model revealed a higher chance of fatal outcome in particular within the age categories 4 years and 45 years , irrespective of malaria parasite coinfection ( table 3 ) . consistent with the data shown in figure 3b , an effect of malaria rdt positivity was only seen in children 514 years of age , who had a higher chance of dying if coinfected with malaria parasites . table 2.crude ( unadjusted ) logistic regression analysis of the association between a fatal outcome and both age and malaria rapid diagnostic test ( rdt ) result among 1047 patients with ebola virus diseasevariablefatal cases / total cases ( % ) crude model , or for fatal outcome ( 95% ci)p valuect of ebov rt - pcr ( increasing , continuous)602/1047 ( 57.5)0.7 ( .7.7)<.001age category , y 0442/55 ( 76.4)2.9 ( 1.45.9).004 51465/123 ( 52.8)1 ( reference ) 1544322/603 ( 53.4)1.0 ( .71.5).91 45173/266 ( 65.0)1.6 ( 1.12.6).02malaria rdt result negative452/798 ( 56.6)1 ( reference ) positive150/249 ( 60.2)1.2 ( .91.5).32abbreviations : ci , confidence interval ; ct , cycle threshold ; ebov , ebola virus ; or , odds ratio ; rt - pcr , reverse transcription polymerase chain reaction . table 3.multivariate logistic regression analysis of the association between age and fatal outcome , by malaria rapid diagnostic test ( rdt ) result , and the effect of malaria per age group ( interaction ) among 1047 patients with ebola virus diseasevariablemalaria rdt negativemalaria rdt positiveinteractionfull model , or for fatal outcome ( 95% ci)p valuefull model , or for fatal outcome ( 95% ci)p valuefull model , or for fatal outcome ( 95% ci)p valuect of ebov rt - pcr ( increasing , continuous)0.7 ( .7.7)<.0010.7 ( .7.7)<.0010.7 ( .7.7)<.001age category , y 0414.3 ( 3.558.5)<.00112.3 ( 3.247.7)<.0010.9 ( .24.8).86 5141 ( reference)4.2 ( 1.710.1).0024.2 ( 1.710.1).002 15443.0 ( 1.55.9).0022.6 ( 1.25.9).020.9 ( .51.5).63 455.0 ( 2.410.5)<.0013.9 ( 1.510.2).0060.8 ( .31.7).52abbreviations : ci , confidence interval ; ct , cycle threshold ; ebov , ebola virus ; or , odds ratio ; rt - pcr , reverse transcription polymerase chain reaction . estimates of the corresponding interaction terms are as follows : age 04 years : or , 0.2 ( 95% ci , .11.4 ; p = .11 ) ; age 514 years : or , 1 ( reference ) ; age 1544 years : or , 0.2 ( 95% ci , .1.6 ; p = .003 ) ; and age 45 years : or , 0.2 ( 95% ci , .1.6 ; p = .006 ) . crude ( unadjusted ) logistic regression analysis of the association between a fatal outcome and both age and malaria rapid diagnostic test ( rdt ) result among 1047 patients with ebola virus disease abbreviations : ci , confidence interval ; ct , cycle threshold ; ebov , ebola virus ; or , odds ratio ; rt - pcr , reverse transcription polymerase chain reaction . multivariate logistic regression analysis of the association between age and fatal outcome , by malaria rapid diagnostic test ( rdt ) result , and the effect of malaria per age group ( interaction ) among 1047 patients with ebola virus disease abbreviations : ci , confidence interval ; ct , cycle threshold ; ebov , ebola virus ; or , odds ratio ; rt - pcr , reverse transcription polymerase chain reaction . estimates of the corresponding interaction terms are as follows : age 04 years : or , 0.2 ( 95% ci , .11.4 ; p = .11 ) ; age 514 years : or , 1 ( reference ) ; age 1544 years : or , 0.2 ( 95% ci , .1.6 ; p = .003 ) ; and age 45 years : or , 0.2 ( 95% ci , .1.6 ; p = .006 ) . during the stay in guckdou , emlab tested specimens from 2741 patients with suspected evd from guinea who either attended a hospital or died in their community . irrespective of whether patients attended a hospital or died in the community , evd was diagnosed in about 50% of all suspected cases . nearly 20% of all evd cases died in the community and were diagnosed on the basis of analysis of swabs . as the ct values appear to be largely normally distributed , the observed curve suggests that the vast majority of throat swabs contain a virus load that can easily be detected in that assay . the reason for this drop is not clear but may be related to the higher median ct value ( 23 ) and lower malaria parasite coinfection rate ( 3% ) , compared with previous months , and to the initiation of the jiki trial in guckdou during this period , which showed a trend toward efficacy of favipiravir in patients with a ct of 20 . we found a difference between the median ct values of evd fatalities and survivors of 5.1 ct units , roughly corresponding to a difference in virus rna concentration of 1.5 log units . patients with evd who have a ct of < 17 have a cfr of 95% , and those with a ct of > 26 have a cfr of 15% . malaria parasite coinfections occur in a significant fraction of patients with evd and seem to codetermine the outcome as expected , we found the highest incidence of malaria in children < 15 years of age . the cfr has a first maximum in children aged < 5 years , followed by a minimum among individuals aged 1019 years and a second maximum among patients aged > 74 years . it may be that both an immature immune system in conjunction with malaria parasite coinfection leads to the increased cfr in young children , while the high cfr in elderly individuals may be due to comorbidities and a generally reduced health and immune status . the uneven malaria distribution among the patients with evd and the age dependency of the effect of malaria parasite coinfection has been taken into account by our full regression model . it revealed that both young age ( 4 years ) and malaria parasite coinfection in children aged 514 years are independent risk factors for a fatal outcome . the lack of significant contribution of malaria parasite coinfection in most age groups may be the result of treatment with antimalarials in the etu ( irrespective of age , all patients received artemisinin - based combination therapy ) .

hollow and concave nanocrystals ( ncs ) are an interesting class of materials with potential applications in catalysis , energy storage , plasmonics , and medicine . they can be synthesized by implementing the kirkendall effect at the nanoscale , by combining the kirkendall effect with anion exchange , via ostwald ripening , by selective etching of a core region initially used as a template for the growth of a shell of another material , or by selective oxidative etching of initial ncs . also , there are several examples of hollow crystals of noble metals synthesized via galvanic replacement reactions , an approach that was recently extended to transition - metal oxides . considerable work has been done on understanding the different mechanisms involved in the creation of voids : for example , the role of defects and dislocations in the formation of hollow nanocrystals was investigated by tang et al . often , hollow particles prepared via templating routes presented pores and cracks due to the large lattice mismatch between the template and the shell material , and the permeability of these ncs was exploited for the encapsulation of various organic and inorganic species . also , reversible switching from solid to hollow particles . an interesting route to hollow metal oxide nanoparticles , recently discovered by jen - la plante and mokari , is based on a melt fracture mechanism . we report in this work an approach to obtain colloidal hollow and concave ncs that exploits several characteristics of two copper chalcogenides of interest here , namely cu2se and cu2s . henceforth , we refer to them as cu2xse and cu2xs , because even in as - synthesized ncs with nominal 2:1 cu : chalcogen ratio , we expect and observe a slight copper deficiency , due to the ease of formation of cu vacancies in these materials . the relevant aspects are ( i ) cu ions ( and electrons ) can be easily extracted from both cu2xse and cu2xs ncs upon reaction with various oxidizing agents ; ( ii ) cu2xse ncs are more prone to oxidation ( i.e. , to formation of additional cu vacancies ) than cu2xs ncs , which we demonstrate here via competitive etching experiments on mixtures of cu2xse ncs and cu2xs ncs ; ( iii ) cu ions have a high diffusivity in both cu2xse and cu2xs . these aspects helped us to rationalize the oxidative etching behavior of ncs composed of a core of cu2xse buried in a shell of cu2xs ( scheme 1 ) . the initial oxidation must occur at the outer cu2xs shell , with transfer of electrons from the ncs to the oxidizing agent and release of cu ions to the solution . however , the formation of a void in the cu2xse core ( scheme 1b ) supports the hypothesis that this loss of cu ions from the shell is counterbalanced by the diffusion of cu ions from the cu2xse core to the shell , with concomitant oxidation of the core , see scheme 1a . in turn , se species diffuse from the initial core toward the cu2xse cu2xs interface , forming a ternary cu prolonged oxidation leads to enlargement of the void , etching of the shell , and partial shell collapse around the core region ( scheme 1c ) . after the region initially occupied by the cu2xse core is vacated , etching continues on the cu2xs shell . the mechanism of void formation can be classified as a limiting case of the nanoscale kirkendall effect : in fact , different from the classical kirkendall effect , here we are seeing the sole out - diffusion of components from the core of a core / shell nc triggered by the oxidation of the particles . ( a ) beginning of the oxidative process : electrons and cu ions start being extracted from the cu2xs shell of an initial cu2xse / cu2xs core / shell nc . the loss of cu ions from the shell is counterbalanced by the diffusion of cu ions from the cu2xse core to the shell . ( b ) early stage of etching : vacancy coalescence creates a void in the core . ( c ) later stage : the core has been dismantled , etching of the shell begins and part of the shell collapses . cadmium oxide ( cdo , 99.99% ) , copper(i ) chloride ( cucl , 99.999% ) , trioctylphosphine oxide ( topo , 99% ) , trioctylphosphine ( top , 97% ) , and selenium powder ( se , 99.99% ) were purchased from strem chemicals . cadmium chloride ( cdcl2 , 99.99% ) , iodine ( i2 , 99.99% ) , iron(iii ) chloride ( fecl3 , 98% ) , oleylamine ( 70% ) , tetrakis(acetonitrile)copper(i ) hexafluorophosphate , and sulfur powder ( s , 99.9% ) were purchased from sigma - aldrich . octadecylphosphonic acid ( odpa ) and hexylphosphonic acid ( hpa ) were purchased from polycarbon industries . cerium(iv ) ammonium nitrate ( > 98.5% ) , nitric acid ( hno3 , 6769% ) , anhydrous toluene and methanol were purchased from carlo erba reagents . cdo ( 50 mg ) , odpa ( 280 mg ) , hpa ( 80 mg ) , and 3 g of topo were loaded in a reaction flask and then heated to 120 c under vacuum for 1 h. nitrogen was pumped into the reaction mixture , and the temperature was raised to 380 c , after which 2 ml of top were added to the flask . the temperature was allowed to recover to 380 c , then 1 g of a stock solution of se : top ( prepared by dissolving 120 mg of se in 10 ml of top ) was injected rapidly into the flask . the reaction was allowed to run for 10 min with the temperature controller set at 380 c . the heating mantle was then removed to allow the solution to cool down , and the resulting particles were transferred into a glovebox , washed by addition of methanol , centrifuged , and finally redissolved in 5 ml of toluene . the concentration of ncs in the solution ( 5.2 10 m ) was estimated by a combination of the geometrical parameters of the crystals extracted from transmission electron microscopy with inductively coupled plasma optical emission spectroscopy ( icp - oes ) . cdo ( 50 mg ) , cdcl2 ( 12 mg ) , odpa ( 280 mg ) , hpa ( 80 mg ) , and topo ( 3 g ) were loaded in a reaction flask and then heated to 120 c under vacuum for 1 h. nitrogen was then pumped into the reaction mixture , and the temperature was raised to 380 c , after which 2 ml of top was added to it , and the temperature was allowed to recover to 380 c . a solution containing cdse ncs in top and s : top was prepared in the following way : 4.3 10 moles of wurtzite bullet - like cdse seeds dissolved in toluene ( prepared in the previous stage , roughly 1/6 of volume of the mother solution was taken ) were precipitated and redispersed in 1 ml top with assistance of ultrasonication . the resulting solution was then mixed with 0.5 g of a stock solution of s : top ( previously prepared by mixing 960 mg of s in 10 ml of top ) . the reaction was allowed to run for 10 min with the temperature controller set at 380 c . the final product was purified by precipitation via addition of methanol , centrifugation , and redispersion in toluene . in a 25 ml round - bottom flask , cdo ( 60 mg ) , cdcl2 ( 6 mg ) , hpa ( 80 mg ) , odpa ( 290 mg ) and topo ( 3 g ) were mixed and degassed under vacuum at a temperature of 120 c for a period of 1 h. the flask was then backfilled with nitrogen , and the temperature was raised to 380 c at which point the red suspension transformed into a transparent solution . two ml of top was then added , and the temperature was allowed to recover . s : top ( 0.25 g ; prepared at a concentration of 90 mg of s per ml of top in a glovebox ) solution was then injected swiftly , and the reaction was allowed to run for 10 min , which led to formation of cds cores , characterized by darkening of the solution . the temperature of the reaction vessel was then reduced to 350 c , and 0.25 g of se : top ( prepared at a concentration of 72 mg of se per ml of top in a glovebox ) was introduced all at once . the reaction mixture was allowed to stir at this temperature for a further 5 min to allow the growth of the cdsxse1x shell around the cds cores , and afterward the heating mantle was removed to let the reaction mixture to cool down . the final brown product was then extracted in toluene and washed using methanol as the antisolvent . cucl ( 100 mg ) and oleylamine ( 10 ml ) were loaded in a reaction flask and then kept at 130 c for 2 h under vacuum . then , nitrogen was fluxed into the reaction mixture , and the temperature was raised up to 240 c , followed by addition of 32 mg of s dissolved in 2 ml of degassed oleylamine . the final product was transferred to the glovebox , precipitated by addition of methanol , centrifuged , and redispersed in toluene . this was performed in an argon - filled glovebox following previously published protocols . in a standard procedure , a stock solution of the cu(i ) complex was prepared by dissolving 40 mg of tetrakis(acetonitrile)copper(i ) hexafluorophosphate in 5 ml of methanol . then 200 l of the core / shell nc stock solution in toluene ( the estimated concentration of ncs was 8.6 10 m ) was injected in 3 ml of the cu solution , under stirring . after 5 min , the particles were precipitated and resuspended in 3 ml of toluene , followed by addition of the remaining 2 ml of the cu(i ) solution . the particles were precipitated again and redissolved in toluene , with the addition of 25 l of previously degassed oleylamine , to further stabilize them . the etching experiments were run using a 2 10 m solution of cucl2 in methanol . the total amount of cu atoms in the initial solution of ncs dispersed in toluene was instead determined by elemental analysis , via icp - oes ( see later ) . the samples were prepared by adding different amounts of the cucl2 solution ( to reach the desired etching stage ) to 250 l of the ncs solution in a vial . in the last step , additional few tens of l of methanol were added to precipitate the particles , and the vial was then centrifuged at 5000 rpm for 10 min . bright - field transmission electron microscopy ( bf - tem ) images and selected area electron diffraction ( saed ) patterns were acquired with a jeol jem-1011 microscope operating at 100 kv . the saed patterns were acquired at constant camera length after mechanically adjusting the height of the sample to the eucentric height and after carefully focusing the nc images . in the same session , the diffraction camera length and the system distortions were calibrated using a nanocrystalline au sputtered film on a standard c - covered cu grid . the elaboration of saed patterns ( beam - stop removal , centering , azimuthal integration , and background subtraction ) was carried out using the pasad software . energy - filtered tem ( eftem ) , high resolution tem ( hrtem ) , high - angle annular dark - field scanning tem ( haadf - stem ) , and energy - dispersive x - ray spectroscopy ( eds ) analyses were carried out by a jeol jem-2200fs instrument operating at 200 kv , equipped with a ceos image aberration corrector , an in - column energy filter ( type ) , and a bruker quantax 400 system with a 60 mm silicon - drift detector ( sdd ) . for octahedron - in - octapod and bullet - in - rod ncs , eftem maps were acquired with the three - window method at the s - l core - loss edge ( 165 ev onset energy , 20 ev slit width ) and at the se - l edge ( 1436 ev onset energy , 100 ev slit width ) . due to higher residual amount of organics for the diamond - in - diamond ncs and for the reversed rod - in - rod ncs for bf - tem and saed analyses , 50 l of the nc suspensions were deposited on carbon - coated cu grids , for hrtem and eftem analyses on commercial ultrathin c - film - coated cu grids , while for eds analyses carbon - coated ni grids and an analytical be cup holder were used . in view of the acquisition of the tilt series for tomography reconstruction , 50 l of each nc solution were deposited onto a 1.5 1.5 mm c - coated copper grid , mounted on a sample holder for tomography ( fischione model 2030 ) . a single - tilt series of haadf - stem images in the widest possible angular range ( maximum allowed in the employed system : 70 to + 70 ) with a 2 step was acquired for each sample , using the jeol jem-2200fs microscope ( at 200 kv ) . alignment based on cross - correlation and sample tomograms ( without fiducial markers ) was applied to the tilt series using the imod software package . the volume reconstruction was then performed starting from the aligned tilt series via a combination of weighted back - projection ( wbp ) and simultaneous iterative reconstruction technique ( sirt ) using the plug - in tomoj of imagej . after the initial wbp , a few tens of sirt iterations were carried out until convergence was reached . the diffractometer was equipped with a cu rotating anode source and a gbel mirror to obtain a parallel beam and to suppress cu k radiation ( 1.392 ) . to acquire data competitive etching experiments were performed using a 3:1 mixture of cu2xs ( 9.0 m ) and cu2xse ( 1.3 m ) ncs solutions , which were drop cast onto a zero background silicon substrate . after a first xrd acquisition on the pristine sample , the so - obtained film was immersed for 1 min in a 0.02 m solution of cucl2 in methanol and thoroughly rinsed with ethanol prior to xrd characterization of the etched ncs . this was carried out via icp - oes , using a icap 6500 thermo spectrometer . samples were dissolved in hcl / hno3 3:1 ( v / v ) ( carlo erba superpure grade ) and left overnight at room temperature , in order to completely digest the ncs . afterward , milli - q grade water ( 18.3 mcm ) was added ( 7 ml ) to the sample . all chemical analyses performed by icp - oes were affected by a systematic error of about 510% . they were performed on a kratos axis ultra dld spectrometer , using a monochromatic al k source ( 15 kv , 20 ma ) . high - resolution narrow scans were performed at constant pass energy of 10 ev and steps of 0.1 ev . the photoelectrons were detected at a takeoff angle of = 0 with respect to the surface normal . the pressure in the analysis chamber was maintained below 7 10 torr for data acquisition . the data were converted to vamas format and processed using casaxps software , version 2.3.15 . the binding energy ( be ) scale was internally referenced to the c 1s peak ( be for c c = 284.8 ev ) . the cu2xse / cu2xs core / shell ncs studied in this work were prepared from cdse / cds core / shell ncs by complete exchange of the cd cations with cu cations ( see experimental section ) . we started from three different geometries for the cdse core and the cds shell and consequently for the corresponding cu - based ncs : bullet - in - rod , diamond - in - diamond , and octahedron - in - octapod . the bullet - in - rod ncs are reported here for the first time ( see experimental section for details on synthesis ) , while the other two sample structures were prepared following procedures published by us and by other groups . as extensively reported in literature , cation exchange from cdse / cds ncs to cu2xse / cu2xs ncs preserves the anion sublattice as well as the size and the shape of the parent ncs . representative bf - tem images ( and sketches ) of the various cu2xse / cu2xs ncs are shown in figure 1a c . we tested different etching agents on these ncs ( cu , fe , ce , i2 , and hno3 ) . the oxidizing effect of these agents on copper chalcogenide ncs is evident through the emergence of a surface plasmon absorption in the nir region , due to collective excitation of free holes in the valence band , created by extraction of cu(i ) ions and electrons from the ncs upon oxidation ( see figure s1 ) . our starting ncs have already a weak nir absorption , due to slight deviation from the 2:1 copper : chalcogen stoichiometry in the cation - exchanged particles . however , this absorption intensifies and shifts to shorter wavelengths as the density of free carriers increases following progressive oxidation of the particles . shapes and geometries for cu2xse / cu2xs core / shell ncs before and after moderate etching . ( a c ) overview bf - tem images of cu2xse / cu2xs core / shell ncs having various shapes and arrangements of both the inner core and the outer shell , namely ( a ) bullet - in - rod , ( b ) diamond - in - diamond , and ( c ) octahedron - in - octapod , schematic sketches shown in the respective lower right insets . the upper right insets in ( a c ) give the combinations of elemental maps of se and s within selected ncs , evidencing the location of the cu2xse core ( see the additional eds line scan in figure s3 for diamond - in - diamond ncs ) . ( d f ) corresponding samples after moderate etching ( = 2.5 for d , = 0.7 for e , and = 3.2 for f ) . scale bars are 100 nm for ( a ) ( 50 nm in the inset ) and ( d ) , 20 nm for ( b ) and ( e ) , 50 nm for ( c ) and ( f ) . in all the experiments , we discuss here the results of tests carried out using cucl2 , an oxidative etching agent typically used to extract cu from various copper sulfides . similar results were obtained using the other agents , as reported in figure s2 . the experiment consisted of room temperature dropwise additions of a solution of cucl2 in methanol to the stirred solution of ncs under inert atmosphere ( see experimental section for details ) . in the following , the reaction conditions will be identified in terms of ratio ( ) of moles of cu(ii ) ions added as cucl2 ( ncucl2 ) to the moles of cu(i ) ions present in the ncs in the solution ( ncu ) , therefore = ncucl2/ncu . after the addition of cucl2 , the solution quickly turned from brown to a color that eventually evolved to light yellow / green at a later stage of etching . the resulting mixture was left stirring for 5 min before the ncs were isolated by precipitation and redispersion in toluene . figure 1d f reports bf - tem images of samples obtained in experiments run at mild etching conditions ( < 4 ) . they evidence a decreased mass density in the regions where the cu2xse core was initially located , suggesting that the core was preferentially dissolved . in all these experiments on cu2xse / cu2xs core - shell ncs , the cores were always preferentially etched , regardless of the size , shape , and crystal structure of the core as well as shape and crystal structure of the shell . for instance , the cu2xs shell in all samples had a chalcocite structure , in the form of hexagonal close - packed ( -chalcocite ) for octahedron - in - octapod and diamond - in - diamond ncs or exhibiting slight structural displacements with respect to it in the bullet - in - rod ncs ( -chalcocite ) . the cu2xse cores , on the other hand , had cubic structure in the octapods , but the other two samples most likely exhibited a hexagonal -chalcocite - like structure , as found by us in a previous work ( see also discussion later ) . despite these differences among the various core / shell samples , they all behaved similarly when etched . a deeper understanding of the processes induced by the exposure of the ncs to oxidizing environments was attained by compositional and structural analyses of the ncs upon progressive addition of cucl2 . in the following , their peculiar design , with the relatively large cu2xse core and the thick ( about 3 nm around the core ) , uniform and elongated cu2xs shell , as well as their high stability under the electron beam , facilitated the study of their chemical and structural transformations . for these ncs , two consecutive stages can be clearly distinguished in the etching process : ( i ) an initial stage ( = 2 ) , when only the core region of the starting particles is consumed ( compare panels a c in figure 2 ) ; ( ii ) a second stage , which sets in after the initial core is emptied , upon further addition of oxidizing agent ( between = 4 and 9 ) . at this stage the central cavity does not appear to expand further , while the extremities of the ncs start being severely consumed ( see figure 2d ) . an analogous evolution is found for the octahedron - in - octapod ncs ( see figures s4 and s5 ) , while a less clear behavior is observed in the diamond - in - diamond ncs , probably due to the comparatively much smaller size of the cu2xse domain in this type of ncs . longitudinal eds line scans along single bullet - in - rod ncs are displayed in figure 2e g , which refer to three ncs , presented in figure 2a , c , and d , respectively . in ncs from the pristine sample ( figure 2a ) , a cu1.9s0.7se0.3 stoichiometry ( the standard deviations on the average eds quantification of each element are reported in the caption of figure 2 ) was obtained from the eds quantification for the central region . this makes sense since both the cu2xse core and the surrounding cu2xs shell are probed in that case . therefore , the overall cu : chalcogen ratio is around 2:1 throughout the nc , as it should be for a pristine nc . the line scans of the other two samples ( figure 2f , g ) clearly show that , as soon as a lower density region is formed in the region initially occupied by the cu2xse core , a dip is visible in both the se and cu signals from the corresponding region ( for instance , compare panels e and f of figure 2 ) . note that a lower density region appears darker in a haadf - stem image . region with slightly higher density than the rest of the rod in the nc sample shown in figure 2f which was obtained after moderate etching ( = 4 ) . at this stage , however , the overall cu : chalcogen ratio had not changed much ( within the experimental error ) with respect to the pristine ncs : it was still cu1.9s0.7se0.3 when averaged over the central regions and cu2.1s at the tips . at a late stage of etching ( around = 9 , figure 2 g ) , the higher density contour surrounding the void had evolved into a thicker , cage - like region . compositional and structural analyses of cu2xse / cu2xs bullet - in - rod ncs upon progressive etching by cucl2 . ( a d ) bf - tem images ( scale bars 50 nm ) of groups of ncs taken from ( a ) a pristine sample ; ( b ) a sample at initial stage of etching ( = 2 ) ; ( c ) a sample at moderate etching stage ( = 4 ) and at ( d ) a late etching stage ( = 9 ) . ( e g ) haadf - stem images of individual ncs taken from the samples in ( a , c , d ) ( scale bars 20 nm ) : longitudinal eds line scans along the axis of individual ncs for cu ( cyan ) , s ( yellow ) , and se ( magenta ) are superimposed on the corresponding images . the compositional change in the central region of the ncs evolved from cu : s : se = 1.9(0.1):0.7(0.1):0.3(0.1 ) ( sample in a ) to cu : s : se = 2.0(0.3):0.7(0.1):0.3(0.1 ) ( sample in c ) to cu : s : se = 0.8(0.1):0.8(0.1):0.2(0.1 ) ( sample in d ) and in the extremities from cu : s = 2.0(0.2 ) ( sample in a ) to cu : s = 2.1(0.3 ) ( sample in c ) to cu : s = 0.9(0.1 ) ( sample in d ) . ( h ) azimuthally integrated saed patterns of groups of ncs from a pristine sample , and from samples at moderate ( = 4 ) and late ( = 9 ) etching stage . these are compared with powder xrd patterns for -chalcocite ( monoclinic cu2s , pdf card no . 03 - 065 - 3556 ) and -chalcocite - like cu2se ( hexagonal cu2se , see figure s6 and related discussion for details ) . ( i ) hrtem image from the tip of a nc before etching and ( j ) magnified region of the image ; this area , being away from the cu2xse core has cu2s composition , with the ( k ) corresponding fourier transform ( ft ) indicating a -chalcocite structure ; ( l ) hrtem image of a survival nanocage at a late stage of etching and ( m ) magnified region of the image ; ( n ) corresponding ft , matching with covellite . nanocages are the only objects surviving at later stages of etching ( see also figure s5 ) . the composition in these severely etched ncs had changed drastically with respect to the previous two samples : it was cu0.8s0.8se0.2 in the center and cu0.9s at the tips , i.e. , the ncs had lost a considerable fraction of the initial cu but also s and se species , as found by elemental analysis ( see experimental section ) . indeed , such analysis performed on solutions of the starting core / shell ncs and on purified solutions of the nanocages indicated a significant loss of copper ( approximately 40% ) and sulfur ( approximately 20% ) in the cages with respect to the starting sample , while the loss of selenium was within the experimental error ( i.e. , < 10% ) . average structural information on the most significant samples was given by saed patterns from groups of ncs ( figures 2h and s7 ) . the saed of the pristine sample ( = 0 , figure 2h , labeled as pristine ) was consistent with a -chalcocite structure ( monoclinic cu2s ) . in this pattern , as in all the others recorded on etched samples , the diffraction signal from the cu2xse cores in the core / shell ncs was not really appreciable . these cores should have a -chalcocite - like cu2se structure , as reported by us in a previous work . when the bullet - shaped cdse ncs used for growing the bullet - in - rod cdse / cds ncs were indeed directly exchanged to cu2xse ncs , their resulting xrd and saed patterns could be matched to such -chalcocite - like structure ( see figure s6 and related caption ) . at the initial stages of etching of the cu2xse / cu2xs bullet - in - rod ncs ( from = 0 to 4 ) , saed indicated an evolution from the initial -chalcocite structure to a structure similar to -chalcocite , but with a shift to smaller lattice plane spacing ( = 4 in figure 2h ) . this lattice contraction can be rationalized on the basis of out - diffusion of a fraction of cu ions from the ncs . the saed pattern observed on ncs at later stages of etching ( = 9 in figure 2h ) when almost only nanocages were left , presents much broader diffraction peaks , most likely due to the defective structure of the strongly etched ncs at this stage and the consequent small size of the crystallites . in addition , the pattern exhibited combined features of substoichiometric -chalcocite and covellite ( cus ) . the transformation from the initial ncs , dominated by a cu2xs shell with -chalcocite structure , to nanocages having a covellite - like structure was further corroborated by hrtem analyses ( see figures 2i k and 2l n , respectively ) . in particular , the identification of the { 204 } planes of -chalcocite as the transversal ones ( i.e. , perpendicular to the elongation direction of the ncs , see figure 2i k ) substantiates the relatively high - intensity peak at about 3 nm observed in saed patterns for = 0 and 4 ( figure 2h ) , due to the strong anisotropy and the observed orientation of ncs , lying with their length parallel to the support film . it is known from previous works that the oxidation of chalcocite in dilute chloride solutions could lead to the formation of covellite . in the present case instead , most of the cu2s -chalcocite shell of the ncs evolved to a substoichiometric cu2xs phase and subsequently dissolved , with the exception of a region surrounding the initial cu2xse core . apparently , this interfacial region had resisted complete dissolution due to its mixed cu / s / se composition . similarly to bullet - in - rod ncs , mixed cu / s / se hollow nanocages were also observed in the final etching stage for octahedron - in - octapod ncs ( see figures s4 and s5 ) . compositional mapping and structural monitoring of the oxidation process do not provide an unequivocal description of the void formation process in the cu2xse core region . one could argue whether , during etching , channels are formed from the cu2xse core to the outer surface of the ncs , which can facilitate the escape of cu species ( and eventually of se species ) . in order to get insights into the morphological evolution of the ncs , the volume of several bullet - in - rod ncs was reconstructed by means of haadf - stem - tomography . a series of samples collected at different stages of etching were analyzed , as reported in figure 3 . for each column in the figure , the upper panel is a haadf - stem image of a representative nc ( these are labeled from i to iv ) . then panel ( a ) is the reconstructed volume of the same nc in the isosurface representation , whereas panel ( b ) is a longitudinal cut - through , and finally panels ( c and d ) are two transversal cut - through from the core region of the reconstructed volume of the same nc . the nc of figure 3i , ( a d ) was collected at an initial stage of etching ( = 2 ) : the reconstructed volume indicated the presence of a void inside the particle , surrounded by a thick continuous shell . the nc of figure 3ii , ( a d ) was collected at the same stage of etching . in this specific nc , a small central void was connected to the outer surface of the nc via a channel . apart from this case which was quite rare at this stage of etching , the channel formation is probably occurring in ncs in which the misfit strain in the cu2xs shell region around the cu2xse core has partially or totally been released by insertion of misfit dislocations . such a more defective structure , under exposure of the ncs to oxidizing environments , probably presents preferential migration avenues , which could facilitate the nucleation of voids and their coalescence . results of haadf - stem tomography analyses on initial and intermediate etching stages . ( i iv , top ) : haadf - stem images of selected cu2xse / cu2xs bullet - in - rod ncs observed at different stages of etching by cucl2 . the ncs in i and ii and the ones in iii and iv are from samples prepared with = 2 and 4 , respectively . for each stage , the insets present the following : ( a ) the isosurface rendering of the corresponding reconstructed volume of the particle ; ( b ) a longitudinal cut - through ; ( c , d ) transversal cut - through , with planes perpendicular to the elongation direction , and corresponding to the positions indicated in ( b ) by dashed lines . in representative ncs collected at moderate etching stages ( = 4 ) , for example , those of figure 3iii , a d ) , the size of the central void was considerably enlarged , and most particles had one or multiple channels connecting the void to the outer surface of the nc ( see the cross sections images of figure 3iv , c , d ) . in many cases , the channels were so large ( figure 3iii , d ) that the ncs could be considered as concave in shape ( see also figure s8 ) . tomography experiments on the diamond - in - diamond ncs ( figure 1b , e ) were not successful , as the particle structure was altered after few minutes of electron beam irradiation , while we did not attempt them on the octapods ( figure 1c , f ) , due to their more complex geometry which would make it more difficult to disentangle the steps of void / channel formation . however , judging from the bf - tem images of figure 1 , it is unlikely that these two nc types would follow a different evolution from the bullet - in - rod geometry . the initial formation of voids in all the ncs studied in this work , upon exposure to oxidizing agents , is therefore compatible with a mechanism of vacancy coalescence following the preferential creation or accumulation of cu vacancies in the central region of the particles , where the cu2xse core was located . as discussed earlier , oxidation in copper chalcogenides leads to the release of cu ions in solution , with concomitant decrease of their cu content , i.e. , formation of cu vacancies . however , in the core / shell ncs the cu2xse core is not directly exposed to the external environment , and obviously the direct oxidation of the ncs must start at the surface of the ncs , which is made of cu2xs . a plausible mechanism operative here could be the following : as soon as the cu2xs shell starts releasing copper ions in solution due to oxidation , thus creating a cu gradient through the nc , a diffusion of copper ions ( and of electrons ) from the central cu2xse core tries to counterbalance this loss in the shell , giving origin to the empty central region . indeed , as reported in figure s9 , we observed via xps measurements that , at an early etching stage , the cu2xse core is already oxidized ( se species present as both selenides , se , and short polyselenides , se ) , while the cu2xs shell is not yet affected by the oxidation process ( s species present in the form of sulfides , s ) . a further evidence in favor of the stronger tendency of cu2xse over cu2xs to oxidation was demonstrated by oxidation experiments on core / shell ncs with reversed geometry , i.e. , cu2xs ( core)/cu2xse ( shell ) ncs , in which instead the shell was preferentially etched , and no hollow or concave structure were observed ( see figure 4 ) . for these experiments , the starting ncs were prepared by direct synthesis of cd1.6s(core)/cd0.97s0.19se0.81(shell ) rod - in - rod ncs ( see figure s10 ) . the synthesis conditions , described in the experimental section , were the only suitable ones that we could identify for the growth of a continuous se - rich shell on top of the starting cds cores . we do not exclude the possibility to optimize this procedure in the future so as to obtain a pure cdse shell , but for the purpose of these experiments we believe the ability to grow a se - rich shell ( atomic ratio se / s = 4 by eds ) was sufficient to prove our mechanism . the as - prepared rod - in - rod ncs were transformed into cu - based counterparts via cation exchange , in analogy to what reported above for cu2xse ( core)/cu2xs ( shell ) ncs . bf - tem micrographs ( figure 4a ) in combination with the eds compositional maps and quantification of the cation - exchanged ncs ( figure 4c ) indicate a cu3.1s / cu1.8s0.17se0.82 rod - in - rod structure with a se / s atomic ratio of 1.2:1 ( as determined by eds ) . these ncs were then subjected to etching , using the same procedure as for the cu2xse ( core)/cu2xs ( shell ) ncs . in this case , no hollow structures were found . instead , at an etching stage corresponding to = 4 , the ncs appeared thinner than the starting particles ( compare bf - tem images of figure 4a , d ) . most importantly , eds mapping clearly shows that the shell is almost completely missing , with the exception of thin residues observed at one end of some of the sulfide rods ( figure 4f ) . correspondingly , a se / s atomic ratio of 0.14 is found in the etched sample . the results on the reversed ncs show that , whatever the arrangement in a core / shell heterostructure is , the se - rich phase is etched preferentially with respect to the s - rich one . ( a ) overview bf - tem image of the ncs , ( b ) haadf - stem image and ( c ) corresponding eds map for s and se for two ncs . ( d ) overview bf - tem image after etching ( = 4 ) , ( e ) haadf - stem image and ( f ) corresponding eds map for s and se for two ncs in the sample . scale bars are 50 nm in ( a , d ) and 20 nm in ( b , c , e , f ) . both types of core / shell structures described in this work , i.e. , cu2xse(core)/cu2xs(shell ) and cu2xs(core)/cu2xsyse1y(shell ) , were obtained by cation exchange from the corresponding cd - based ncs . elemental analysis ( both by icp - oes and eds ) indicates that there are residual cd ions in the cation - exchanged ncs , although the cd / cu atomic ratio was low ( around 1% or less ) . as the ncs are not entirely cd - free , one might argue if the residual cd ions are preferentially located in one region of the ncs ( for example in the core or in the shell ) , and consequently if this would explain the higher rate of dissolution of the se - rich regions . however , the unambiguous localization of cd in a specific region of the ncs ( core or shell ) was not possible due to the low signal / noise ratio for cd in the corresponding eds spectra . therefore , in order to rule out any possible role of residual cd on the differential etching behavior observed here , we carried out competitive etching experiments on a simpler system , i.e. a mixture of cu2xse and cu2xs ncs , each prepared following standard literature protocols ( with minor modifications , see experimental section ) and not obtained by cation exchange . in these experiments , the as - synthesized cu2xse ncs had cubic structure , while the cu2xs ncs had monoclinic structure , both materials exhibiting slightly substoichiometric compositions . fine details on the shape or crystal structure as well as exact initial composition of these ncs should have little relevance in the etching process , similarly to the case of the core / shell ncs discussed earlier . in the competitive etching experiments , the mixtures of cu2xse and cu2xs ncs were subjected to the same oxidizing treatment as the various core / shell samples discussed above . ( a , b ) haadf - stem images of a mixture of cu2xse and cu2xs ncs ( a ) as - synthesized and ( b ) after exposure of the same grid to cucl2 methanol solution for 10 s ( the inset represents eds mapping of se ( magenta ) and s ( yellow ) for the region within the box ) . ( c ) comparison of xrd spectra for a mixture of cu2se and cu2xs ncs before and after 1 min immersion in a cucl2 solution , compared with berzelianite cu2se ( pdf card no . 230959 ) reference patterns . starting from the non - oxidized samples and at any stage of the etching , combination of haadf - stem imaging and eds compositional mapping allowed us to unambiguously distinguish the cu2xse ncs from the cu2xs ncs in a mixture of them . upon oxidation , the initial cu2xse ncs , much brighter than cu2xs ncs , due to both the higher thickness and the higher atomic weight of se over s ( figure 5a ) , were dissolved , while cu2xs ncs did not undergo an appreciable size variation ( figure 5b ) , as additionally supported by eds elemental mapping ( inset of figure 5b ) . a rather unequivocal proof was additionally provided by xrd analyses of a film containing a mixture of cu2xs and cu2xse ncs before and after immersion of the film in a solution of cucl2 in methanol for 1 min ( figure 5c ) . two main effects are evident from the comparison of the pristine sample and the one immersed in the oxidizing solution : ( i ) the diffraction peaks corresponding to both phases shift to higher 2 values , due to cell contraction , which can be ascribed to cu extraction ; and ( ii ) the intensity of the features related to cu2xse ncs decreases more than the one of the cu2xs - related features . one reason for the stronger tendency of cu2xse ncs toward oxidation with respect to cu2xs ncs might reside in a lower electron affinity of cu2xse ncs over cu2xs ncs . this is supported by xps investigation of the valence band ( vb ) in films of cu2xse and cu2xs ncs , reported in figure s11 : the vb edge for cu2xse ncs lies at slightly higher energy ( + 0.3 ev ) than that of cu2xs ncs , in agreement with previous works on films of the same materials . this is indeed expected for copper chalcogenides , considering that in these materials the top of the valence band has a strong contribution from the chalcogen p orbitals , and that their energy increases following the s 3p se 4p trend . on the other hand , even in pristine core / shell cu2xse / cu2xs nc structures , the free carriers already present should quickly equilibrate the fermi levels of the two domains , leveling off differences in electron affinities between the two domains . a convincing explanation for preferential cu depletion in cu2xse over cu2xs might be sought in the lower average energy of the cu se bond over that of the cu s bond , which should entail a lower formation energy of cu vacancies in cu2xse than in cu2xs . in diatomic gaseous cu - e ( e = s , se ) species , the bond dissociation energy ( i.e. , for the cu e cu + e reaction ) is 255 kjmol for cuse , and 275 kjmol for cus also , the standard enthalpies of formation at room temperature of cu2s and cu2se are 79.5 and 59.4 kjmol , respectively . direct calculations of vacancy formation energies in cu2s and cu2se are made difficult by the many possibilities for both cu and vacancy occupation sites in these materials and by the variety of crystal structures for both cu2s and cu2se ( recently , a cu vacancy formation energy of 1.6 ev in cu2s was experimentally derived by bekenstein et al . ) . on the other hand , vacancy formation energies have been calculated for compound such as cu2znsns4 ( czts ) and cu2znsnse4 ( cztse ) , for cuins2 , cuinse2 , and their corresponding s se alloys as well as for cugas2 and cugase2 . in all cases , cu vacancies were always found to have lower formation energy in the se - based ( or se - rich ) semiconductors than in the s - based ( or s - rich ) ones . we have demonstrated an approach to synthesize hollow and concave nanoparticles based on the preferential oxidation of the cu2xse core region in colloidal core / shell cu2xse / cu2xs ncs . our experiments indicate that the initial effect of oxidation is the creation of a void in the core , due to diffusion of cu ions and electrons from the core to the shell , in an effort to counterbalance the loss of cu and electrons from the shell to the solution phase . this mechanism of void formation is rationalized in terms of a stronger tendency of cu2xse over cu2xs towards oxidation and of the fast cu diffusion in copper chalcogenides . further oxidation of the ncs leads to an expansion of the inner void , until it erodes the initial cu2xse core . at the same time , even the shell starts being etched and partially dismantled , leaving concave particles with cu2xsyse1y composition . future developments in this direction will include the study of this mechanism in other combinations of materials having a high diffusion cation in common . apart from the synthesis of hollow and concave particles , another interesting extension could be the selective etching of a given domain in a segmented heterostructured nc / nanowire , with the consequence of disassembling it into smaller components at precisely defined cutting regions .

hollow and concave nanocrystals find applications in many fields , and their fabrication can follow different possible mechanisms . we report a new route to these nanostructures that exploits the oxidation of cu2xse / cu2xs core / shell nanocrystals with various etchants . even though the cu2xse core is encased in a thick cu2xs shell , the initial effect of oxidation is the creation of a void in the core . this is rationalized in terms of diffusion of cu+ ions and electrons from the core to the shell ( and from there to the solution ) . differently from the classical kirkendall effect , which entails an imbalance between in - diffusion and out - diffusion of two different species across an interface , the present mechanism can be considered as a limiting case of such effect and is triggered by the stronger tendency of cu2xse over cu2xs toward oxidation and by fast cu+ diffusion in copper chalcogenides . as the oxidation progresses , expansion of the inner void erodes the entire cu2xse core , accompanied by etching and partial collapse of the shell , yielding cu2xsyse1y concave particles .

Introduction Experimental Section Results and Discussion Conclusions

hollow and concave nanocrystals ( ncs ) are an interesting class of materials with potential applications in catalysis , energy storage , plasmonics , and medicine . they can be synthesized by implementing the kirkendall effect at the nanoscale , by combining the kirkendall effect with anion exchange , via ostwald ripening , by selective etching of a core region initially used as a template for the growth of a shell of another material , or by selective oxidative etching of initial ncs . considerable work has been done on understanding the different mechanisms involved in the creation of voids : for example , the role of defects and dislocations in the formation of hollow nanocrystals was investigated by tang et al . we report in this work an approach to obtain colloidal hollow and concave ncs that exploits several characteristics of two copper chalcogenides of interest here , namely cu2se and cu2s . the relevant aspects are ( i ) cu ions ( and electrons ) can be easily extracted from both cu2xse and cu2xs ncs upon reaction with various oxidizing agents ; ( ii ) cu2xse ncs are more prone to oxidation ( i.e. the initial oxidation must occur at the outer cu2xs shell , with transfer of electrons from the ncs to the oxidizing agent and release of cu ions to the solution . however , the formation of a void in the cu2xse core ( scheme 1b ) supports the hypothesis that this loss of cu ions from the shell is counterbalanced by the diffusion of cu ions from the cu2xse core to the shell , with concomitant oxidation of the core , see scheme 1a . in turn , se species diffuse from the initial core toward the cu2xse cu2xs interface , forming a ternary cu prolonged oxidation leads to enlargement of the void , etching of the shell , and partial shell collapse around the core region ( scheme 1c ) . after the region initially occupied by the cu2xse core is vacated , etching continues on the cu2xs shell . the mechanism of void formation can be classified as a limiting case of the nanoscale kirkendall effect : in fact , different from the classical kirkendall effect , here we are seeing the sole out - diffusion of components from the core of a core / shell nc triggered by the oxidation of the particles . ( a ) beginning of the oxidative process : electrons and cu ions start being extracted from the cu2xs shell of an initial cu2xse / cu2xs core / shell nc . the loss of cu ions from the shell is counterbalanced by the diffusion of cu ions from the cu2xse core to the shell . ( b ) early stage of etching : vacancy coalescence creates a void in the core . ( c ) later stage : the core has been dismantled , etching of the shell begins and part of the shell collapses . s : top ( 0.25 g ; prepared at a concentration of 90 mg of s per ml of top in a glovebox ) solution was then injected swiftly , and the reaction was allowed to run for 10 min , which led to formation of cds cores , characterized by darkening of the solution . then 200 l of the core / shell nc stock solution in toluene ( the estimated concentration of ncs was 8.6 10 m ) was injected in 3 ml of the cu solution , under stirring . the cu2xse / cu2xs core / shell ncs studied in this work were prepared from cdse / cds core / shell ncs by complete exchange of the cd cations with cu cations ( see experimental section ) . as extensively reported in literature , cation exchange from cdse / cds ncs to cu2xse / cu2xs ncs preserves the anion sublattice as well as the size and the shape of the parent ncs . representative bf - tem images ( and sketches ) of the various cu2xse / cu2xs ncs are shown in figure 1a c . the oxidizing effect of these agents on copper chalcogenide ncs is evident through the emergence of a surface plasmon absorption in the nir region , due to collective excitation of free holes in the valence band , created by extraction of cu(i ) ions and electrons from the ncs upon oxidation ( see figure s1 ) . shapes and geometries for cu2xse / cu2xs core / shell ncs before and after moderate etching . ( a c ) overview bf - tem images of cu2xse / cu2xs core / shell ncs having various shapes and arrangements of both the inner core and the outer shell , namely ( a ) bullet - in - rod , ( b ) diamond - in - diamond , and ( c ) octahedron - in - octapod , schematic sketches shown in the respective lower right insets . the upper right insets in ( a c ) give the combinations of elemental maps of se and s within selected ncs , evidencing the location of the cu2xse core ( see the additional eds line scan in figure s3 for diamond - in - diamond ncs ) . in the following , the reaction conditions will be identified in terms of ratio ( ) of moles of cu(ii ) ions added as cucl2 ( ncucl2 ) to the moles of cu(i ) ions present in the ncs in the solution ( ncu ) , therefore = ncucl2/ncu . they evidence a decreased mass density in the regions where the cu2xse core was initially located , suggesting that the core was preferentially dissolved . in all these experiments on cu2xse / cu2xs core - shell ncs , the cores were always preferentially etched , regardless of the size , shape , and crystal structure of the core as well as shape and crystal structure of the shell . for instance , the cu2xs shell in all samples had a chalcocite structure , in the form of hexagonal close - packed ( -chalcocite ) for octahedron - in - octapod and diamond - in - diamond ncs or exhibiting slight structural displacements with respect to it in the bullet - in - rod ncs ( -chalcocite ) . in the following , their peculiar design , with the relatively large cu2xse core and the thick ( about 3 nm around the core ) , uniform and elongated cu2xs shell , as well as their high stability under the electron beam , facilitated the study of their chemical and structural transformations . for these ncs , two consecutive stages can be clearly distinguished in the etching process : ( i ) an initial stage ( = 2 ) , when only the core region of the starting particles is consumed ( compare panels a c in figure 2 ) ; ( ii ) a second stage , which sets in after the initial core is emptied , upon further addition of oxidizing agent ( between = 4 and 9 ) . an analogous evolution is found for the octahedron - in - octapod ncs ( see figures s4 and s5 ) , while a less clear behavior is observed in the diamond - in - diamond ncs , probably due to the comparatively much smaller size of the cu2xse domain in this type of ncs . the line scans of the other two samples ( figure 2f , g ) clearly show that , as soon as a lower density region is formed in the region initially occupied by the cu2xse core , a dip is visible in both the se and cu signals from the corresponding region ( for instance , compare panels e and f of figure 2 ) . compositional and structural analyses of cu2xse / cu2xs bullet - in - rod ncs upon progressive etching by cucl2 . ( i ) hrtem image from the tip of a nc before etching and ( j ) magnified region of the image ; this area , being away from the cu2xse core has cu2s composition , with the ( k ) corresponding fourier transform ( ft ) indicating a -chalcocite structure ; ( l ) hrtem image of a survival nanocage at a late stage of etching and ( m ) magnified region of the image ; ( n ) corresponding ft , matching with covellite . indeed , such analysis performed on solutions of the starting core / shell ncs and on purified solutions of the nanocages indicated a significant loss of copper ( approximately 40% ) and sulfur ( approximately 20% ) in the cages with respect to the starting sample , while the loss of selenium was within the experimental error ( i.e. in this pattern , as in all the others recorded on etched samples , the diffraction signal from the cu2xse cores in the core / shell ncs was not really appreciable . at the initial stages of etching of the cu2xse / cu2xs bullet - in - rod ncs ( from = 0 to 4 ) , saed indicated an evolution from the initial -chalcocite structure to a structure similar to -chalcocite , but with a shift to smaller lattice plane spacing ( = 4 in figure 2h ) . this lattice contraction can be rationalized on the basis of out - diffusion of a fraction of cu ions from the ncs . in particular , the identification of the { 204 } planes of -chalcocite as the transversal ones ( i.e. in the present case instead , most of the cu2s -chalcocite shell of the ncs evolved to a substoichiometric cu2xs phase and subsequently dissolved , with the exception of a region surrounding the initial cu2xse core . compositional mapping and structural monitoring of the oxidation process do not provide an unequivocal description of the void formation process in the cu2xse core region . one could argue whether , during etching , channels are formed from the cu2xse core to the outer surface of the ncs , which can facilitate the escape of cu species ( and eventually of se species ) . then panel ( a ) is the reconstructed volume of the same nc in the isosurface representation , whereas panel ( b ) is a longitudinal cut - through , and finally panels ( c and d ) are two transversal cut - through from the core region of the reconstructed volume of the same nc . apart from this case which was quite rare at this stage of etching , the channel formation is probably occurring in ncs in which the misfit strain in the cu2xs shell region around the cu2xse core has partially or totally been released by insertion of misfit dislocations . for each stage , the insets present the following : ( a ) the isosurface rendering of the corresponding reconstructed volume of the particle ; ( b ) a longitudinal cut - through ; ( c , d ) transversal cut - through , with planes perpendicular to the elongation direction , and corresponding to the positions indicated in ( b ) by dashed lines . in representative ncs collected at moderate etching stages ( = 4 ) , for example , those of figure 3iii , a d ) , the size of the central void was considerably enlarged , and most particles had one or multiple channels connecting the void to the outer surface of the nc ( see the cross sections images of figure 3iv , c , d ) . in many cases , the channels were so large ( figure 3iii , d ) that the ncs could be considered as concave in shape ( see also figure s8 ) . the initial formation of voids in all the ncs studied in this work , upon exposure to oxidizing agents , is therefore compatible with a mechanism of vacancy coalescence following the preferential creation or accumulation of cu vacancies in the central region of the particles , where the cu2xse core was located . however , in the core / shell ncs the cu2xse core is not directly exposed to the external environment , and obviously the direct oxidation of the ncs must start at the surface of the ncs , which is made of cu2xs . a plausible mechanism operative here could be the following : as soon as the cu2xs shell starts releasing copper ions in solution due to oxidation , thus creating a cu gradient through the nc , a diffusion of copper ions ( and of electrons ) from the central cu2xse core tries to counterbalance this loss in the shell , giving origin to the empty central region . indeed , as reported in figure s9 , we observed via xps measurements that , at an early etching stage , the cu2xse core is already oxidized ( se species present as both selenides , se , and short polyselenides , se ) , while the cu2xs shell is not yet affected by the oxidation process ( s species present in the form of sulfides , s ) . a further evidence in favor of the stronger tendency of cu2xse over cu2xs to oxidation was demonstrated by oxidation experiments on core / shell ncs with reversed geometry , i.e. we do not exclude the possibility to optimize this procedure in the future so as to obtain a pure cdse shell , but for the purpose of these experiments we believe the ability to grow a se - rich shell ( atomic ratio se / s = 4 by eds ) was sufficient to prove our mechanism . the results on the reversed ncs show that , whatever the arrangement in a core / shell heterostructure is , the se - rich phase is etched preferentially with respect to the s - rich one . as the ncs are not entirely cd - free , one might argue if the residual cd ions are preferentially located in one region of the ncs ( for example in the core or in the shell ) , and consequently if this would explain the higher rate of dissolution of the se - rich regions . however , the unambiguous localization of cd in a specific region of the ncs ( core or shell ) was not possible due to the low signal / noise ratio for cd in the corresponding eds spectra . fine details on the shape or crystal structure as well as exact initial composition of these ncs should have little relevance in the etching process , similarly to the case of the core / shell ncs discussed earlier . in the competitive etching experiments , the mixtures of cu2xse and cu2xs ncs were subjected to the same oxidizing treatment as the various core / shell samples discussed above . ( a , b ) haadf - stem images of a mixture of cu2xse and cu2xs ncs ( a ) as - synthesized and ( b ) after exposure of the same grid to cucl2 methanol solution for 10 s ( the inset represents eds mapping of se ( magenta ) and s ( yellow ) for the region within the box ) . starting from the non - oxidized samples and at any stage of the etching , combination of haadf - stem imaging and eds compositional mapping allowed us to unambiguously distinguish the cu2xse ncs from the cu2xs ncs in a mixture of them . two main effects are evident from the comparison of the pristine sample and the one immersed in the oxidizing solution : ( i ) the diffraction peaks corresponding to both phases shift to higher 2 values , due to cell contraction , which can be ascribed to cu extraction ; and ( ii ) the intensity of the features related to cu2xse ncs decreases more than the one of the cu2xs - related features . one reason for the stronger tendency of cu2xse ncs toward oxidation with respect to cu2xs ncs might reside in a lower electron affinity of cu2xse ncs over cu2xs ncs . this is indeed expected for copper chalcogenides , considering that in these materials the top of the valence band has a strong contribution from the chalcogen p orbitals , and that their energy increases following the s 3p se 4p trend . on the other hand , even in pristine core / shell cu2xse / cu2xs nc structures , the free carriers already present should quickly equilibrate the fermi levels of the two domains , leveling off differences in electron affinities between the two domains . a convincing explanation for preferential cu depletion in cu2xse over cu2xs might be sought in the lower average energy of the cu se bond over that of the cu s bond , which should entail a lower formation energy of cu vacancies in cu2xse than in cu2xs . we have demonstrated an approach to synthesize hollow and concave nanoparticles based on the preferential oxidation of the cu2xse core region in colloidal core / shell cu2xse / cu2xs ncs . our experiments indicate that the initial effect of oxidation is the creation of a void in the core , due to diffusion of cu ions and electrons from the core to the shell , in an effort to counterbalance the loss of cu and electrons from the shell to the solution phase . this mechanism of void formation is rationalized in terms of a stronger tendency of cu2xse over cu2xs towards oxidation and of the fast cu diffusion in copper chalcogenides . further oxidation of the ncs leads to an expansion of the inner void , until it erodes the initial cu2xse core . apart from the synthesis of hollow and concave particles , another interesting extension could be the selective etching of a given domain in a segmented heterostructured nc / nanowire , with the consequence of disassembling it into smaller components at precisely defined cutting regions .

melanocortin 1 ( mc1 ) receptor is an attractive molecular target for melanoma imaging because of its overexpression on both murine and human melanoma cells . recently , we have identified a class of tc - labeled -melanocyte stimulating hormone ( -msh ) peptides to target mc1 receptors for melanoma imaging . specifically , the cyclic rxd motifs { arg - x - asp - dtyr - asp , x = gly , ala , val , thr , ser , nle , phe , and dphe } were attached to [ cys , d - phe , arg]-msh313 via a lysine linker to yield rxd - lys-(arg)ccmsh peptides . interestingly , single amino acid at the x position yielded a profound impact on the melanoma targeting and clearance properties of tc - rxd - lys-(arg)ccmsh peptides . for instance , the substitution of gly in tc - rgd - lys-(arg)ccmsh with ala , thr , val , and ser improved the mc1 receptor binding affinities and enhanced the melanoma uptake in b16/f1 melanoma - bearing c57 mice . on the other hand , the substitution of gly in tc - rgd - lys-(arg)ccmsh with nle decreased the mc1 receptor binding affinity . although the substitution of gly in tc - rgd - lys-(arg)ccmsh with phe and dphe increased the mc1 receptor binding affinities , both tc - rfd - lys-(arg)ccmsh and tc - rfd - lys-(arg)ccmsh exhibited much higher liver uptake as compared to tc - rgd - lys-(arg)ccmsh . despite the promising melanoma targeting results , extremely high renal uptake ( 67135% id / g at 2 h postinjection ) is a common issue associated with tc - rxd - lys-(arg)ccmsh peptides . thus , it is desirable to reduce the nonspecific renal uptake of tc - rxd - lys-(arg)ccmsh peptides to facilitate their potential therapeutic applications . in our previous reports , l - lysine co - injection significantly reduced the renal uptake of tc - rxd - lys-(arg)ccmsh peptides by 37%51% at 2 h postinjection without affecting their tumor uptake.l - lysine is a positively charged amino acid . the effect of l - lysine co - injection in reducing the renal uptake indicated that the overall positive charges of tc - rxd - lys-(arg)ccmsh peptides contributed to their nonspecific renal uptake . obviously , the substitution of the positively charged lys linker with a neutral amino acid can decrease the overall charges of tc - rxd - lys-(arg)ccmsh peptides . according to the effect of l - lysine co - injection in decreasing the renal uptake , we hypothesized that the substitution of the lys linker with a neutral -ala linker would decrease the renal uptake of tc - rxd - lys-(arg)ccmsh peptides . to examine our hypothesis , we synthesized six peptides with -ala linkers , namely , peptides 16 . the mc1 receptor binding affinities of these six peptides were examined in b16/f1 melanoma cells . we further determined the biodistribution properties in b16/f1 melanoma - bearing c57 mice for these six tc - peptides . thereafter , we determined the imaging property of tc-4 in b16/f1 melanoma - bearing c57 mice . the peptides were synthesized and purified by reverse phase - high performance liquid chromatography ( rp - hplc ) according to our previously published procedures . the chemical purities of the peptides were greater than 95% after the hplc purification ( table 1 ) . the measured molecular weight was 2123 da for peptide 1 , 2137 da for peptide 2 , 2135 da for peptide 3 , 2107 da for peptide 4 , 2064 da for peptide 5 , and 2080 da for peptide 6 ( table 1 ) . the ic50 value was 2.76 0.51 nm for peptide 1 , 1.56 0.63 nm for peptide 2 , 1.99 0.16 nm for peptide 3 , 0.35 0.01 nm for peptide 4 , 3.34 0.28 nm for peptide 5 , and 3.84 0.71 nm for peptide 6 in b16/f1 melanoma cells , respectively . competitive binding curves of peptide 1 ( , pink ) , peptide 2 ( , black ) , peptide 3 ( , orange ) , peptide 4 ( , blue ) , peptide 5 ( , green ) , and peptide 6 ( , red ) in b16/f1 murine melanoma cells . the ic50 values were 2.76 0.51 nm for peptide 1 , 1.56 0.63 nm for peptide 2 , 1.99 0.16 nm for peptide 3 , 0.35 0.01 nm for peptide 4 , 3.34 0.28 nm for peptide 5 , and 3.84 0.71 nm for peptide 6 . the tc - peptides were separated from their excess nonlabeled peptides by rp - hplc . the specific activities of tc-1 , tc-2 , tc-3 , tc-4 , tc-5 , and tc-6 were 8.62 10 , 8.57 10 , 8.57 10 , 8.68 10 , 8.85 10 , 8.79 10 mbq / g , respectively . the retention times of tc-1 , tc-2 , tc-3 , tc-4 , tc-5 , and tc-6 were 13.1 , 13.0 , 14.1 , 13.7 , 17.0 , and 14.2 min , respectively . all six tc - peptides were stable in mouse serum at 37 c for 24 h ( figure 3 ) . radioactive hplc profiles of tc-1 ( a ) , tc-2 ( b ) , tc-3 ( c ) , tc-4 ( d ) , tc-5 ( e ) , and tc-6 ( f ) in mouse serum after incubation at 37 c for 24 h. the arrows denote the original retention times of tc-1 ( 13.1 min ) , tc-2 ( 13.0 min ) , tc-3 ( 14.1 min ) , tc-4 ( 13.7 min ) , tc-5 ( 17.0 min ) , and tc-6 ( 14.2 min ) , prior to the incubation in mouse serum . the melanoma targeting and pharmacokinetic properties of tc-1 , tc-2 , tc-3 , tc-4 , tc-5 , and tc-6 are shown in tables 27 . all six tc - peptides exhibited similar tumor uptake pattern in b16/f1 melanoma - bearing c57 mice . the highest tumor uptake appeared either at 2 or 4 h postinjection . among these six tc - peptides , tc-4 showed the highest tumor uptake of 15.66 6.19% id / g at 2 h postinjection . the tumor uptake of tc-4 gradually decreased to 14.67 3.81 and 7.79 2.68% id / g at 4 and 24 h postinjection . co - injection of 10 g ( 6.1 nm ) of nonradiolabeled ndp - msh with tc-4 decreased the tumor uptake to 2.43 0.53% id / g at 2 h postinjection , demonstrating that the tumor uptake was mc1 receptor - mediated . kidneys were the excretion routes for all six tc - peptides . among these six tc - peptides , tc-4 showed the lowest renal uptake of 20.18 3.86% id / g at 2 h postinjection . the renal uptake of tc-4 gradually decreased to 19.83 6.34 and 3.92 0.99% id / g at 4 and 24 h postinjection . co - injection of 10 g ( 6.1 nm ) of nonradiolabeled ndp - msh with tc-4 did not significantly reduce the renal uptake ( p > 0.05 ) at 2 h postinjection , indicating that the renal uptake was nonspecific . the substitution of the positively charged lys linker with the neutral -ala dramatically decreased the renal uptake of tc - rxd--ala-(arg)ccmsh peptides . interestingly , further reduction of the overall positive charges of tc-5 and tc-6 did not decrease the renal uptake further as compared to tc-4 . approximately 6878% of tc - peptides cleared through the urinary system by 2 h postinjection , whereas approximately 7786% of tc - peptides washed out through the urinary system by 4 h postinjection . the effect of l - lysine co - injection on the renal uptake of tc-4 at 2 h postinjection is presented in figure 4 . co - injection of 15 mg of l - lysine significantly ( p < 0.05 ) reduced the renal uptake of tc-4 from 20.18 3.86% id / g to 13.06 3.62% id / g without significantly affecting the tumor uptake at 2 h postinjection . the data are presented as percent injected dose / gram or as percent injected dose ( mean sd , n = 4 ) . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-1 with or without ndp - msh peptide blockade at 2 h postinjection . the data are presented as percent injected dose / gram or as percent injected dose ( mean sd , n = 4 ) . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-2 with or without ndp - msh peptide blockade at 2 h postinjection . the data are presented as percent injected dose / gram or as percent injected dose ( mean sd , n = 4 ) . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-3 with or without ndp - msh peptide blockade at 2 h postinjection . the data are presented as percent injected dose / gram or as percent injected dose ( mean sd , n = 4 ) . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-4 with or without ndp - msh peptide blockade at 2 h postinjection . the data are presented as percent injected dose / gram or as percent injected dose ( mean sd , n = 4 ) . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-5 with or without ndp - msh peptide blockade at 2 h postinjection . the data are presented as percent injected dose / gram or as percent injected dose ( mean sd , n = 4 ) . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-6 with or without ndp - msh peptide blockade at 2 h postinjection . effect of l - lysine co - injection on the tumor and kidney uptake of tc-4 at 2 h postinjection . the blue and green columns represent the tumor and renal uptake of tc-4 without and with l - lysine co - injection : ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-4 without and with l - lysine co - injection . because tc-4 showed the highest tumor uptake and the lowest renal uptake than the other five tc - peptides at 2 h postinjection , we further determined the tumor imaging property , specificity of tumor uptake , and urinary metabolites of tc-4 in b16/f1 melanoma - bearing c57 mice . whole - body spect / ct image at 2 h postinjection is presented in figure 5 . flank b16/f1 melanoma lesions were clearly visualized by spect using tc-4 as an imaging probe . the spect image of tumor accurately matched its anatomical location obtained in the ct image . the spect image showed high contrast of tumor to normal organ except for kidneys , which was consistent with the biodistribution results . as shown in figure 5 , the tumor uptake was blocked by unlabeled ndp - msh , demonstrating that the tumor uptake was receptor - mediated . the urinary metabolites of tc-4 at 2 h postinjection are shown in figure 6 . representative whole - body spect / ct image of b16/f1 melanoma - bearing c57 mice 2 h after injection of tc-4 without ( a ) and with ( b ) peptide blockade . flank melanoma lesions ( t ) are highlighted with an arrow on the image . the arrow denotes the original retention time of tc-4 prior to tail vein injection . in our previous reports , we have found the importance of single amino acid at the x position in the tumor targeting properties of tc - rxd - lys-(arg)ccmsh peptides in b16/f1 melanoma - bearing c57 mice . specifically , the substitution of gly in tc - rgd - lys-(arg)ccmsh with ala , thr , val , and ser improved the mc1 receptor binding affinities and enhanced the melanoma uptake in b16/f1 melanoma - bearing c57 mice . despite the promising melanoma targeting results associated with tc - rxd - lys-(arg)ccmsh peptides , it is desirable to reduce the nonspecific renal uptake ( 67135% id / g at 2 h postinjection ) of tc - rxd - lys-(arg)ccmsh peptides to facilitate their potential therapeutic applications . in this study , we substituted the positively charged lys linker with the neutral -ala linker to determine whether such linker change could reduce the renal uptake of tc - rxd--ala-(arg)ccmsh peptides . furthermore , we replaced the rad moiety with nad and ead moieties to examine whether the further reduction of the overall positive changes of tc - nad--ala-(arg)ccmsh and tc - ead--ala-(arg)ccmsh could decrease their renal uptake further . the substitution of lys linker with -ala linker slightly affected the receptor binding affinities of rxd--ala-(arg)ccmsh peptides . the receptor binding affinities of rxd--ala-(arg)ccmsh peptides were approximately 2-fold weaker than rxd - lys-(arg)ccmsh peptides , respectively . despite the fact that the -his - dphe - arg - trp- motif is the binding moiety for the mc1 receptor , the decrease in receptor binding affinity with the -ala linker substitution indicated that the linker might somehow interact with the receptor binding moiety . the decrease in receptor binding affinities of rxd--ala-(arg)ccmsh peptides also resulted in the reduction in tumor uptake of tc - rxd--ala-(arg)ccmsh peptides by 2145% in b16/f1 melanoma - bearing c57 mice . specifically , the tumor uptake of tc-4 was 79% of the tumor uptake of tc - rad - lys-(arg)ccmsh at 2 h postinjection . the substitution of lys linker with -ala linker dramatically decreased the renal uptake of tc - rxd--ala-(arg)ccmsh peptides by 6479% in b16/f1 melanoma - bearing c57 mice . for instance , the renal uptake of tc-4 was only 22% of the renal uptake of tc - rad - lys-(arg)ccmsh at 2 h postinjection . it is worthwhile to note that there is a positively charged arg residue in the rad moiety of tc-4 . thus , we were interested in whether the replacement of arg with nle ( neutral ) and glu ( negatively charged ) could further decrease the renal uptake of tc-5 and tc-6 as compared to tc-4 . interestingly , the replacement of arg with nle and glu did not further decrease the renal uptake of tc-5 and tc-6 as compared to tc-4 . clearly , the tumor targeting and clearance properties of tc-4 were more favorable than the other tc - peptides investigated in this study . . the b16/f1 melanoma lesions could be clearly visualized by spect / ct using tc-4 as an imaging probe . at the present time , tc-(arg)ccmsh and tc(edda)-hynic - ggnle - cycmshhex were reported as promising cyclic imaging probes for melanoma . remarkably , tc-4 displayed comparably high melanoma uptake ( 14.67 3.81% id / g ) as tc-(arg)ccmsh and tc(edda)-hynic - ggnle - cycmshhex at 4 h postinjection . however , the renal uptake of tc-4 was higher than those of tc-(arg)ccmsh ( 11.66 1.44% id / g ) and tc(edda)-hynic - ggnle - cycmshhex ( 7.52 0.96% id / g ) . the difference in renal uptake was likely due to the structural differences among these tc - peptides . interestingly , we found that l - lysine co - injection significantly reduced the renal uptake of tc-4 by 35% at 2 h postinjection without affecting its tumor uptake significantly ( figure 4 ) . therefore , l - lysine co - injection could be utilized to further decrease the renal uptake of re - rad - lys-(arg)ccmsh to facilitate its potential therapeutic application . the substitution of the lys linker with the -ala linker dramatically decreased the renal uptake of tc - rxd--ala-(arg)ccmsh peptides . among these six tc - peptides , tc-4 exhibited the highest tumor uptake and the lowest renal uptake at 2 h postinjection . the replacement of arg with nle and glu did not further decrease the renal uptake of tc-5 and tc-6 as compared to tc-4 . the tumor targeting and clearance properties of tc-4 highlighted it as a lead peptide for future studies . i - tyr-[nle , dphe]--msh { i-(tyr)-ndp - msh } was obtained from perkinelmer , inc . all other chemicals used in this study were purchased from thermo fischer scientific ( waltham , ma ) and used without further purification . b16/f1 murine melanoma cells were obtained from american type culture collection ( manassas , va ) . six new peptides were synthesized using fluorenylmethyloxycarbonyl ( fmoc ) chemistry according to our previously published procedures with slight modification on sieber amide resin by an advanced chemtech multiple - peptide synthesizer ( louisville , ky ) . briefly , 70 mol of sieber amide resin and 210 mol of fmoc - protected amino acids were used for the synthesis . intermediate scaffolds of h2n - arg(pbf)-ser / thr / val - asp(otbu)-dtyr(tbu)-asp(o-2-phenylisopropyl)--ala - cys(trt)-cys(trt)-glu(otbu)-his(trt)-dphe - arg(pbf)-trp(boc)-cys(trt)-arg(pbf)-pro - val and h2n - arg(pbf)/nle / glu(otbu)/-ala - asp(otbu)-dtyr(tbu)-asp(o-2-phenylisopropyl)--ala - cys(trt)-cys(trt)-glu(otbu)-his(trt)-dphe - arg(pbf)-trp(boc)-cys(trt)-arg(pbf)-pro - val were synthesized on sieber amide resin . the protecting group of 2-phenylisopropyl of each scaffold was removed , and each peptide was cleaved from the resin treating with a mixture of 2.5% of trifluoroacetic acid ( tfa ) and 5% of triisopropylsilane . after the precipitation with ice - cold ether and characterization by ms , each protected peptide was dissolved in h2o / ch3cn ( 50:50 ) and lyophilized to remove the reagents such as tfa and triisopropylsilane . each protected peptide was further cyclized by coupling the carboxylic group from the asp with the amino group from the arg , nle , or glu at the n - terminus . the cyclization reaction was achieved by overnight reaction in dimethylformamide ( dmf ) using benzotriazole-1-yl - oxy - tris - pyrrolidinophosphonium hexafluorophosphate ( pybop ) as a coupling agent in the presence of n , n - diisopropylethylamine ( dipea ) . the protecting groups were totally removed by treating with a mixture of tfa , thioanisole , phenol , water , ethanedithiol , and triisopropylsilane ( 87.5:2.5:2.5:2.5:2.5:2.5 ) for 2 h at room temperature ( 25 c ) . each peptide was precipitated and washed with ice - cold ether four times , purified by rp - hplc , and characterized by liquid chromatography mass spectrometry ( lc ms ) . the chemical purity of each peptide was determined by waters rp - hplc instrument ( milford , ma ) on a grace vydac c-18 reverse phase analytic column ( deerfield , il ) using a 20 min gradient of 1828% acetonitrile in 20 mm hcl aqueous solution at a flow rate of 1 ml / min . the ic50 values of the peptides for the mc1 receptor were determined in b16/f1 melanoma cells . the b16/f1 cells were seeded into a 24-well cell culture plate at a density of 2.5 10 cells / well and incubated at 37 c overnight . after being washed with binding medium { modified eagle s medium with 25 mm n-(2-hydroxyethyl)piperazine - n-(2-ethanesulfonic acid ) ( hepes ) , ph 7.4 , 0.2% bovine serum albumin ( bsa ) , 0.3 mm 1,10-phenathroline } , the cells were incubated at 25 c for 2 h with approximately 30 000 counts per minute ( cpm ) of i-(tyr)-ndp - msh in the presence of increasing concentrations ( 1010 m ) of each peptide in 0.3 ml of binding medium . the cells were rinsed twice with 0.5 ml of ice - cold , ph 7.4 , 0.2% bsa/0.01 m phosphate buffered saline ( pbs ) to remove any unbound radioactivity and lysed in 0.5 ml of 1 m naoh for 5 min . the activities associated with the cells were measured in a wallac 2480 automated counter ( perkinelmer , nj ) . the ic50 value for each peptide was calculated using prism software ( graphpad software , la jolla , ca ) . the standard deviation of ic50 value was generated by two independent experiments in triplicate for each peptide . the peptides were labeled with tc via a direct reduction reaction using sncl2 as a reducing agent . briefly , 10 l of 1 mg / ml sncl2 in 0.1 m hcl , 40 l of 0.5 m nh4oac ( ph 5.2 ) , 100 l of 0.2 m na2 tartate ( ph 9.2 ) , 100 l of fresh tco4 solution ( 3774 mbq ) , and 10 l of 1 mg / ml of each peptide in aqueous solution were added into a reaction vial and incubated at 25 c for 20 min to form the tc - labeled peptide . each tc - peptide was purified to a single species by waters rp - hplc ( milford , ma ) on a grace vydac c-18 reverse phase analytic column ( deerfield , il ) using a 20 min gradient of 1828% acetonitrile in 20 mm hcl aqueous solution at a flow rate of 1 ml / min . each purified peptide was purged with n2 gas for 20 min to remove the acetonitrile . the ph of final peptide solution was adjusted to 7.4 with 0.1 n naoh and sterile normal saline for stability , biodistribution , and imaging studies . the serum stabilities of tc-1 , tc-2 , tc-3 , tc-4 , tc-5 , and tc-6 were determined by incubation in mouse serum at 37 c for 24 h and monitored for degradation by rp - hplc . briefly , 100 l of each hplc - purified peptide solution ( 7.4 mbq ) was added into 100 l of mouse serum ( sigma - aldrich corp , st . louis , mo ) and incubated at 37 c for 24 h. after the incubation , 200 l of a mixture of ethanol and acetonitrile ( v : v = 1:1 ) was added to precipitate the serum proteins . the resulting mixture was centrifuged at 16000 g for 5 min to collect the supernatant . the supernatant was purged with n2 gas for 30 min to remove the ethanol and acetonitrile . the resulting sample was mixed with 500 l of water and injected into rp - hplc for analysis using the gradient described above . all the animal studies were conducted in compliance with institutional animal care and use committee approval ( 12 - 100851-hsc ) . the biodistribution properties of the tc - peptides were determined in b16/f1 melanoma - bearing c57 female mice ( harlan , indianapolis , in ) . each c57 mouse was subcutaneously inoculated on the right flank with 1 10 b16/f1 cells . the weight of tumors reached approximately 0.2 g at 10 days after cell inoculation . each melanoma - bearing mouse was injected with 0.037 mbq of each tc - peptide via the tail vein . groups of four mice were sacrificed at 0.5 , 2 , 4 , and 24 h postinjection , and tumors and organs of interest were harvested , weighed , and counted . the specificity of tumor uptake was determined by co - injecting each tc - peptide with 10 g ( 6.1 nmol ) of unlabeled ndp - msh at 2 h postinjection . the effect of l - lysine co - injection on the renal uptake of tc-4 was examined in b16/f1 melanoma - bearing c57 . a group of four mice were injected with an aqueous mixture of 0.037 mbq of tc-4 and 15 mg of l - lysine . the mice were sacrificed at 2 h postinjection , and tumor and kidneys were harvested , weighed , and counted . tc-4 was the lead peptide because of its highest tumor uptake and the lowest renal uptake . thus , we further determined the melanoma imaging property of tc-4 and the specificity of melanoma uptake . approximately 3.74.1 mbq of tc-4 with or without 10 g ( 6.1 nmol ) of unlabeled ndp - msh was injected into b16/f1 melanoma - bearing c57 mice via the tail vein , respectively . the mice were euthanized for small animal spect / ct ( nano - spect / ct , bioscan , washington , dc ) imaging 2 h postinjection . the 9 min ct imaging was immediately followed by the spect imaging of whole body . reconstructed data from spect and ct were visualized and co - registered using invivoscope ( bioscan , washington , dc ) . approximately 3.7 mbq of tc-4 was injected into a b16/f1 melanoma - bearing c57 mouse via the tail vein to determine the urinary metabolites . the collected urine sample was centrifuged at 16000 g for 5 min before the hplc analysis . a 20 min gradient of 1626% acetonitrile/20 mm hcl with a flow rate of 1 ml / min was used for urine analysis . statistical analysis was performed using the student s t test for unpaired data to determine the significance of differences in tumor and kidney uptake with / without peptide blockade in biodistribution studies described above .

the purpose of this study was to examine whether the substitution of the lys linker with the -ala could reduce the renal uptake of 99mtc - labeled arg - x - asp - conjugated and x - ala - asp - conjugated -melanocyte stimulating hormone ( -msh ) peptides . rsd--ala-(arg11)ccmsh ( 1 ) { c[arg - ser - asp - dtyr - asp]--ala - cys - cys - glu - his - dphe - arg - trp - cys - arg - pro - val - nh2 } , rtd--ala-(arg11)ccmsh ( 2 ) , rvd--ala-(arg11)ccmsh ( 3 ) , rad--ala-(arg11)ccmsh ( 4 ) , nad--ala-(arg11)ccmsh ( 5 ) , and ead--ala-(arg11)ccmsh ( 6 ) peptides were synthesized and evaluated for their melanocortin 1 ( mc1 ) receptor binding affinities in b16/f1 melanoma cells . the biodistribution of their 99mtc - conjugates were determined in b16/f1 melanoma - bearing c57 mice . the substitution of the lys linker with -ala linker dramatically reduced the renal uptake of all six 99mtc - peptides . 99mtc-4 exhibited the highest melanoma uptake ( 15.66 6.19% id / g ) and the lowest kidney uptake ( 20.18 3.86% id / g ) among these 99mtc - peptides at 2 h postinjection . the b16/f1 melanoma lesions could be clearly visualized by single photon emission computed tomography ( spect)/ct using 99mtc-4 as an imaging probe .

Introduction Results Discussion Conclusions Experimental Section

melanocortin 1 ( mc1 ) receptor is an attractive molecular target for melanoma imaging because of its overexpression on both murine and human melanoma cells . recently , we have identified a class of tc - labeled -melanocyte stimulating hormone ( -msh ) peptides to target mc1 receptors for melanoma imaging . specifically , the cyclic rxd motifs { arg - x - asp - dtyr - asp , x = gly , ala , val , thr , ser , nle , phe , and dphe } were attached to [ cys , d - phe , arg]-msh313 via a lysine linker to yield rxd - lys-(arg)ccmsh peptides . for instance , the substitution of gly in tc - rgd - lys-(arg)ccmsh with ala , thr , val , and ser improved the mc1 receptor binding affinities and enhanced the melanoma uptake in b16/f1 melanoma - bearing c57 mice . on the other hand , the substitution of gly in tc - rgd - lys-(arg)ccmsh with nle decreased the mc1 receptor binding affinity . although the substitution of gly in tc - rgd - lys-(arg)ccmsh with phe and dphe increased the mc1 receptor binding affinities , both tc - rfd - lys-(arg)ccmsh and tc - rfd - lys-(arg)ccmsh exhibited much higher liver uptake as compared to tc - rgd - lys-(arg)ccmsh . despite the promising melanoma targeting results , extremely high renal uptake ( 67135% id / g at 2 h postinjection ) is a common issue associated with tc - rxd - lys-(arg)ccmsh peptides . thus , it is desirable to reduce the nonspecific renal uptake of tc - rxd - lys-(arg)ccmsh peptides to facilitate their potential therapeutic applications . in our previous reports , l - lysine co - injection significantly reduced the renal uptake of tc - rxd - lys-(arg)ccmsh peptides by 37%51% at 2 h postinjection without affecting their tumor uptake.l - lysine is a positively charged amino acid . obviously , the substitution of the positively charged lys linker with a neutral amino acid can decrease the overall charges of tc - rxd - lys-(arg)ccmsh peptides . according to the effect of l - lysine co - injection in decreasing the renal uptake , we hypothesized that the substitution of the lys linker with a neutral -ala linker would decrease the renal uptake of tc - rxd - lys-(arg)ccmsh peptides . the mc1 receptor binding affinities of these six peptides were examined in b16/f1 melanoma cells . we further determined the biodistribution properties in b16/f1 melanoma - bearing c57 mice for these six tc - peptides . thereafter , we determined the imaging property of tc-4 in b16/f1 melanoma - bearing c57 mice . the peptides were synthesized and purified by reverse phase - high performance liquid chromatography ( rp - hplc ) according to our previously published procedures . the chemical purities of the peptides were greater than 95% after the hplc purification ( table 1 ) . the ic50 value was 2.76 0.51 nm for peptide 1 , 1.56 0.63 nm for peptide 2 , 1.99 0.16 nm for peptide 3 , 0.35 0.01 nm for peptide 4 , 3.34 0.28 nm for peptide 5 , and 3.84 0.71 nm for peptide 6 in b16/f1 melanoma cells , respectively . competitive binding curves of peptide 1 ( , pink ) , peptide 2 ( , black ) , peptide 3 ( , orange ) , peptide 4 ( , blue ) , peptide 5 ( , green ) , and peptide 6 ( , red ) in b16/f1 murine melanoma cells . all six tc - peptides were stable in mouse serum at 37 c for 24 h ( figure 3 ) . all six tc - peptides exhibited similar tumor uptake pattern in b16/f1 melanoma - bearing c57 mice . the highest tumor uptake appeared either at 2 or 4 h postinjection . among these six tc - peptides , tc-4 showed the highest tumor uptake of 15.66 6.19% id / g at 2 h postinjection . the tumor uptake of tc-4 gradually decreased to 14.67 3.81 and 7.79 2.68% id / g at 4 and 24 h postinjection . co - injection of 10 g ( 6.1 nm ) of nonradiolabeled ndp - msh with tc-4 decreased the tumor uptake to 2.43 0.53% id / g at 2 h postinjection , demonstrating that the tumor uptake was mc1 receptor - mediated . among these six tc - peptides , tc-4 showed the lowest renal uptake of 20.18 3.86% id / g at 2 h postinjection . the renal uptake of tc-4 gradually decreased to 19.83 6.34 and 3.92 0.99% id / g at 4 and 24 h postinjection . co - injection of 10 g ( 6.1 nm ) of nonradiolabeled ndp - msh with tc-4 did not significantly reduce the renal uptake ( p > 0.05 ) at 2 h postinjection , indicating that the renal uptake was nonspecific . the substitution of the positively charged lys linker with the neutral -ala dramatically decreased the renal uptake of tc - rxd--ala-(arg)ccmsh peptides . interestingly , further reduction of the overall positive charges of tc-5 and tc-6 did not decrease the renal uptake further as compared to tc-4 . approximately 6878% of tc - peptides cleared through the urinary system by 2 h postinjection , whereas approximately 7786% of tc - peptides washed out through the urinary system by 4 h postinjection . the effect of l - lysine co - injection on the renal uptake of tc-4 at 2 h postinjection is presented in figure 4 . co - injection of 15 mg of l - lysine significantly ( p < 0.05 ) reduced the renal uptake of tc-4 from 20.18 3.86% id / g to 13.06 3.62% id / g without significantly affecting the tumor uptake at 2 h postinjection . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-1 with or without ndp - msh peptide blockade at 2 h postinjection . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-2 with or without ndp - msh peptide blockade at 2 h postinjection . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-3 with or without ndp - msh peptide blockade at 2 h postinjection . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-4 with or without ndp - msh peptide blockade at 2 h postinjection . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-5 with or without ndp - msh peptide blockade at 2 h postinjection . ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-6 with or without ndp - msh peptide blockade at 2 h postinjection . effect of l - lysine co - injection on the tumor and kidney uptake of tc-4 at 2 h postinjection . the blue and green columns represent the tumor and renal uptake of tc-4 without and with l - lysine co - injection : ( ) p < 0.05 for determining the significance of differences in tumor and kidney uptake between tc-4 without and with l - lysine co - injection . because tc-4 showed the highest tumor uptake and the lowest renal uptake than the other five tc - peptides at 2 h postinjection , we further determined the tumor imaging property , specificity of tumor uptake , and urinary metabolites of tc-4 in b16/f1 melanoma - bearing c57 mice . whole - body spect / ct image at 2 h postinjection is presented in figure 5 . flank b16/f1 melanoma lesions were clearly visualized by spect using tc-4 as an imaging probe . the urinary metabolites of tc-4 at 2 h postinjection are shown in figure 6 . representative whole - body spect / ct image of b16/f1 melanoma - bearing c57 mice 2 h after injection of tc-4 without ( a ) and with ( b ) peptide blockade . in our previous reports , we have found the importance of single amino acid at the x position in the tumor targeting properties of tc - rxd - lys-(arg)ccmsh peptides in b16/f1 melanoma - bearing c57 mice . specifically , the substitution of gly in tc - rgd - lys-(arg)ccmsh with ala , thr , val , and ser improved the mc1 receptor binding affinities and enhanced the melanoma uptake in b16/f1 melanoma - bearing c57 mice . despite the promising melanoma targeting results associated with tc - rxd - lys-(arg)ccmsh peptides , it is desirable to reduce the nonspecific renal uptake ( 67135% id / g at 2 h postinjection ) of tc - rxd - lys-(arg)ccmsh peptides to facilitate their potential therapeutic applications . in this study , we substituted the positively charged lys linker with the neutral -ala linker to determine whether such linker change could reduce the renal uptake of tc - rxd--ala-(arg)ccmsh peptides . furthermore , we replaced the rad moiety with nad and ead moieties to examine whether the further reduction of the overall positive changes of tc - nad--ala-(arg)ccmsh and tc - ead--ala-(arg)ccmsh could decrease their renal uptake further . the substitution of lys linker with -ala linker slightly affected the receptor binding affinities of rxd--ala-(arg)ccmsh peptides . the receptor binding affinities of rxd--ala-(arg)ccmsh peptides were approximately 2-fold weaker than rxd - lys-(arg)ccmsh peptides , respectively . despite the fact that the -his - dphe - arg - trp- motif is the binding moiety for the mc1 receptor , the decrease in receptor binding affinity with the -ala linker substitution indicated that the linker might somehow interact with the receptor binding moiety . the decrease in receptor binding affinities of rxd--ala-(arg)ccmsh peptides also resulted in the reduction in tumor uptake of tc - rxd--ala-(arg)ccmsh peptides by 2145% in b16/f1 melanoma - bearing c57 mice . specifically , the tumor uptake of tc-4 was 79% of the tumor uptake of tc - rad - lys-(arg)ccmsh at 2 h postinjection . the substitution of lys linker with -ala linker dramatically decreased the renal uptake of tc - rxd--ala-(arg)ccmsh peptides by 6479% in b16/f1 melanoma - bearing c57 mice . for instance , the renal uptake of tc-4 was only 22% of the renal uptake of tc - rad - lys-(arg)ccmsh at 2 h postinjection . thus , we were interested in whether the replacement of arg with nle ( neutral ) and glu ( negatively charged ) could further decrease the renal uptake of tc-5 and tc-6 as compared to tc-4 . interestingly , the replacement of arg with nle and glu did not further decrease the renal uptake of tc-5 and tc-6 as compared to tc-4 . the b16/f1 melanoma lesions could be clearly visualized by spect / ct using tc-4 as an imaging probe . remarkably , tc-4 displayed comparably high melanoma uptake ( 14.67 3.81% id / g ) as tc-(arg)ccmsh and tc(edda)-hynic - ggnle - cycmshhex at 4 h postinjection . however , the renal uptake of tc-4 was higher than those of tc-(arg)ccmsh ( 11.66 1.44% id / g ) and tc(edda)-hynic - ggnle - cycmshhex ( 7.52 0.96% id / g ) . the difference in renal uptake was likely due to the structural differences among these tc - peptides . interestingly , we found that l - lysine co - injection significantly reduced the renal uptake of tc-4 by 35% at 2 h postinjection without affecting its tumor uptake significantly ( figure 4 ) . therefore , l - lysine co - injection could be utilized to further decrease the renal uptake of re - rad - lys-(arg)ccmsh to facilitate its potential therapeutic application . the substitution of the lys linker with the -ala linker dramatically decreased the renal uptake of tc - rxd--ala-(arg)ccmsh peptides . among these six tc - peptides , tc-4 exhibited the highest tumor uptake and the lowest renal uptake at 2 h postinjection . the replacement of arg with nle and glu did not further decrease the renal uptake of tc-5 and tc-6 as compared to tc-4 . intermediate scaffolds of h2n - arg(pbf)-ser / thr / val - asp(otbu)-dtyr(tbu)-asp(o-2-phenylisopropyl)--ala - cys(trt)-cys(trt)-glu(otbu)-his(trt)-dphe - arg(pbf)-trp(boc)-cys(trt)-arg(pbf)-pro - val and h2n - arg(pbf)/nle / glu(otbu)/-ala - asp(otbu)-dtyr(tbu)-asp(o-2-phenylisopropyl)--ala - cys(trt)-cys(trt)-glu(otbu)-his(trt)-dphe - arg(pbf)-trp(boc)-cys(trt)-arg(pbf)-pro - val were synthesized on sieber amide resin . the protecting groups were totally removed by treating with a mixture of tfa , thioanisole , phenol , water , ethanedithiol , and triisopropylsilane ( 87.5:2.5:2.5:2.5:2.5:2.5 ) for 2 h at room temperature ( 25 c ) . the ic50 values of the peptides for the mc1 receptor were determined in b16/f1 melanoma cells . after being washed with binding medium { modified eagle s medium with 25 mm n-(2-hydroxyethyl)piperazine - n-(2-ethanesulfonic acid ) ( hepes ) , ph 7.4 , 0.2% bovine serum albumin ( bsa ) , 0.3 mm 1,10-phenathroline } , the cells were incubated at 25 c for 2 h with approximately 30 000 counts per minute ( cpm ) of i-(tyr)-ndp - msh in the presence of increasing concentrations ( 1010 m ) of each peptide in 0.3 ml of binding medium . briefly , 10 l of 1 mg / ml sncl2 in 0.1 m hcl , 40 l of 0.5 m nh4oac ( ph 5.2 ) , 100 l of 0.2 m na2 tartate ( ph 9.2 ) , 100 l of fresh tco4 solution ( 3774 mbq ) , and 10 l of 1 mg / ml of each peptide in aqueous solution were added into a reaction vial and incubated at 25 c for 20 min to form the tc - labeled peptide . the biodistribution properties of the tc - peptides were determined in b16/f1 melanoma - bearing c57 female mice ( harlan , indianapolis , in ) . the specificity of tumor uptake was determined by co - injecting each tc - peptide with 10 g ( 6.1 nmol ) of unlabeled ndp - msh at 2 h postinjection . the effect of l - lysine co - injection on the renal uptake of tc-4 was examined in b16/f1 melanoma - bearing c57 . the mice were sacrificed at 2 h postinjection , and tumor and kidneys were harvested , weighed , and counted . tc-4 was the lead peptide because of its highest tumor uptake and the lowest renal uptake . approximately 3.74.1 mbq of tc-4 with or without 10 g ( 6.1 nmol ) of unlabeled ndp - msh was injected into b16/f1 melanoma - bearing c57 mice via the tail vein , respectively . approximately 3.7 mbq of tc-4 was injected into a b16/f1 melanoma - bearing c57 mouse via the tail vein to determine the urinary metabolites .